Guide to Research
Techniques in
Neuroscience
Second Edition
Matt Carter
Jennifer Shieh
	
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xiii
Foreword to the Second Edition
When Guide to Research Techniques in Neuroscience was published in 2010,
the field was already vibrant with an abundance of experimental approaches
and research methods for studying the brain. Amazingly, the ingenuity of
neuroscientists to develop more diverse and more powerful techniques in the
past 5 years has already rendered the first edition somewhat out of date. New
technologies allow neuroscientists to measure and manipulate neural activity
with unprecedented spatial and temporal precision, to create brain–machine
interfaces in a variety of animals, including humans, and even to turn the
brains of animals completely transparent for remarkably detailed anatomical
characterization.
The enthusiasm for advancing techniques in neuroscience has never been
stronger. In 2013, US President Barack Obama launched the BRAIN (Brain
Research through Advancing Innovative Neurotechnologies) Initiative, a
bold enterprise spanning multiple federal agencies and private sector partners
to accelerate the development and application of new, sophisticated
tools to understand the brain. We have enormous potential to continue to
build on technology in imaging and electrophysiology to better examine the
collective actions of thousands or even tens of thousands of neurons; what
we have seen to date is only the tip of the iceberg. Additionally, the ability
to manipulate neural activity in highly specific populations of neurons is
allowing for remarkable advances in basic neuroscience research in animal
models as well as potentially therapeutic applications in humans. Like the
US, a number of other countries are also pursuing exciting neuroscience initiatives.
We are at a unique point in the history of neuroscience—in the next
several years, we have an opportunity to amplify our previous achievements
by combining exciting new technologies with important research questions.
In the face of these exciting new techniques, classical approaches to brain
research still have an important role. New techniques are not replacing traditional
methods so much as they are standing on their solid foundation. Excellent
studies continue to rely on behavioral, histological, and electrophysiological
methods that have been the hallmark of neuroscience research for dozens of
years. Therefore, this book is even more important now than when it was first
published. Future neuroscience studies will depend on a common framework of
techniques, and this guide provides that solid foundation. As before, this book
serves as an excellent resource for anyone from the beginning graduate student
to the seasoned faculty member who could use an efficient, cogent guidebook
xiv  Foreword to the Second Edition
by their side. Here’s to the amazing advances we have made in the past and the
exciting possibilities we have for the future!
William T. Newsome, PhD
Professor, Department of Neurobiology
Stanford University
Investigator, Howard Hughes Medical Institute
xv
Foreword to the First Edition
Understanding how the brain works is perhaps the greatest challenge facing
contemporary science. How mental life is rooted in the biology of the brain
is intrinsically fascinating, and researchers from remarkably diverse scientific
backgrounds are being drawn increasingly to the field of neuroscience. Psychologists,
molecular biologists, physiologists, physicists, engineers, computer
scientists (and more) are all contributing importantly to the richness of modern
neuroscience research. The brain remains the most complex and enigmatic
entity in the known universe, and all relevant scientific techniques and perspectives
must ultimately be brought to bear to solve its mysteries.
The quickening pace and diversity of neuroscience research pose substantial
challenges, however, for new students and established researchers seeking to
enter the field for the first time. The techniques used to measure and manipulate
the nervous system are dizzying in their scope and complexity, and some of
the important new approaches are accompanied by clouds of jargon that are all
but impenetrable to outsiders. Fortunately for those of us who are perplexed, a
concise, no-nonsense guidebook has now arrived. The Guide to Research Techniques
in Neuroscience presents the central experimental techniques in contemporary
neuroscience in a highly readable form. It is all there—from brain
imaging to electrophysiology to microscopy to transgenic technologies. Matt
Carter and Jennifer Shieh take us on a very practical cook’s tour of research
techniques, all the while providing concise overviews of exactly where and
how particular techniques fit into the grand scheme of the basic questions being
asked of the nervous system.
As a neurophysiologist, I read quickly through the electrophysiology and
brain imaging chapters, noting with appreciation the efficient presentation of
basic information that should be in the intellectual repertoire of any aspiring
neuroscientist. But I read avidly the chapters on cloning, gene delivery, and use
of transgenic animals—the exotica (to me!) of molecular biological approaches
made clean, simple, and pleasingly devoid of specialist vocabulary. I rather suspect
that most neuroscience professionals will have the same experience reading
this book, encountering high-yield veins of useful information interspersed
with much they already know. For the student or postdoc just entering the field,
however, the majority of the book will be very high yield.
This book is an essential resource for anyone—from the beginning graduate
student to the seasoned faculty member—who could use an efficient
­guidebook at his or her side while reading papers written by any of their
xvi  Foreword to the First Edition
colleagues, irrespective of the level of analysis, from small molecules to spike
trains. It may not render the mysterious raster plots of systems lectures immediately
clear, but it will orient you to the key types of information you want
to take home from that talk. The book is a little gem. Read it, rely on it, and
pass it on to others.
William T. Newsome, PhD
Professor, Department of Neurobiology
Stanford University
Investigator, Howard Hughes Medical Institute
xvii
Preface to the Second Edition
It has been only 5 years since the publication of the first edition of Guide to
Research Techniques in Neuroscience, yet the rapid pace of biological discovery,
enabled by finer tools and more powerful techniques, has already made a
second edition necessary. In this new edition, we have added recently developed
methods as well as older methods that have been more widely adopted among
neuroscience labs. Many of these methods are used to stimulate or inhibit neural
activity, and therefore we decided to add an entirely new chapter, Manipulating
Neural Activity. In addition to including new information, we have made this
latest edition a full-color book and redesigned most of the nearly 200 figures to
increase detail and clarity.
In the past 5 years, we have also come a long way in our professional
lives, leaving graduate school, and attaining full-time scientific positions.
Matt completed a postdoc in the lab of Richard Palmiter at the University of
Washington and is now an assistant professor of biology at Williams College
in Williamstown, MA. Jennifer advanced to become an AAAS Science and
Technology Policy Fellow in Washington, DC and is now a program officer
at the National Institutes of Health. While we look ahead to our futures, we
are excited that “Understanding Techniques in Neuroscience,” the course we
started at Stanford almost a decade ago, lives on among the current graduate
students, with versions being taught at many other colleges and universities
throughout the country.
Thank you to the Elsevier team for their support and patience: Natalie Farra,
for seeing that it was time to write the second edition, and Kathy Padilla, for
making sure that our project stayed on track. Obviously, our amazing contributors
to the first edition still earn our gratitude. For updates to this second edition,
we especially thank Moriah Thomason, Matt Kaufman, Astra Bryant, and
Matthew Goodman for their valuable input. Jennifer would also like to thank
the AAAS Science and Technology Policy Fellow community and its vibrant
Neuropolicy group for expansive discussions, helpful advice, and allowing her
to stay connected to her first love—the brain.
Finally, nothing would be possible without the love, encouragement, and
support from our significant others. Jennifer offers a big thank you to Steve,
while Matt owes his wife Ali big time. And 4-year-old Liam and baby Hadley
as well! Thank you!
The next 5 years are bound to be even more exciting, especially because of
new funding efforts such as the BRAIN (Brain Research through Advancing
xviii  Preface to the Second Edition
Innovative Neurotechnologies) Initiative to spur the advancement of new technologies
to better understand the nervous system at multiple levels. If the pace
of the next 5 years matches that of the preceding 5 years, we are in for a fun
ride!
Matt Carter, Williamstown, MA
Jennifer C. Shieh, Washington, DC1
September 2014
1. Contributions provided by Jennifer C. Shieh to this work were written in a personal capacity
and do not necessarily represent the opinions or endorsement of the National Institutes of Health,
Department of Human Health Services, or the Federal Government.
xix
Preface to the First Edition
The story of why we decided to write this book can essentially be summarized
as follows: We tried to find a book just like it, could not find one anywhere, and
ultimately decided the book would be so useful that we would write it ourselves.
When we were advanced undergraduates and beginning graduate students,
we found the number of techniques used in neuroscience research daunting. In
no other biological field are students required to learn biochemistry, molecular
biology, genetics, electrophysiology, microscopy, histology, behavioral assays,
and human imaging technologies, not to mention the basic biological and physical
properties that allow these methodologies to work. It is true that each neuroscientist
only practices a handful of these general techniques. However, all
neuroscientists must learn to understand and appreciate each other’s research,
even if they do not perform the specific experiments themselves.
For example, consider the study of the auditory system. Auditory research
can be performed in humans using functional magnetic resonance imaging
(fMRI) and other methods of whole-brain imaging. Auditory research can also
be performed in animal models, from standard laboratory animals (mice, rats,
flies) to animals that have unique auditory properties that make them particularly
well-suited for auditory research (bats, barn owls). These animal models
can be studied using in vivo electrophysiology while they are anesthetized or
while performing behavioral tasks. Additionally, the cells responsible for hearing
and interpreting auditory stimuli can be examined in vitro, for example, by
recording electrical signals from isolated hair cells. Histological and biochemical
techniques can highlight the specific ion channels and other proteins that
provide auditory cells their physiological properties. Genetic methods can be
used to investigate the role of molecules that make hearing possible or cause
auditory deficits. Thus, anyone interested in the auditory system, or any other
specific subfield of neuroscience, must be prepared to read about and analyze
the contributions of studies using a wide spectrum of techniques.
As students new to neuroscience research, we were overwhelmed with the
expectation that we understand all of these methods. Moreover, we were surprised
by the number of advanced students who seemed to pretend that they
understood all these techniques, when they really only understood the specific
techniques they used in their own studies. Sometimes, these students and even
some postdocs and faculty would confess to us, “I should have learned that long
ago but I just don’t really understand it.” We would hear scientists who use electrophysiology
and fMRI techniques sometimes say, “Who cares about a blob on
xx  Preface to the First Edition
a gel?” Likewise, we would hear geneticists and molecular biologists confess
“I don’t know what the squiggly lines in electrophysiology studies mean,” or
“I don’t know why fMRI experiments are so hard—can’t you just put someone
in a scanner and turn on the machine?” In short, it became obvious that there
should be a guide to neuroscience techniques. So we tried to find one.
Finding a guide to neuroscience techniques turned out to be more difficult
than we imagined. During our first trips to the annual Society for Neuroscience
conference, we tried to find a simple techniques book among the hundreds of
books being promoted by different publishers. Amazingly, there was no single
book that aimed to explain the breadth of neuroscience techniques at a basic
level. There were detailed books written about single techniques (“PCR Methods”
or “Making Transgenic Animals”) and recipe books filled with protocols,
but no book addressed techniques as a subject themselves. Even the almighty
Internet did not provide thorough insight into standard techniques. On the Internet,
you can find protocols for doing a western blot and learn that it is a technique
that measures the amount of protein in a sample. But you cannot find
information about other ways a scientist can measure protein expression, or
why you might choose a western blot over these other methods. There are no
example figures from the literature, nor are there suggestions of what to look for
in these figures. Our dream book would contain a description of western blots, a
comparison of western blots with similar methods, and an example of what the
data looked like in the literature.
No such book existed, and we wanted one. With the encouragement of faculty
members and students at Stanford, we decided to write this book. We also
decided that the information would make an excellent seminar course. Along
with a talented student colleague of ours, Saul Villeda, we created a 9-week
techniques course called “Understanding Techniques in Neuroscience.” This
class surveys neuroscience techniques and provides examples from the literature.
We wrote a 110+ page course reader to accompany the class and quickly
decided that this text should also form the basis of a future book that could exist
independently of the class.
The class was amazingly well received. The first year, our class had 15 students.
Through word of mouth, the class doubled in size the next year. The
third year, over 100 students attended our lectures, including undergraduates,
graduate students, postdocs, and occasionally a faculty member. We believe the
number of people taking the course demonstrates that neuroscientists are interested
in learning about each other’s methods, as well as the need for formalized
education about neuroscience techniques.
Therefore, we decided to adapt our course reader into this book. It is the
book that we so desperately wanted when we were beginning neuroscience
students, and we are happy that it is now possible to find at Society for Neuroscience
meetings. We learned a tremendous amount of information while
researching and writing this book, and we hope that you find it helpful for your
own education.
Preface to the First Edition  xxi
We would like to thank the editorial staff at Elsevier/Academic Press for
making this book a reality. From Susan Lee, who initially saw the potential of
our course reader, to Melissa Turner and Mica Haley, who managed to get the
final product with only minor hair pulling.
This book would not have been possible without the guidance and help of
the incredible faculty at Stanford. In particular, Bill Newsome provided tremendous
encouragement and support throughout the writing process. As promised,
he will receive a fine bottle of Woodford Reserve upon the successful publication
of this book. We must also acknowledge the understanding of our research
advisors Luis de Lecea and Sue McConnell. As promised, they will receive our
theses without further delay. Finally, we thank Mehrdad Shamloo and Mehrdad
Faizi at Stanford’s Behavioral and Functional Neuroscience Laboratory and Jon
Mulholland of the Cell Sciences Imaging Facility for their expert advice.
Our teaching partner Saul Villeda researched many of the methods that are
detailed in this book and helped prepare the original course reader. Saul is an
amazing teacher and it has been our pleasure to work with him over the years,
both as colleagues and friends.
We also received substantial guidance and support from the Stanford neuroscience
community. So many students and postdocs spent their valuable
time and energy to help us understand techniques, edit our chapters, and provide
encouragement as we finished the book. Special thanks to Raag Airan,
Bjorn Brembs, Brittany Burrows, Laurie Burns, Kelsey Clark, Emily Drabant,
Lydia-Marie Joubert, Rachel Kalmar, Jocelyn Krey, Dino Leone, Jon Mulholland,
Scott Owen, Georgia Panagiotakos, Chris Potter, Elizabeth Race, Victoria
Rafalski, Andreas Rauschecker, Magali Rowan, Rory Sayres, Bob Schafer, Jana
Schaich Borg, John Wilson, and Sandra Wilson, who provided substantial suggestions
to improve the content and text. We completed this book with our neuroscience
colleagues in mind, and we hope that all future neuroscience students
find this book helpful to their education and throughout their careers.
Finally, we thank our significant others, Vishal Srivastava and Alison Cross.
Thank you SO much for giving us time, love, and encouragement.
Matt Carter
Jennifer C. Shieh
Stanford, CA, 2009
xxiii
Introduction
The human mind has been studied for thousands of years, but the human brain,
as well as the brains of other species, has only been studied for about a century.
Only 150 years ago, the ability to study the nervous systems of humans and
other animals was limited to direct observation and by examining the effects of
brain damage in people and other organisms. With the advent of histology came
the ability to visualize and differentiate between neurons based on morphology.
The great neuroscientist Santiago Ramón y Cajal used a method called
Golgi staining to visualize the morphology and architecture of neurons and their
circuits throughout the brain. Cajal used the Golgi stain to propel the field of
neuroscience into its modern state.
Indeed, in the history of neuroscience, each leap forward in knowledge has
been based on a leap forward in techniques and technology. Just as Ramón y
Cajal used Golgi staining to greatly advance our understanding of the structure
of the nervous system, scientists throughout the twentieth century used
more and more advanced techniques to contribute to our understanding of the
function of the nervous system: Eccles, Hodgkin, and Huxley used intracellular
recording technology to investigate the ionic basis of membrane potentials;
Hubel and Wiesel used extracellular recording technology to investigate how
information is processed and recorded in the visual system; Neher and Sakmann
used patch-clamp technology to investigate the physiology of single-ion
channels. In the latter half of the twentieth century, the explosion of molecular
biology techniques and methods of genetically manipulating model organisms
allowed neuroscientists to study individual genes, proteins, and cell types.
Technology has progressed so far in the past 100 years that the Golgi stain
itself seems to have been reinvented through powerful technologies (Chapter 12)
that allow investigators to turn specific neurons different colors to further investigate
the structure and connectivity of the nervous system. In fact, technologies
are developing at such a rapid pace that in 2013, U.S. President Barack
Obama announced the BRAIN (Brain Research through Advancing Innovative
Neurotechnologies) Initiative to double federal funding for new techniques and
technologies to accelerate our understanding of the nervous system.
The modern neuroscientist now has hundreds of techniques that can be used
to answer specific scientific questions. This book contains 15 chapters that provide
an overview of the most commonly used techniques. Although there are
dozens of techniques that seem very different at first glance, many of them
xxiv Introduction
attempt to study the nervous system in the same way. For example, transcranial
magnetic stimulation (Chapter 1), physical lesions (Chapter 8), optogenetic
inhibition (Chapter 8), and genetic knockdown or knockouts (Chapter 13) are
all attempts to test the effect of a loss-of-function of some aspect of the nervous
system on another aspect of the nervous system. For each level of investigation
(whole brains to individual genes), research strategies can be similar in concept
even if the techniques used are very different in practice.
LEVELS OF INVESTIGATION
Something immediately obvious to all students of neuroscience is that the nervous
system is exceptionally complicated and can be examined at multiple
levels of investigation. The basic functional unit of the nervous system is the
neuron. The human brain is composed of approximately 100 billion neurons
that are connected into circuits via approximately 100 trillion synapses. Neural
circuits are organized into anatomical structures and larger networks of neurons
that can integrate information across modalities from many different parts of the
brain. These networks process sensory information from the external and internal
environment and provide the neural basis of cognition—learning, memory,
perception, decision-making, emotion, and other higher-order processes. The
final output of the nervous system is a behavior composed of a coordinated
motor action. This behavior can be either extremely simple, such as a motor
reflex, or incredibly complicated, such as dancing, typing, or playing a musical
instrument. Behavior is usually defined not just by what an organism does, but
what it chooses to do. Therefore, except in rare circumstances of lesion or disease,
cognition and behavior are inseparably linked, and in animals other than
humans, behavior is used as a readout of animal cognition.
Just as one can start with a neuron and scale up toward circuits, cognition,
and behavior, a scientist can also scale down and examine the components that
make up a neuron. A neuron is itself defined as having a cell body (soma), axon,
and dendrites. These neuronal components contain subcellular specializations
that make the neuron unique among other cell types. Specialized organelles in
a neuron, such as vesicles containing neurotransmitters, provide the cell with
the ability to signal to other neurons. Specialized cytoskeletal elements allow a
neural process to extend great distances throughout the brain and body. Several
proteins provide neurons with their intercellular signaling abilities and physiological
characteristics. For example, biosynthetic enzymes produce neurotransmitters,
while other proteins serve as receptors for these signaling molecules.
One of the most important types of proteins in the nervous system form ion
channels, the transmembrane structures that allow neurons to become electrically
active under certain conditions. All of these proteins are the products of
genes, the functional units of an organism’s genome. The human genome contains
approximately 30,000 genes, with each neural subtype expressing its own
subset of these genes.
Introduction  xxv
The complexity of the nervous system is awesome in scope. It is amazing
that a mutation in a single gene, such as a gene that codes for a transmembrane
ion channel, can produce effects that alter the electrical properties of a neuron,
in turn altering the normal firing patterns of a neural circuit and thus causing an
abnormal behavior.
A neuroscientist can approach the study of the nervous system through any
of these levels of organization. The 15 chapters of this book provide a guide to
the types of experiments that can be performed at each level. However, irrespective
of technique, the basic scientific approach one can use to study the nervous
system is consistent from level to level, whether the subject is human cognition
or axon guidance in cell culture.
METHODS OF STUDYING THE NERVOUS SYSTEM
There are four general methods of studying the nervous system:
	1.	Examining case studies—identifying interesting events that have occurred
naturally and using these events to develop hypotheses that can be tested in
future experiments
	2.	Screens—performing unbiased searches for anatomical structures, neurons,
proteins, or genes that could play a role in a subject of interest
	3.	Description—using techniques that allow a scientist to observe the nervous
system without manipulating any variables
	4.	Manipulation—testing hypotheses by determining the effect of an independent
variable on a dependent variable
Each of these four methods is described in more detail below.
Examining Case Studies
A case study is an example of an event that happened to a subject (most
often a human or group of humans) that demonstrates an important role for
an aspect of the nervous system. The circumstances surrounding the event
are usually nonrepeatable and cannot be precisely recreated in a laboratory
setting. Such demonstrations are, therefore, not true experiments in that no
variables are deliberately controlled by a scientist. However, these events
can often reveal substantial information about an aspect of neural function
that was previously unknown.
For example, consider the case of Phineas Gage, a railroad worker who was
involved in an accident in 1848 that caused an iron rod to pass through his
skull. The rod entered the left side of his face, passed just behind his left eye,
and exited through the top of his head, completely lesioning his frontal lobes.
This is an amazing event, not only because Gage survived (and lived for another
12 years), but also because it informed scientists about the function of the frontal
lobe of the brain. The event allowed investigators to retrospectively ask the
question: “What is the effect of removing the frontal lobe on consciousness and
behavior?” According to Gage’s friends, family, and coworkers, he was “no
xxvi Introduction
longer Gage.” He retained the ability to learn, remember, sense, and perceive
his environment, to execute motor functions, and to live a fairly normal life,
but it seemed to people who knew him that his personality had changed completely.
After the accident, Gage was described as impolite, erratic, unreliable,
and offensive to others. He wound up losing his job at the railroad, not because
of any physical or mental incapacity, but because he was simply so disrespectful
and offensive that people could not stand to work with him.
This case study is not a true experiment; no scientist decided to test the
removal of the frontal lobe on personality. But the incident, and others like it,
allows neuroscientists to form hypotheses based on naturally occurring events.
Because of Gage’s story, neuroscientists could hypothesize about the contribution
of the frontal lobe to human personality. Future experiments could test
these hypotheses on animal models (that share certain human personality traits)
and even attempt to identify neural circuits that contribute to human behaviors.
Screens
A screen is a method that allows an investigator to determine which nuclei, neurons,
or genes/proteins may be involved in a particular biological process. Such
experiments are not necessarily driven by a hypothesis, but the experiment identifies
candidates that can form the basis for future hypothesis-driven research. For
example, a neuroscientist who wants to identify genes involved in body weight
regulation may compare gene expression profiles in central feeding centers of the
brain in both fed and starved animals: genes expressed in starved animals relative
to fed animals may be important for generating the motivation to eat.
Screens can be performed at multiple levels of investigation. When a cognitive
neuroscientist places a human subject into an fMRI scanner and examines which
brain areas show increased activation in response to a specific stimulus or task, the
scientist is essentially performing a screen of different brain regions. When a fly
geneticist examines thousands of mutagenized flies for deficiency in a behavioral
task, the scientist is attempting to identify genes necessary for that behavior to
occur. Such genes can then be tested in future experiments. Thus, screens can be
performed to identify interesting molecules or entire brain regions.
Description
Descriptive science is the act of observing properties of the nervous system
without manipulation. This type of research is usually the first step in acquiring
knowledge about a newly discovered gene, protein, or neuronal subtype. For
example, an investigator could describe the sequence of a gene and where in
the brain the gene is expressed. Likewise, in the case of proteins, an investigator
could describe the amino acid sequence of the protein and where in the brain the
protein is expressed. Neurons can be described in terms of what genes/proteins
they express, their morphology, how many neurons make up a population of
neurons, and their electrophysiological properties.
Introduction  xxvii
It is important to note that just because a study may be descriptive does not
mean that it is necessarily easier than other types of experiments. Observation
and description form the foundation for understanding the relationship between
structure and function, as well as providing insight about what elements to
manipulate in future experiments. Thus, descriptive neuroscience plays just as
important a role in modern research as when Ramón y Cajal observed the structure
of neurons 100 years ago.
Manipulation
Manipulating an aspect of the nervous system or environment and examining
the effect this perturbation has on a separate aspect of the nervous system is
the only way to test a hypothesis in neuroscience. A manipulation experiment
tests the effect of X onY. The variable that is manipulated, X, is referred to as the
independent variable. The part of the system that is measured, Y, is referred to
as the dependent variable.
Two of the most common types of manipulation experiments are loss-offunction
and gain-of-function experiments. In a loss-of-function (also called
“necessity”) experiment, a part of the nervous system is diminished or removed
in an attempt to determine if it is necessary for a certain process to occur. The
following questions are all loss-of-function questions:
	l	Is a normal copy of the gene Fezf2 necessary for the proper development of
the cerebral cortex?
	l	Is the receptor for the hypocretin neuropeptide necessary for normal sleep/
wake transitions in mammals?
	l	Is electrical activity in the medial geniculate nucleus required for auditorydriven
spikes in the auditory cortex?
	l	Can human patients with damage to the cerebellar vermis perform as well as
healthy controls on a verbal-memory task?
In all of these experiments, an aspect of the nervous system is partially
or totally disrupted, whether it is a gene, a protein, electrical activity, or an
entire brain structure. The independent variable is the loss of the structure, and
the dependent variable is the effect on another aspect of the nervous system.
Sometimes, a good follow-up for a loss-of-function experiment is a rescue
experiment in which the aspect of the nervous system that is lost is deliberately
returned. For example, if it is found that a certain line of fruit flies lacks a gene
that is necessary for proper development of an eye, the specific gene can be
reintroduced to the flies in transgenic experiments to see if the functional gene
can rescue the aberrant phenotype.
In a gain-of-function (also called “sufficiency”) experiment, an aspect
of the nervous system is increased relative to normal. This may include an
increased expression of a gene or protein, an increase in electrical activity in a
brain region, or an increase of a particular neurotransmitter in the extracellular
medium. The aspect of the nervous system that is increased is the independent
xxviii Introduction
variable, and the effect on another part of the nervous system is the dependent
variable. The following questions are all gain-of-function questions:
	l	Can an increase in the gene TrpA1 cause mice to be hypersensitive to cold
temperatures?
	l	Can an intracerebroventricular injection of neuropeptide Y cause an increase
in feeding behavior in rats?
	l	Can electrical microstimulation of the lateral geniculate nucleus increase
spike frequencies over time in area V4 of the visual cortex?
	l	Can stimulation of the motor cortex using transcranial magnetic stimulation
induce motor behaviors?
In both loss-of-function and gain-of-function experiments, it is important not
to overstate the conclusions of the experiments. For example, consider a loss-offunction
experiment in which a mouse that lacks a gene is unresponsive to painful
stimuli. The investigator could conclude that this gene is necessary for proper performance
on an assay for pain detection. However, an inappropriate conclusion
would be that this gene regulates pain detection. Perhaps this gene is responsible
for normal development of the spinal cord and the mouse lacks all peripheral sensation.
Alternatively, this gene may code for a protein that is necessary for normal
development of the thalamus; if improper development of the part of the thalamus
that receives information about painful stimuli causes the stimuli not to reach the
somatosensory cortex, the animal will not perform normally on the pain-detection
task. Careful controls are necessary to reach appropriate conclusions.
UNDERSTANDING TECHNIQUES IN NEUROSCIENCE
We hope that these 15 chapters serve as a useful guide to studying the nervous
system. There are two important points to keep in mind while reading these
chapters:
	1.	All of the techniques described throughout this book depend on the principles
just mentioned. For each level of investigation that a scientist may
choose to study, the same four general categories of methods exist: examining
case studies, screens, description, and manipulation. The same principles
that are used to study brain activity in awake human subjects are used
to study genes and proteins in tissue samples. The methods may vary, but the
principles remain the same.
	2.	It is also important to remember that techniques and methods should
never be the guiding force behind doing experiments in research. Ideally,
experiments should be performed to answer an interesting question, not
the other way around. A technique should not be used for its own sake but
because it is the best technique available to answer a particular research
question.
We hope this book answers your questions about what techniques are available
in modern neuroscience research, but more importantly, we hope these
techniques answer your specific research questions as well!
1
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00001-0
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 1
Whole-Brain Imaging
After Reading This Chapter, You Should be Able to:
	l	Compare the relative strengths and limitations of different structural and functional
brain imaging techniques
	l	Explain the physical and physiological basis of (MRI/fMRI) technology
	l	Describe the design of a functional brain imaging experiment: formulating a
hypothesis, choosing task paradigms, performing the experiment, acquiring and
analyzing data, and constructing figures
Techniques covered:
	l	Structural techniques: cerebral angiography, computerized tomography (CT), MRI,
and diffusion MR imaging
	l	Functional techniques: fMRI, positron emission tomography (PET), single-proton
emission computerized tomography (SPECT), electroencephalography (EEG), magnetoencephalography
(MEG), and optical imaging
	l	Techniques used to investigate the necessity and sufficiency of a specific brain
region for a cognitive function: transcranial magnetic stimulation (TMS), ultrasonic
neuromodulation (USNM), and case studies
Modern brain imaging technology can seem like magic. The ability to produce
detailed images of the human brain without physically penetrating the skull is
a technological marvel that has saved thousands of lives and allowed scientists
to study the structure of the brain throughout development, disease, and
aging. Furthermore, the ability to image neural activity in the brain during cognition
has provided scientists the opportunity to correlate activity in distinct
brain regions with specific mental operations, a truly remarkable achievement.
Colorful figures depicting activity in the human brain dazzle scientists and nonscientists
alike.
Of course, brain imaging technology is not magic. The technology that
produces detailed images of the brain depends on complex physics, expensive
equipment, and skilled technicians. As with all scientific experiments, brain
imaging studies must be well designed, the data accurately analyzed, and the
results carefully interpreted. The purpose of this chapter is to explain the ostensible
magic of whole-brain imaging technology and provide insight into how
experiments are designed and interpreted.
2  Guide to Research Techniques in Neuroscience
Whole-brain imaging technology can essentially be divided into two categories:
structural and functional. Structural techniques produce images of the
anatomical architecture of the brain, whereas functional techniques produce
images of the physiological processes that underscore neural activity. This
chapter will survey both classifications of techniques and describe how they can
be used in modern neuroscience research. We focus on MRI and fMRI technology
in humans due to the widespread use of these techniques in the neuroscience
literature. After reviewing these techniques, we will survey the essential
components of a functional imaging experiment: forming hypotheses, choosing
appropriate task paradigms, performing experiments, acquiring and analyzing
data, and producing figures for publication.
STRUCTURAL BRAIN IMAGING TECHNIQUES
Structural brain imaging techniques are used to resolve the anatomy of the brain
in a living subject without physically penetrating the skull. These techniques can
be used in combination with functional brain imaging techniques to correlate
neural activity in specific anatomical regions with behavioral or cognitive functions.
Structural techniques can also be used to measure anatomical changes that
occur over time, such as a decrease in brain mass that occurs with aging or the
progression of disease. Most often, these techniques are used in clinical neuroscience
and neurology to diagnose diseases such as tumors and vascular disorders.
Brain imaging technologies take advantage of the different biochemical
compositions of brain regions and use these differences to form the basis of an
image (Figure 1.1). Neural cell bodies contain many biomolecules, including
proteins and carbohydrates. Axons and fiber tracts are relatively fatty due to
the insulation provided by myelin. Cerebrospinal fluid (CSF) in the ventricles
and surrounding the brain is essentially a saline solution. The microanatomy
and composition of individual neural structures cause distinct regions of the
brain to appear different when examined with the naked eye. For example, when
examining slices of a brain, tissue mostly composed of cell bodies appears gray
compared with other areas and therefore is referred to as “gray matter.” Brain
tissue mostly composed of axons and fiber tracts appears white and therefore is
referred to as “white matter.” Often, the most informative structural images of
the brain show the contrast between gray and white matter. Thus, the ultimate
goal of structural imaging technologies is to differentiate between proteins and
carbohydrates (cell bodies), fat (axon tracts), and salt water (CSF), as this contrast
reveals the most information about brain architecture.
Until the early 1970s, there was no technology that could differentiate
between these substances within the brain. Conventional X-ray technology is
essentially useless for this purpose. During an X-ray procedure, an X-ray beam is
passed through an object and then onto a photographic plate (Figure 1.2A). Each
of the molecules through which the beam passes absorbs some of the radiation,
so only the unabsorbed portions of the beam reach the photographic plate. X-ray
Whole-Brain Imaging Chapter | 1  3
photography is, therefore, only effective in characterizing internal structures that
differ substantially from their surroundings in the degree to which they absorb
X-rays, such as bone in flesh (Figure 1.2B). By the time an X-ray beam passes
through the relatively soft consistency of the brain (not to mention the relatively
hard consistency of the skull!), little information about individual brain structures
can be discerned (Figure 1.2C). Therefore, in the 1960s and 1970s, there was
strong motivation to discover better ways of imaging the brain. The techniques
described in the following sections represent 30–40 years worth of innovation
in technology ultimately designed to show contrast within the soft tissue of the
brain.
Cerebral Angiography
A cerebral angiogram is an enhanced X-ray that uses dyes to make up for the
relatively poor soft-tissue contrast of conventional X-rays. A radio-opaque dye
that absorbs X-rays better than surrounding tissue is injected into an artery that
delivers blood to the brain. This substance heightens the contrast between the
cerebral circulatory system and surrounding brain tissue during an X-ray (Figure
1.3A). Thus, the most prominent aspect of the central nervous system imaged in a
cerebral angiogram is the brain vasculature. Angiograms can show vascular damage
and indicate the presence of a tumor or aneurysm, an abnormal ballooning
of a portion of an artery due to weakening of a blood vessel wall (Figure 1.3B).
Axon terminals
Cell body
Dendrites
Axon
Myelin
(A)
Gray matter
White matter
Ventricles (with CSF)
(B)
FIGURE 1.1  The composition of the brain. (A) A microscopic view of a neuron. Each
neuron is composed of a cell body, dendrites, and an axonal process. The cell bodies and dendrites
are rich in proteins and carbohydrates. Axons are surrounded by a myelin insulation made
of fats. (B) A macroscopic view of the brain. Gray matter is rich in cell bodies and, therefore,
in proteins and carbohydrates. White matter is composed of axon tracts and is, therefore, rich
in fatty myelin. Cerebrospinal fluid (CSF) in the ventricles is a saline solution. To produce an
image, brain imaging technologies must differentiate among proteins/carbohydrates, fats, and
saline.
4  Guide to Research Techniques in Neuroscience
Computerized Tomography
Another method that improves upon conventional X-ray technology to image
the brain and body is computerized tomography (the “CT scan”—sometimes
also called computerized axial tomography, or “CAT scan”).A patient or subject
lies with his or her head positioned in the center of a cylinder (Figure 1.4A). A
narrow beam of X-rays is aimed through the person’s head and hits a detector
on the opposite side. The beam and detector rotate in a slow arc, taking many
individual X-ray scans at the same axial plane. As mentioned previously, a single
X-ray scan would supply little information about the structure of the brain.
However, multiple scans taken from different angles can be combined to provide
information about small differences in radiodensity between different brain
(A)
(B)
(C)
FIGURE 1.2  Standard X-ray technology alone cannot produce detailed images of the brain.
(A) During an X-ray procedure, an X-ray beam is passed through an object and onto a photographic
plate. Only the unabsorbed portions of the beam reach the plate, creating an image. (B) The contrast
between the soft consistency of skin and muscle compared with the hard consistency of bone is sufficient
to form an X-ray image. However, (C) the contrast between the soft consistency of different
tissues within the nervous system is insufficient to form an X-ray image of the brain.
Whole-Brain Imaging Chapter | 1  5
structures. These data are entered into a computer algorithm that constructs a
composite picture based on the X-ray scans from all the different angles. With
this information, a slice, or tomogram (tomo means cut or slice), can be generated.
Typically, 8–10 images of axial brain sections are obtained for analysis.
FIGURE 1.3  Cerebral angiography. (A) A cerebral angiogram depicting the vasculature of the
right hemisphere of the brain. (B)A cerebral angiogram indicating the presence of a brain aneurysm.
(A, B: Reprinted from Nolte, J. and Angevine, J. B. (2007). The Human Brain in Photographs and
Diagrams, 3rd ed. with permission from Mosby/Elsevier: Philadelphia. Courtesy of (A) Dr Joachim
F. Seeger and (B) Dr Raymond F. Carmody.)
Ventricles
White matterGray matter
Rotating X-ray source
X-ray detector
CT scanner
(A) (B)
FIGURE 1.4  Computerized tomography (CT). (A) During an imaging session, a narrow beam
of X-rays is slowly rotated around a subject’s head to hit a detector on the opposite side. Signals
from around the head are combined using a computer algorithm that constructs a composite picture
based on the various X-ray angles. (B) A modern CT scan can distinguish between gray and
white matter, differentiate ventricles, and depict structures with a spatial resolution of millimeters.
(B: Reprinted from Nolte, J. and Angevine, J. B. (2007). The Human Brain in Photographs and
Diagrams, 3rd ed. with permission from Mosby/Elsevier: Philadelphia. Courtesy of Dr Raymond
F. Carmody.)
6  Guide to Research Techniques in Neuroscience
The quality of a CT scan depends on the width of the X-ray beam (narrower
is better), the sensitivity of the X-ray detector, and the ability of the computer
to construct an image from the data. Modern CT scans can distinguish between
gray matter, white matter, and ventricles with a spatial resolution of millimeters
(Figure 1.4B). They are particularly useful for identifying fluid boundaries in
human patients, such as when blood collects on the brain surface in a hematoma,
or detecting hard objects in soft tissue, such as a tumor or calcification.
CT scanners are faster, cheaper to operate, and less prone to motion artifacts
than MRI scanners; therefore, they tend to be the first tool used to diagnose a
patient with a potential head injury.
Magnetic Resonance Imaging
MRI technology produces highly detailed structural images of the brain and
body. The resolution of a modern MR image is far superior to a CT image,
typically less than a millimeter. Thus, MRI technology has largely superseded
computerized tomography as the method of imaging the brain in both
clinical and research settings. The technology that makes MRI possible is
complex, but necessary to understand to fully appreciate MR images of the
brain.
The Electromagnetic Basis of MRI Technology
As the name suggests, magnetic resonance imaging takes advantage of the magnetic
properties of biological tissue to produce an image. Most often, MRI utilizes
the magnetic properties of hydrogen protons, as they are highly abundant in the
fluids and organic compounds of the brain and body. The main function of an
MRI scanner is to artificially excite these hydrogen protons and then measure
their relaxation properties over time.
An MRI scanner is composed of a long tube-like chamber, where a subject
is placed, surrounded by electric coils hidden within the MRI apparatus
(Figure 1.5). As current passes through the coils in a clockwise rotation, a
magnetic field is produced longitudinal to the subject, in the direction of the
feet to head. The purpose of putting the subject in a magnetic field is to affect
the hydrogen protons in the subject’s tissues. Protons can be thought of as
miniature magnets: they spin about an axis, and their spinning positive charge
induces a tiny magnetic field (Figure 1.6A). Normally, the magnetic fields
of individual protons orient in random directions (Figure 1.6B). However,
when a subject is placed inside the strong magnetic field of an MRI machine,
the magnetic fields of individual protons align in the axis of the field. Some
protons align parallel to the magnetic field, toward the subject’s head, while
others align in the opposite, antiparallel direction, toward the subject’s feet
(Figure 1.6C). It is slightly more energetically favorable for the protons to orient
in the parallel rather than antiparallel direction, so there is a net magnetic
field vector in the parallel direction (Figure 1.6D).
Whole-Brain Imaging Chapter | 1  7
There is one more important detail to know about the protons in a magnetic
field: they do not simply stay stationary, aligned parallel (or antiparallel)
to the magnetic field lines. Instead, they precess around their axis,
spinning like a top (Figure 1.7). The frequency with which they spin is
dependent on the strength of the external magnetic field (generated by the
MRI machine). The larger the external magnetic field, the higher the precession
frequency. The strength of a magnetic field is measured in Tesla (T).
Most conventional MRI machines create external magnetic fields at 1.5–3 T.
Newer, more powerful MRI scanners create fields at 7 T. Higher magnetic
field strengths increase the signal-to-noise ratio and give higher contrast and
spatial resolution. However, these powerful magnets are also more expensive
and more likely to cause physiological discomfort in subjects, such as nausea
or dizziness.
The MRI scanner contains
electric coils that produce a
static magnetic field in the
long axis and perpendicular
axes of the subject
A subject in an MRI
scanner rests comfortably
inside a narrow chamber
A radiofrequency coil (not
shown) is placed around
the head to perturb the
static magnetic fields and
generate the MRI signal
FIGURE 1.5  A human subject in a magnetic resonance imaging (MRI) scanner. The subject
lies inside a chamber surrounded by electric coils. Current passing through these coils induces a
strong magnetic field.
8  Guide to Research Techniques in Neuroscience
Generating an Image
Prior to the beginning of an imaging session, there is a net magnetic field ­vector
in the parallel direction, longitudinal to the subject’s body (Figure 1.8A). To
collect data for an MR image, the subject is briefly exposed to pulses of electromagnetic
energy, referred to as radiofrequency (RF) pulses (Figure 1.8B).
Applying an RF pulse that has the same frequency as the proton precession
frequency causes two effects: (1) some protons in the parallel phase absorb
energy, reverse polarity to the antiparallel phase, and therefore decrease the net
(A)
+
(C) (D)
(B)
North
South South
North
FIGURE 1.6  Protons align with the magnetic field. (A) As a proton spins around its axis, the
rotating positive charge induces a magnetic field. Therefore, a proton can be thought of as a tiny
magnet. (B) Before an external magnetic field is applied, protons orient in random directions. (C)
However, in the presence of a strong magnetic field, protons align in either a parallel or antiparallel
direction. (D) In a magnetic resonance imaging scanner, protons aligned in the parallel direction are
directed toward the subject’s head, while protons aligned in the antiparallel direction are directed
toward the subject’s feet. Slightly more protons are aligned in the parallel direction, so there is a net
magnetic force toward the subject’s head.
Whole-Brain Imaging Chapter | 1  9
longitudinal magnetization; (2) some protons get in sync and start to precess in
phase. Their vectors now add up in a direction that is transverse to the external
magnetic field (perpendicular to the subject’s body). Thus, a new transversal
magnetization is established (Figure 1.8C).
After the RF pulse is switched off, the high-energy nuclei begin to relax and
realign (Figure 1.8D). Eventually, the longitudinal magnetization increases to its
North
South
FIGURE 1.7  Protons precess around an axis in a pattern that can resemble a spinning top.
Transversal
Longitudinal
RF pulse
FIGURE 1.8  A radiofrequency (RF) pulse alters the net magnetic field. (A) Before the RF
pulse is applied, there is a net magnetic force in the longitudinal direction toward the subject’s
head. (B, C) After the RF pulse is applied, the net longitudinal field decreases, as some protons
reorient to the antiparallel orientation. Also, a new net magnetic field is created in the transverse
direction. (D) After the RF pulse is switched off, the longitudinal field increases to normal and
the transverse field decreases until the magnetic field returns to the state before the RF pulse
was applied.
10  Guide to Research Techniques in Neuroscience
original value, while the transversal magnetization decreases to zero. The time
(in milliseconds) required for a certain percentage of the protons to realign in the
longitudinal direction is termed T1. The transversal relaxation time is termed T2.
Both the information acquired in the longitudinal direction (T1) as well as
the information acquired in the transversal direction (T2) are measured by an
antenna inside the MRI scanner. Recall that the goal of brain imaging technology
is to resolve differences in tissues made of proteins and carbohydrates, fat,
and salt water. What makes MRI technology useful for producing images of the
brain is that these substances exhibit different values for both T1 and T2 relative
to each other. For any given point in time during the relaxation phase, the
T1 white matter signal is stronger than that of gray matter, and the gray matter
signal is stronger than that of CSF (Figure 1.9A). These differences in signal
intensity are exactly opposite for a T2 measurement: the CSF signal is strongest,
followed by gray matter and then white matter (Figure 1.9B). By examining the
contrast in signal from different points in space, it is possible to differentiate
between different substances and form an image.
An image of the brain formed from T1 data is referred to as a T1-weighted
image, while an image formed from T2 data is said to be T2-weighted. These
Time
T1SignalIntensity
White matter
CSF
Gray matter
Time
T2SignalIntensity
White matter
Gray matter
CSF
(A)
(C)
(B)
(D)
FIGURE 1.9  Different substances exhibit different T1 and T2 time constants.
(A) For any given longitudinal magnetization relaxation time (T1), the white matter signal
intensity will be greater than that of cerebrospinal fluid (CSF). (B) In contrast, for any given
transversal magnetization relaxation time (T2), the CSF signal intensity is greater than that for white
matter. (C) Therefore, white matter will be bright and CSF dark on a T1-weighted image, while
(D) white matter will be dark and CSF bright on a T2-weighted image. (C, D: Courtesy of
Dr Rory Sayres.)
Whole-Brain Imaging Chapter | 1  11
images appear different from one another because of the differences in signal
intensity among substances in T1- vs. T2-weighted images (Figures 1.9C and D).
As a rule of thumb, if the CSF is black, you are looking at a T1-weighted image,
as CSF exhibits the lowest relative T1 signal intensity. If, on the other hand,
the CSF appears bright white, you are looking at a T2-weighted image, as CSF
exhibits the highest relative T2 signal intensity. In the literature, most anatomical
data are presented as T1-weighted images, as these images usually show ­better
contrast between brain structures. However, sometimes T2-weighted images are
optimal—for example, lesions of white matter that occur due to the rupturing
of blood vessels are more easily detectable on a T2-weighted image. Therefore,
T2-weighted images may be most beneficial when examining patients following
trauma or stroke.
Selecting a Slice of Brain to Image
How does an investigator select a slice to examine? Recall that an RF pulse will only
excite protons with the same precession frequency as the frequency of the pulse.
The precession frequency varies with the strength of the external magnetic field, so
to select a single slice of the brain to image, an additional magnetic field is applied to
the external magnetic field at a gradient (Figure 1.10A). Because the field strength
is not equal at all planes in the longitudinal direction, an RF pulse at a specific frequency
will only affect the protons, and thus the signal, at a specific plane. This is
the slice of the brain that will be presented as a two-dimensional image.
To measure signal from individual points within the slice, two additional
magnetic gradients are applied in the other two axes (Figure 1.10B
and C). Therefore, each point in space will have its own unique magnetic
signature. Each point has a specific volume and is termed a voxel, a threedimensional
version of a pixel that represents a cubic volume of brain space.
The resolution of each voxel is determined by the values of the gradients applied
to the subject. With greater magnetic field strengths, more dramatic gradients
can be established. This is why a 3 T MRI scanner can produce an image with
higher spatial resolution than a 1.5 T scanner.
MRI has a number of features that have made it an especially valuable
research tool for both diagnostic and research studies.
	l	MRI is entirely noninvasive, as no substances need to be injected into human
subjects. Sometimes, a contrast agent is injected that enhances the visibility
of water-rich regions. However, for most brain imaging procedures, no additional
contrast is necessary.
	l	Slices of the brain can be obtained from any angle. CT scanners can only
image slices of the brain based on the axis of rotation of the X-ray emitter and
detector within the apparatus.
	l	By varying the gradient and RF pulse parameters, MRI scanners can be used
to generate images that highlight certain brain regions and provide contrast
between specific kinds of neural tissue.
	l	No X-ray radiation is applied to the subject in the scanner.
12  Guide to Research Techniques in Neuroscience
Even with these relative advantages, MRI has not completely replaced CT
imaging. CT imaging is better for visualizing bony or calcified structures in the
head, and also remains the imaging technique of choice for subjects who might
not be able to enter a strong magnetic field (for instance, due to a pacemaker)
or subjects with claustrophobia. Additionally, CT scanners have much lower
operating costs than MRI scanners, making their use relatively less expensive.
Diffusion MRI
Diffusion MRI is an application of MRI that is used to examine the structure of
axon fiber tracts in the brain. Traditional MRI images present white matter as a
homogeneous structure (as in Figures 1.9C and D). In reality, fiber tracts originate
from various sources, radiate in different orientations, and travel to distinct
regions of the brain. Diffusion MRI provides investigators the opportunity to
Magnetic field strength
Proton precession frequency
RF pulse
(A)
Magnetic field gradient in
anterior–posterior direction
Magnetic field gradient in
medial–lateral direction
(B) (C)
FIGURE 1.10  How a slice is selected in magnetic resonance imaging. (A) To image a brain
slice, an external magnetic field is applied at a gradient. An RF pulse will only excite protons in a
particular slice, as the pulse can only excite atoms with the same precession frequency. To measure
the signal intensity for each point within the slice, two additional gradients are applied, one in the
medial–lateral direction (B) and one in the anterior–posterior direction (C).
Whole-Brain Imaging Chapter | 1  13
visualize these different white matter pathways and study the complexities of
axonal architecture (Figure 1.11).
The term diffusion refers to the fact that water molecules, like all other
molecules, randomly move through a medium over time. For example, in a
glass of water, any particular molecule of water will move around randomly
in any direction, limited only by the walls of the container. This type of diffusion
is referred to as isotropic—diffusion along all directions. However, water
molecules within brain tissue diffuse quite differently due to the physical environment
of the brain. They tend to diffuse most rapidly along parallel bundles
of fibers with coherent orientations. This type of diffusion is referred to as
anisotropic—diffusion that is not equal in all directions but instead tends to
move randomly along a single axis.
Diffusion MRI is able to measure the anisotropic diffusion of water along
fiber bundles, highlighting the connectivity between brain regions. The physics
and mathematics behind this technology are too complex for this text, but
the goal is to use MRI technology to analyze the magnitude and direction of
the diffusion of water molecules for each voxel of tissue, thus creating a threedimensional
image of fiber tracts. There are various kinds of diffusion MRI
methods, the most commonly used called diffusion tensor imaging.
Diffusion MRI can provide information about which areas of the brain
are connected, but it is not able to determine the direction of this connectivity
(which endpoint is the source and which endpoint is the target). However, it is
possible to combine diffusion MRI with functional MRI, allowing researchers
to identify temporal correlations between activity in distinct regions of the brain
to make conclusions about functional connectivity between brain structures.
FIGURE 1.11  Diffusion tensor imaging. An example of an image of white matter tracts in the
corpus callosum. (Reprinted from Van Hecke, W. et al. (2008). On the construction of an intersubject
diffusion tensor magnetic resonance atlas of the healthy human brain. Neuroimage 43(1),
69–80, with permission from Elsevier.)
14  Guide to Research Techniques in Neuroscience
FUNCTIONAL BRAIN IMAGING TECHNIQUES
Functional brain imaging techniques are used to measure neural activity in
the central nervous system without physically penetrating the skull. The ultimate
goal of these techniques is to determine which neural structures are active
during certain mental operations. Although these techniques cannot demonstrate
that a brain region causes certain actions or is the specific structure that
regulates a cognitive process, they can provide other useful information. For
example, functional brain imaging techniques can show that activity in specific
brain regions is often correlated with a particular stimulus, emotional state, or
behavioral task. They can show that information is represented in certain places
within the brain, that information may be present in the brain without being
consciously perceived, and that diseased brains may process information abnormally
compared to healthy brains. Importantly, these techniques have allowed
neuroscientists the opportunity to study the neural basis of cognition, emotion,
sensation, and behavior in humans, a feat that cannot be achieved by most other
techniques in this book.
Functional Magnetic Resonance Imaging
Functional magnetic resonance imaging uses the same physical principles as
MRI to produce high-resolution representations of neural activity over time. As
with MRI technology, fMRI detects signals from excited hydrogen protons in a
magnetic field. Recall that different substances within the brain exhibit different
T1 and T2 values following stimulation with an RF pulse. In a structural imaging
experiment, these values are used to measure differences in signal intensity
between gray matter, white matter, and CSF. Functional magnetic resonance
imaging technology takes advantage of the signal intensity of another substance
within the brain: hemoglobin, the protein in the blood that carries oxygen to
cells. On a T2 image, oxyhemoglobin (the oxygen-carrying form of hemoglobin)
has a relatively stronger magnetic resonance signal than deoxyhemoglobin
(the oxygen-depleted form of hemoglobin). Thus, fMRI allows an investigator
the opportunity to examine changes in the oxygenation state of hemoglobin
over time.
The ability to examine changes in oxygen metabolism over time in the brain
is useful because it serves as an indirect measure of neural activity. Active
neurons will consume more oxygen relative to when they are at rest. Initially,
this activity decreases the levels of oxyhemoglobin and increases the levels
of deoxyhemoglobin. However, within seconds, the brain microvasculature
responds to this local oxygen depletion by increasing the flow of oxygen-rich
blood to the active area. This phenomenon is referred to as the blood oxygen
level-­dependent (BOLD) effect (Figure 1.12), and it forms the basis of the
fMRI signal. The precise physiological nature of the relationship between neural
activity and the BOLD effect is the subject of much current research.
Whole-Brain Imaging Chapter | 1  15
Functional magnetic resonance imaging depends on T2-weighted images
because the contrast in signal intensity between deoxyhemoglobin and oxyhemoglobin
is greatest on these kinds of images. In a typical experiment,
a T2-weighted image of the brain is obtained prior to the presentation of a
stimulus. After a stimulus is presented or the subject performs a task, additional
T2-weighted images are obtained. As neurons become active due to the
stimulus or task, the BOLD effect causes a relative increase in oxyhemoglobin
to the microenvironment, and thus an increase in T2-weighted signal. The
difference in T2 signal between the prestimulus and poststimulus time points
and is measured color coded for visualization of the data. These data are
typically superimposed over a T1-weighted image that more clearly depicts
the underlying anatomy of the brain. The end result is a colorful statistical
representation of neural activity superimposed on an anatomical image of the
brain—a depiction of the BOLD response over time (Figure 1.13). It is important
to understand that fMRI data in a two-dimensional figure are actually
four-dimensional: the x, y, z coordinate planes for each voxel in space, as well
as the fourth dimension of time (the before and after time points during which
the stimulus is presented).
Although fMRI technology provides a powerful tool to study the neural
basis of cognition, there are important limitations. One of the biggest challenges
in fMRI research is that the actual T2 signal change for a given voxel of brain
space, before and after BOLD changes, can be as low as 0.2%. This change is
very difficult to detect, especially given that the noise of the system can be as
high as 0.3–0.4%. Therefore, fMRI stimuli must be repeated several times for
a single subject, and a series of statistical tests must confirm the presence of a
Hemoglobin
Oxygen
(A) (B)
FIGURE 1.12  The blood oxygen level-dependent (BOLD) effect. (A) A group of neurons at
rest are supplied by blood from capillaries. (B) When these neurons become active, they increase
their metabolic demand for oxygen. The microvasculature responds by supplying more oxygenrich
blood to the local area. The relative increase in the oxygenated form of hemoglobin causes an
increase in the T2 signal.
16  Guide to Research Techniques in Neuroscience
(A)
(C)
(E) (F)
(B)
(D)
FIGURE 1.13  Example of a functional magnetic resonance imaging (fMRI) experiment.
This experiment examines the difference in blood oxygen level-dependent (BOLD) signal intensity
between subjects exposed to light or no light. (A) First, the investigators select a slice to examine.
(B) A T1-weighted image is obtained that provides structural data. T2-weighted images are used
for the actual data collection in the conditions of (C) light and (D) no light. (E) The data analysis
compares BOLD signal intensity between (C) and (D). This result is superimposed on the structural
data to produce an image (F) suitable for publication. (A–F: Reprinted with permission of
Springer Science + Business Media and Jens Frahm from Windhorst, U. and Johansson, H. (eds.),
1999. Modern Techniques in Neuroscience Research, Chapter 38: Magnetic Resonance Imaging of
Human Brain Function, p. 1064, Figure 5.)
Whole-Brain Imaging Chapter | 1  17
reproducible signal. Another limitation is the temporal delay: it can take 6–10 s
after the presentation of a stimulus for oxygenated blood to flow to an active
region, so there can be a long time delay between the stimulus or task and the
measurement of neural activity. The temporal resolution, the ability to resolve
neural activity into discrete events, is about 6 to 10 s, relatively poor compared
to other techniques such as electroencephalography or magnetoencephalography
(described below). Finally, fMRI cannot identify the neurochemistry of
neural events, such as which neurotransmitters or neuromodulators mediate a
change in neural activity. Inferences about the neurochemical makeup of neural
activity must be based on prior knowledge of brain anatomy or other forms
of whole-brain imaging, such as positron emission tomography (PET) (see the
­following section).
Even with these limitations, fMRI remains a powerful technique for correlating
neural activity with specific mental operations. A well-designed experiment
can reveal much about the human brain and animal brains (Box 1.1), allowing
investigators to noninvasively examine the physiology of cognition. See the last
part of this chapter for a thorough discussion of the design of a functional imaging
experiment.
Positron Emission Tomography
PET provides a representation of neural activity but no information about brain
structure. This technology was developed in the 1970s and 1980s as a novel
method of functional imaging, but has largely been superseded by fMRI technology
for most cognitive experiments. In a PET experiment, an unstable positron-emitting
isotope is injected into a subject’s carotid artery (a neck artery
that feeds the ipsilateral cerebral hemisphere). As the isotope decays, it emits
a positron, an antimatter counterpart of an electron. When a positron comes
into contact with an electron, an annihilation event occurs, resulting in a pair of
gamma photons that move in opposite directions (Figure 1.14A). These photons
pass through the body and can be measured by a gamma-detecting device that
circles the subject’s head. The detector identifies a pair of gamma photons that
arrive at opposite sides of the subject’s head at the same time (within a few
nanoseconds; Figure 1.14B). As the detector rotates around the subject’s head,
these signals can be used to derive the source of the annihilation events within
the subject (Figure 1.14C).
A PET experiment can utilize a variety of positron-emitting isotopes. One
of the most commonly used is fluorodeoxyglucose (FDG), a radioactive form
of glucose. As metabolically active neurons require an increase in the uptake of
glucose from the blood, the presence of FDG can be used as an indirect marker
of neural activity. In addition to FDG, radioactive water can be injected into the
brain’s circulatory system. Because there is an increase in blood flow to active
areas of the brain, the PET scan will indicate the areas where blood flow is
increased during activity.
18  Guide to Research Techniques in Neuroscience
A particularly useful aspect of PET imaging is the ability to use positronemitting
isotopes that can bind to specific receptors in the brain. For example, a
radioactive ligand that binds to serotonin receptors can indicate the locations and
binding potential of these receptors in the brain, providing information about
the relative metabolism of serotonin in human subjects. The ability to image the
metabolism of specific bioactive molecules makes PET imaging unique among
functional imaging techniques and provides a utility that fMRI cannot.
There are a number of limitations to PET. Compared to fMRI, PET has
about the same temporal resolution (6–10 s), a lower spatial resolution, and
BOX 1.1  Functional Magnetic Resonance Imaging (fMRI) Experiments
in Animals
A major benefit of functional brain imaging technologies is the ability to noninvasively
study neural activity in humans. However, this technology can also be
used with other animals, such as primates and rodents. The major benefits of using
animal subjects include the ability to validate the use of animal models and to
bridge human fMRI experiments and animal electrophysiology experiments. The
ability to use both fMRI and electrophysiology in the same animal (and even at the
same time) greatly aids our understanding of the functional activity of individual
neurons and entire brain regions in the same experiment. Furthermore, fMRI studies
in primates that screen neural activity across the entire brain can inform future
electrophysiological studies about brain regions containing neurons of interest to
an investigator.
Functional magnetic resonance imaging studies using primates present additional
limitations and challenges compared to traditional human studies. The horizontal
position of most MRI scanners is not ideal for primate studies, so vertical
scanners have been created to accommodate the special chambers that support
and brace a conscious primate. The animal’s head must be fixed in place with a
head post so there is no head movement during an experiment. The animal must
also be acclimated to these experimental conditions so it is comfortable in its
environment and can perform the task. A further challenge is that during the actual
experimental sessions, a primate may lose motivation to perform a task or attend to
a stimulus. Unlike an electrophysiology experiment in which an animal occasionally
receives a juice reward (Chapter 2), juice must be consistently provided so that
the animal stays attentive and completes the scanning session.
MRI scanners with higher magnetic field strengths allow high-resolution,
detailed imaging in small animals such as rats and mice. Obviously, these animals
cannot perform complicated cognitive tasks and most likely need to be anesthetized
during an imaging session. However, the benefit of doing fMRI in rodents is
the possibility of injecting psychoactive drugs during an imaging session or performing
lesion studies. Thus, it is possible to study the global effects of a pharmacological
agent, such as a receptor antagonist or the loss of a brain region over time.
Furthermore, a scientist can follow up functional imaging data with histological
studies after the completion of an experiment.
Whole-Brain Imaging Chapter | 1  19
cannot generate anatomical data. PET images are often combined with CT or
MRI images to present functional data in an anatomical context. The cost of
doing PET is very expensive. Because most isotopes used for PET studies have
extremely short half-lives, positron-emitting isotopes must be synthesized on
site using a room-sized device called a cyclotron, which itself is expensive
to purchase and maintain. The half-life of FDG is 110 min, so this compound
cannot be ordered and delivered from a remote location. Finally, PET requires
the injection of radioactive substances into subjects. Therefore, multiple PETimaging
sessions are not recommended for a single subject. Because of these
limitations, most modern functional imaging experiments utilize fMRI technology
instead of PET, with the exception of those experiments that focus on the
metabolism of bioactive substances, such as neurotransmitters.
Single-Proton Emission Computerized Tomography
Single-proton emission computerized tomography (SPECT) imaging is very
similar to PET imaging, producing functional images of neural activity but no
structural data. Like PET, a radioactive probe is injected (or inhaled) into the circulatory
system. The probes bind red blood cells to be carried throughout the body.
Because blood flow is increased in active brain structures, the radioactive signal is
used to assess an increase in neural metabolism. As the label undergoes radioactive
decay, it emits high-energy photons that can be detected using a gamma camera.
The camera is rapidly moved around the head of a subject to collect photons
from many different angles, permitting a three-dimensional reconstruction.
Although SPECT is very similar to PET in concept, it is not as costly because
the radiolabeled probes are usually commercially available and do not require
(C)Annihilation event
Gamma ray
detectors
(B)
Positron
Electron
(A)
FIGURE 1.14  Positron emission tomography (PET) imaging. (A) An unstable
positron-emitting isotope will decay over time and emit a positron. When a
positron comes into contact with an electron, an annihilation event occurs and a pair of gamma
photons are released. (B) If this unstable isotope is injected into a subject, the annihilation event can
be detected by a PET scanner. This scanner consists of a series of gamma ray detectors arranged
around the subject’s head. (C) Unstable isotopes for many metabolic substances, such as glucose or
neuropeptides, can be imaged in a PET experiment. The increase in signal over time can map sites
of metabolic activity caused by a specific neural stimulus or task.
20  Guide to Research Techniques in Neuroscience
an on-site cyclotron. Therefore, SPECT can be thought of as a cheaper alternative
to PET. The disadvantages to using SPECT as an imaging technique are
similar to the disadvantages of using PET: the technology has a relatively low
spatial resolution compared to fMRI (about 8 mm), and radioactive substances
must be injected into a subject.
Electroencephalography
EEG is a measure of the gross electrical activity of the surface of the brain. It is
not truly a brain imaging technique, as no meaningful images of the brain can
be produced using this technique alone. However, EEG is noninvasive and can
be used to ascertain particular states of consciousness with a temporal resolution
of milliseconds. EEG combined with other imaging techniques, such as
fMRI, can provide an excellent temporal and spatial representation of neural
activity.
To produce an electroencephalogram, several disk-shaped electrodes, about
half the size of a dime, are placed on the scalp (Figure 1.15). The scalp EEG
reflects the sum of electrical events at the surface of the brain. These events
include action potentials and postsynaptic potentials, as well as electrical signals
from scalp muscles and skin. An interesting way to think about EEG technology
is to imagine what it would be like to place a microphone above a large
crowd of people, such as above Times Square during New Year’s Eve. It would
be impossible to make out the signal from an individual person in the crowd;
however, it would be possible to determine when a meaningful event occurred,
such as when the crowd counted down to midnight. Likewise, it is impossible to
record the electrical activity from a single neuron with EEG, but it is possible to
ascertain when a meaningful event occurs in the brain, such as when a subject
detects a salient stimulus in an experiment. Thus, combining the temporal resolution
of EEG with the spatial resolution of fMRI provides a powerful method
to detect the precise timing and location of neural activity within the brain.
A powerful application of EEG is the study of event-related potentials
(ERPs) in humans and animals. An ERP is a distinct, stereotyped waveform
in the EEG that corresponds to a specific sensory, cognitive, or motor event.
For example, if a human subject abruptly hears an alarm, the perception of the
sound may be represented as an ERP in the EEG waveform. Thus, neuroscientists
can study the moment that the brain processes a stimulus with subsecond
temporal resolution, allowing a noninvasive means of evaluating brain function
in experimental subjects or patients with cognitive diseases.
Magnetoencephalography
MEG measures changes in magnetic fields on the surface of the scalp that are
produced by changes in underlying patterns of neural electrical activity (Figure
1.16). About 50000 neurons are required to produce a detectable signal with
Whole-Brain Imaging Chapter | 1  21
MEG, a number that may seem large but is actually much smaller than what
is required for an EEG signal, which may require millions of neurons. MEG
offers relatively poor spatial resolution but excellent temporal resolution compared
with PET, SPECT, and fMRI. Therefore, MEG can be thought of as a
compromise technique: it offers excellent temporal resolution and much better
spatial resolution compared with EEG, but not as good spatial resolution as
other imaging techniques. In particular, MEG in combination with fMRI allows
for excellent temporal and spatial resolution of neural activity. Unfortunately,
MEG is a very expensive technique, requiring a room that can obstruct magnetic
fields from outside sources; even something as small as a coffee machine in a
neighboring building can be detected if the room is not adequately insulated.
1
2
3
4
5
6
FIGURE 1.15  A human subject wearing electrodes for electroencephalography recordings.
The electrodes measure the global electrical activity of millions of neurons in the brain. Each electrode
placed on the scalp records a unique trace of activity based on its location.
Subject in
MEG scanner
Within scanner,
the subject wears
a cap with dozens
of magnetic field
sensors
(A) (B)
FIGURE 1.16  A magnetoencephalography (MEG) setup. (A) A subject wears a cap with
magnetic field sensors and (B) is placed inside an imaging chamber. MEG equipment is expensive
and requires a room with strong insulation.
22  Guide to Research Techniques in Neuroscience
Like EEG, the temporal precision of MEG allows for the study of eventrelated
changes in brain activity. The MEG equivalent of an event-related potential
is referred to as an event-related field (ERF).
Optical Imaging
Optical imaging techniques produce images of neural activity by measuring
changes in blood flow and metabolism from the surface of the brain. Rather
than detecting changes in the magnetic or electrical properties of neurons, as
in fMRI, EEG, or MEG, optical imaging detects changes in light reflectance
from the surface of the brain due to changes in the amount of blood flowing to
neural tissue. In animal preparations or during human surgery, light is shined
on an exposed portion of the brain. Any light that is reflected off the surface
is detected by a sensitive camera and recorded by a computer. When neurons
are more active, changes in blood volume, blood oxygenation, and the lightscattering
properties of neural tissue (resulting from ion and chemical movements)
all cause small (0.1–3.0%) changes in the reflectance of light from the
brain’s surface. For each experiment, the investigator images a baseline amount
of light reflectance, and then compares this baseline parameter to changes in
light reflectance that arise due to the presentation of a stimulus. Optical imaging
technologies allow for spatial resolutions of <1 mm and temporal resolutions of
2–8 s. These technologies have been used to produce high-resolution functional
maps of the visual cortex in both animals and humans.
Earlier in this chapter, we defined functional imaging techniques as methods
that allowed investigators to measure changes in neural activity over time
without physically penetrating the skull. Obviously, many optical imaging techniques
are an exception to this definition, as the brain surface must be exposed
to allow light to penetrate and reflect back to a camera. However, diffuse optical
imaging (DOI) and near-infrared spectroscopy (NIRS) are noninvasive
alternatives that utilize the same basic principles as invasive optical imaging
but record light reflectance through the scalp (Figure 1.17). The signal is much
weaker than invasive optical imaging, as light must pass through the superficial
layers of the head to the brain, and then from the brain to optical electrodes,
known as optrodes or optodes, placed on the surface of the scalp. However,
these techniques are sensitive enough to detect large changes in neural activity
and can be useful in clinical applications as an alternative to fMRI or PET
because of their low cost and portability. For example, long-term monitoring of
cerebral oxygenation in a patient following a stroke is a potential use of DOI
that would be practically impossible with fMRI or PET.
Table 1.1 compares the spatial and temporal resolutions, cost, and invasiveness
of the various functional brain imaging techniques described previously.As
mentioned before, many imaging laboratories now combine multiple techniques
to make up for the inadequacies of any single technique. For example, fMRI
offers excellent spatial resolution and is noninvasive, but it does not offer good
Whole-Brain Imaging Chapter | 1  23
temporal resolution of neural activity during experiments because the BOLD
effect occurs over a period of 6–10 s. Therefore, some laboratories combine
fMRI with MEG or EEG, in which signals can be detected within a fraction of
a second.
So far, this chapter has focused almost exclusively on the technology of
structural and functional brain imaging. Of course, the utility of these technologies
depends entirely on how they are used: the research hypotheses,
experimental designs, and methods of data analysis. In the last part of this
Subject with light
sources/detectors
placed above the
frontal cortex
Scalp
Light sourceLight detector
FIGURE 1.17  Optical imaging. Light is shined onto the surface of the brain. A portion of this
light is reflected off the brain and detected by multiple optrodes. Changes in neural activity produce
changes in the amount of light that is absorbed and reflected by the brain. Therefore, optical
imaging can be used to indirectly detect changes in neural activity. These techniques can be either
invasive (the skull is opened to reveal the surface of the brain) or noninvasive.
TABLE 1.1  Comparison of Functional Imaging Techniques
Spatial
resolution
Temporal
resolution Cost Invasiveness
fMRI 1 mm 6–10 s Expensive Noninvasive
PET 4 mm 1 min Very
expensive
Radioactive
injection
SPECT 8 mm 2–8 s Expensive Radioactive
injection
EEG 1 cm 1 ms Moderate Noninvasive
MEG 1 mm 1 ms Very
expensive
Noninvasive
Optical
imaging
1 mm 2–8 s Inexpensive Can be invasive
24  Guide to Research Techniques in Neuroscience
chapter, we will examine the approaches that can be taken in the design and
analysis of functional imaging experiments.
FUNCTIONAL IMAGING EXPERIMENTAL DESIGN
AND ANALYSIS
Functional imaging technologies are particularly well suited to cognitive
neuroscience, the field of neuroscience dedicated to elucidating the neural
basis of thought and perception. Cognitive operations routinely studied in
the literature include attention, learning and memory, executive function, language,
emotion, and higher-order sensory processing, such as the enjoyment
of art or music. Determining the neural basis of cognitive processes, especially
processes that seem unique to humans, absolutely depends on brain imaging
techniques.
In addition to answering scientific questions about cognitive neuroscience,
fMRI has added important information to other neuroscience fields, such as
sensory or motor systems neuroscience. These studies identify regions of the
human brain that are active in the presence of certain sensory stimuli or motor
actions. Some studies reproduce findings in humans that were previously demonstrated
in other animals, as well as add important insights that enhance our
understanding of these systems. For example, fMRI research has demonstrated
that visual pathways in humans are anatomically consistent with visual pathways
found in other mammals. However, our understanding of the visual system
has been greatly enhanced by the fact that human subjects can report experiences
and receive verbal instructions that other animals cannot. For example,
researchers have found that activity in the visual cortex increases not only when
human subjects are exposed to visual stimuli, but also when subjects are told
to imagine visual stimuli. Furthermore, fMRI research has demonstrated that
different areas of the visual cortex process different aspects of visual stimuli,
such as color, shape, and texture. Thus, fMRI contributes meaningfully to our
understanding of both cognitive as well as systems neuroscience.
Unfortunately, the conception and design of functional imaging experiments
are not always well understood by scientists who do not use these methods.
This is probably because these technologies are unlike most other techniques
used in neuroscience, as well as the fact that they are usually only performed
by the dedicated researchers who use them regularly. A common complaint
among brain imaging specialists is the misconception that you can simply place
a human subject into a scanner, tell them to look at some stimulus, and then
publish the results. Like any other technique, whole-brain imaging experiments
must be carefully designed and interpreted, more than nonspecialists may sometimes
appreciate. The next section describes some of the scientific considerations
in the design and execution of a functional imaging experiment. We will
focus on fMRI, as this technique dominates the field, but the same principles
can be applied to other functional imaging techniques as well.
Whole-Brain Imaging Chapter | 1  25
Planning the Experiment
Before any human subject is placed in a scanner, an investigator conceives and
designs an experiment months or even years in advance. There are many practical
considerations that must be taken into account in the design of the experiment
that may affect the ambitions of the investigator. Once these limitations are understood,
the investigator designs the experiment to answer a specific question or test
a specific hypothesis. Finally, the investigator designs a proper task paradigm and
tests efficacy of the stimuli so that the results are appropriate and accurate.
Practical Considerations
Perhaps the most important practical consideration of an fMRI experiment is
its cost. The actual scanning machine, as well as its support apparatus, costs
millions of dollars and is almost always shared by multiple labs. Routine maintenance
of the scanner is also expensive, with specialized technicians on call to
fix any potential physical problems that may arise. Most research institutions
charge an individual lab based on the number of hours the scanner is used.
These institutions may also charge money based on the time of day, with scanner
use during the day costing more than scanner use late at night. It is not
unusual for scanner time to cost between $100 and $1000 per hour, depending
on the institution’s operating costs. Therefore, an fMRI experiment must be
well designed so that scanner time is not wasted. fMRI is too expensive for simply
playing around and putting humans in the scanner for no defined purpose.
Finally, scanner time is usually reserved well in advance due to the number of
scientists who wish to perform experiments.
Another important practical concern of fMRI is that a subject must keep
his or her head completely motionless during a scanning session. Even small
movements can disrupt the magnetic field, causing artifacts in the images. Also,
if a subject moves during a scan, the acquired images will not line up with each
other, making them uninterpretable. This immobility means the subject will be
unable to perform certain activities while inside the scanner, such as speaking,
exercising, or moving facial muscles. However, subjects can be trained to
perform simple hand movements to complete a task or respond to a stimulus,
such as pressing a button on a hand device. Some specialized devices have been
invented to deliver olfactory or gustatory stimuli to subjects.
A practical concern for an investigator who wishes to study sleep, attention,
or the auditory system is that an fMRI scanner can be very noisy. The RF pulse
sequences are loud and last the entire duration of the experiment. Therefore,
many subjects do not sleep normally in a scanner, and the changes in attention
due to a surprising stimulus may actually be due to the surprise of a noisy scanner
turning on and off. Auditory stimuli can also be very difficult to work with,
for obvious reasons. These limitations are not insurmountable, but require extra
planning by the scientist to make sure that a sporadically loud environment does
not bias the results.
26  Guide to Research Techniques in Neuroscience
Structure of a Functional Imaging Experiment
Like any other experiment in neuroscience, functional imaging experiments
examine the effect of an independent variable on a dependent variable. The
independent variable is the experimental variable that is intentionally manipulated
by the researcher and is hypothesized to cause a change in the dependent
variable. In a functional imaging experiment, the independent variable can be
a stimulus, task, or even a difference in the subjects being tested such as their
age, gender, or disease state. The dependent variable is the quantifiable variable
measured by the researcher to determine the effect of the independent variable.
This variable is different for each brain imaging technique. In the case of fMRI
research, the dependent variable is the BOLD signal intensity for a particular
part of the brain. In the case of PET or SPECT technology, the dependent variable
is the intensity of gamma radiation. All of these dependent variables are
used as proxies for neural activity.
The specific hypotheses for a functional imaging experiment are usually
framed in the following manner:
	l	Activity in brain region X is correlated with stimulus/taskY.
	l	For a given stimulus or task, activity in brain region X precedes activity in
brain regionY.
	l	Activity in brain region X is higher under conditionY than condition Z.
	l	Activity in brain region X is higher in human populationY than human population
Z.
	l	Activity in brain region X changes across time as a subject learns a task.
These hypotheses depend upon the investigator knowing of the existence
of “brain region X” and having a good rationale for studying that region. The
amount of data generated during an fMRI experiment is quite large, and processing
fMRI data can be easier if the investigator has an idea of where to look.
Despite large amounts of data, fMRI can also be used to screen the entire
brain to find brain regions that increase neural activity in response to a stimulus/
condition, or to examine distributed networks of neural activity in a resting state.
The increasing availability of powerful computational resources and techniques has
enabled these more data-driven, exploratory approaches to analyze the large quantities
of data produced in brain imaging. Machine learning-based techniques, such
as multivariate pattern analysis, can be used to extract patterns from large datasets,
allowing the advancement of studies that look at brain-wide functional networks.
Itisalsoimportanttorecognizewhetherafunctionalimagingstudyisbetween
subjects or within subjects. In a study that is between subjects, the independent
variable is the different populations of subjects, such as male vs. female, old vs.
young, healthy vs. diseased, genotype A vs. genotype B, etc. These differences
in subjects can potentially create problems in analysis. For example, it is known
that the BOLD response can change with age; therefore, comparing a younger
group of subjects with an older group of subjects using fMRI may require additional
controls to ensure that changes in brain activation are not simply due to
Whole-Brain Imaging Chapter | 1  27
age-related changes in the BOLD effect. In a study that is within subjects, each
subject participates in all experimental conditions and the independent variable
is something other than the identity of the particular subject.
Task Paradigms
After developing a specific research hypothesis, an investigator determines an
appropriate task paradigm, a strategy for presenting stimuli to subjects during
an experiment. These paradigms are usually divided into two major categories:
blocked designs or event-related designs.
In a blocked design, a subject is presented with two or more categories of stimuli
that alternate every 1–2 min. For example, consider an experiment that features
two categories of stimuli, X and Y. Category X might represent something such
as smiling faces while category Y could represent neutral faces. In the first block
of stimuli, the subject is presented with different images made up entirely of category
X:X1, X2, X3, X4, and so on.A typical block may last between 10 and 60 s,
with each individual stimulus presented from 1 to 10 s. After the end of the block,
there may or may not be a brief period when no stimuli are presented. Then, a
second block of stimuli is presented made up entirely of category Y:Y1, Y2, Y3,
Y4, and so on. These blocks of stimuli are repeated in cycles, alternating X andY,
until a sufficient amount of data is collected from a subject. Some blocked designs
contain a third category, alternating between blocks of X,Y, and Z.
Blocked designs are useful for obtaining relatively high signal-to-noise
information from active brain regions. Because stimuli of the same experimental
category are presented repeatedly for 1–2 min, the hemodynamic BOLD
response has sufficient time to increase and a strong signal is produced. These
paradigms are often chosen for experiments determining which brain regions
are active for a given stimulus or task. They are also good for neural processes
that last a relatively long time, such as changes in cognitive or emotional state.
A blocked design is not as useful for examining a change in brain activity during
relatively short events that last 2–5 s, such as when a subject is required to make
a decision inside a scanner.
In an event-related design, stimuli are presented as isolated, individual
events of short duration. This paradigm is useful for discrete tasks in which a
subject must recognize an event, detect a novel stimulus, or make a decision, all
processes that take place in short time intervals. The hemodynamic properties
of the BOLD effect may return to baseline between events, or for fast-paced
stimuli, the BOLD response may not return to baseline, and so the effects of
two stimuli on neural activity can be examined within a small time window.
For example, stimulus A may indicate the nature of stimulus B, and the BOLD
signal can be measured for B with and without the presence of A. Event-related
designs are not usually used in experiments that affect a subject’s emotional
state over time, as these states usually last over relatively longer time periods.
Blocked designs and event-related designs are not mutually exclusive, and
indeed some studies take advantage of both designs and employ what is referred
28  Guide to Research Techniques in Neuroscience
to as a mixed design. The relative advantages and disadvantages of all three
task paradigms are compared in Table 1.2.
Pilot Experiments
Because MRI scanner time is so expensive, it is often necessary to test the delivery
of stimuli or characterize the behavior of subjects during a task before any
imaging experiments take place. These pilot experiments take place outside of
the MRI facility and demonstrate the efficacy of stimulus delivery in producing
an appropriate human response. Statistical analysis of how subjects perform
their tasks is sometimes necessary to gain approval before the actual experiments
take place in the scanner. Only after the investigator characterizes a cognitive
task in terms of the efficacy of the stimuli and behavior of human subjects
will it be appropriate to begin to associate neural correlates with that behavior.
Planning an Experiment that Uses Human Subjects
Once an experiment is designed, a lab must be given permission to use humans
as research subjects by an Institutional Review Board (IRB). An IRB is a
TABLE 1.2  Relative Advantages and Disadvantages of Different Functional
Magnetic Resonance Imaging (fMRI) Task Paradigms
Advantages Disadvantages
Blocked 	l	More statistical power for
detecting subtle differences
across different conditions
	l	Tend to be simpler to implement
and analyze
	l	Good for examining cognitive
state changes
	l	Predictability of stimuli
may confound results
	l	Information about the
time course of the activation
response is lost
within a block
	l	Not applicable to
certain types of tasks
(e.g., novelty)
Event-related 	l	Reduces confounds of predictable
stimulus order, because
stimuli can be presented
randomly
	l	Can sort trials after the experiment
according to specific
behavioral outcomes
	l	Useful for examining temporal
characteristics of responses
	l	Flexible analysis strategies
	l	More complex design
and analyses than
blocked design
	l	Lower signal-to-noise
ratio than block designs
	l	Must perform longer
scan runs to compensate
for loss in statistical
power
Mixed 	l	Can compare short-term transient
activity with long-term
sustained activity
	l	Most complicated
analyses
Whole-Brain Imaging Chapter | 1  29
committee of about 10 individuals, typically composed of physicians and
research faculty, as well as nonfaculty members such as a nurse, minister, graduate
student, or lawyer. The purpose of the IRB is to review studies that use
human subjects and determine if the study meets ethical, safety, and scientific
obligations. An IRB typically meets anywhere from 2 to 12 times per year to
review and vote on the submitted applications. Additionally, any research project
involving human subjects must meet the standards of the Health Insurance
Portability and Accountability Act’s Privacy Rule, which regulates the protection,
security, and confidentiality of private health information.
Conducting the Experiment
After an experiment is designed, discussed, and approved, a scientist proceeds
with recruiting human subjects and collecting data in the scanner. These scientists
must be familiar with the scanner technology and experimental procedures
before any actual data collection begins. The difficulty in recruiting human subjects
and the high cost of scanner time makes it essential that experiments are
conducted properly and without many mistakes.
Working with Human Subjects
Though the exact number varies based on the experimental design, usually
about 10–20 subjects are necessary for a typical imaging experiment to reach
an appropriate, statistically significant conclusion. Recruiting human subjects
can be relatively easy or difficult depending on the type of subjects needed.
If the only requirement for a subject is that he or she is an average, healthy
adult, then recruiting subjects can be relatively straightforward. At academic
institutions, undergraduates and graduate students are often targeted for recruitment,
with compensation given in the form of extra credit for a course or a cash
payment. When subjects must represent a particular population of individuals
(e.g., individuals with posttraumatic stress disorder, elderly subjects, or individuals
diagnosed with depression) it can be much harder to recruit a necessary
number of subjects. Consider a study in which the hypothesis is that depressed
individuals have a significantly different level of activity in a particular brain
region than nondepressed individuals. In this case, the investigator must find
individuals who have been formally diagnosed with depression. Additionally,
because psychotropic drugs can confound the results, it is necessary that none
of these subjects received medication after their diagnosis. Therefore, the investigator
must identify 10–20 subjects who have been diagnosed with depression
but chose not to undergo pharmacological treatment (or who have not yet started
treatment). In studies like this, recruiting subjects can be the bottleneck in the
time it takes to complete an experiment. Investigators might need to formally
recruit individuals from other institutions, such as nursing homes, hospitals, or
secondary schools. It might be necessary to formally collaborate with clinical
neurologists to recruit patients with various mental health disorders.
30  Guide to Research Techniques in Neuroscience
Once a subject is recruited, the investigator schedules a specific session
time in the scanner. Prior to the start of an experiment, the investigator must
inform the subject about the nature of the experiment (although details of the
experiment can be withheld for experimental purposes). The subject must sign
a release form to indicate that he or she is a willing participant in the study and
that any data gathered can be used for publication. Finally, the subject must
complete a brief survey about his or her personal health to ensure that there
are no confounding variables such as history of mental health disease, cardiovascular
disorders, or heavy use of alcohol or other drugs. The subject is also
requested to remove any metal objects before entering the scanner, such as loose
change, belt buckles, earrings, and so on. Often the investigators ask the subjects
to change into a disposable gown to ensure that there are no metal objects
placed inside the scanner.
During the experiment, the investigator must ensure that the subject is as
comfortable as possible inside the scanner. To reduce head movement, the subject’s
head is surrounded by soft padding to hold it firmly in place inside a
head coil. The investigator provides the subject with a squeeze ball that, when
pressed, can stop the scans at any time if the subject becomes uncomfortable.
For experiments that require the subject to make a choice or respond to a stimulus,
the investigator also provides a nonmetallic handheld device with buttons
that the subject can press to provide feedback. Visual stimuli can be projected
through goggles or to a mirror inside the scanner to allow for video presentations
from a nonmetallic surface.
After the experiment is complete, an investigator usually debriefs the subject
on the experiment and pays the subject for his or her time. Depending on the
experiment, follow-up sessions can be necessary, and these are also scheduled
well in advance.
Data Acquisition
In a typical experiment, it is common for about 10–20 human subjects to participate
(twice that for a between-subjects study). Each subject usually participates
in one to three sessions, the scheduled time when an actual experiment
is conducted. Each session contains many runs in which the brain is repeatedly
scanned and the hypothesis is tested. Within each run, functional data are
accumulated and stored as volumes—three-dimensional constructions of brain
space. A complete volume is typically acquired every 1–3 s. Each volume contains
a complete set of slices, two-dimensional representations of a plane of
the brain that are only one voxel thick. A voxel is the smallest functional unit
of brain space that can be analyzed for changes in signal intensity over time.
In fMRI, a voxel is usually 1–5 mm3. Remember that a voxel of fMRI data
is essentially four-dimensional: three dimensions represent the location of the
voxel in space (x, y, z) and the fourth dimension is the time in which the signal
intensity was recorded. Thus, an fMRI experiment generates huge quantities
of data: 10–20 subjects, each with one to three sessions, each with several
Whole-Brain Imaging Chapter | 1  31
experimental runs, each with several volumes of brain space, each with several
brain slices, and each with a grid of voxels that represent the data! Therefore,
computers with ample storage space are required to record and store data until
the results can be analyzed after the collection is complete.
Manipulating Neural Activity during an Experiment
One of the limitations of studying the human nervous system is that it is very
difficult or even impossible to functionally perturb the brain during an experiment.
For example, if activity in a specific part of the brain is correlated
with performance on a task, it would be interesting to determine if a loss of
that part of the brain caused a deficit in the ability of the subject to perform
the task. While such a loss-of-function experiment could be performed on
another model organism with a lesion study, this is, of course, impossible in
humans. Likewise, it would also be interesting to stimulate neural activity
in a particular brain region to determine if performance improves on a task.
Such gain-of-function experiments are also possible in model organisms, with
stimulating microelectrodes or overexpressed genes driving activity in a specific
population of neurons. Again, this is impossible in typical, noninvasive
fMRI experiments.
Two technologies make it possible to reversibly modulate neural activity
in humans. Transcranial magnetic stimulation (TMS) reversibly activates or
inactivates regions of the human brain. In this technique, a coil that generates
magnetic field pulses is positioned near a subject’s head (Figure 1.18). The
changing magnetic fields induce weak electric currents at a specific focal point
TMS coil
Induced
electrical activity
FIGURE 1.18  Transcranial magnetic stimulation (TMS). A coil is held near a person’s head,
and a magnetic field stimulates electrical activity in a superficial brain region. Depending on the
strength of the magnetic field and the brain region, this electrical activity can cause either a temporary
loss-of-function or gain-of-function in that brain region.
32  Guide to Research Techniques in Neuroscience
on the surface of the brain. Depending on the brain region and the strength of
the magnetic field impulse, the electrical activity may either stimulate neural
activity or cause a hyperpolarized state in which neural activity is temporarily
inactivated. Therefore, it is theoretically possible to perform a loss- or gain-offunction
experiment using this technology. One of the limitations of this technology
is that TMS can only induce electrical activity on the outer surface of the
brain—it is currently impossible to stimulate or inactivate deep brain structures
that may be of interest to an investigator. However, there are thousands of publications
that have used TMS to selectively activate and inactivate activity in the
human cerebral cortex. This technology can also potentially be used for therapeutic
treatment of diseases and is being heavily researched in the treatment of
depression and motor diseases.
Another technology, ultrasonic neuromodulation (USNM), reversibly
activates brain regions of interest through the delivery of transcranial ultrasound.
These waves subtly increase the temperature of the brain, causing a
transient increase in neural activity at a greater spatial resolution than TMS
technologies.
An additional strategy for investigators to perform loss-of-function experiments
is to attempt to find patients with lesions (caused by an accident or stroke)
to specific brain regions. Because many institutions that utilize fMRI scanners
are associated with hospitals, identifying and recruiting patients with lesions can
be coordinated between researchers and neurologists. However, these patients
may present lesions that are not tightly localized to the brain area of interest,
there may be compensatory mechanisms that develop over time, and it may be
more difficult for these patients to perform specific tasks. All of these caveats
must be considered before including these patients in experiments, as well as
when evaluating these experiments in the literature.
Postexperimental Data Analysis
The end result of most fMRI experiments is a colorful figure depicting the activation
or inactivation of a particular region of the brain that is correlated with
a stimulus or task. These figures can seem deceptively simple to produce, yet
require rigorous methods of data analysis and interpretation.
Data Analysis
What is an investigator to do with the huge quantity of data acquired from multiple
research subjects? In most analyses, the data from each subject are analyzed
separately first and then pooled together into a common subject pool.
Because there is variability in every subject’s brain structure, data must first
be fit into a common three-dimensional brain template. Structural data from
each individual’s brain are therefore stretched and warped to fit specific anatomical
landmarks in a precise mapping system. One of the most widely used
coordinate systems for normalizing fMRI data is called Talairach space, based
Whole-Brain Imaging Chapter | 1  33
on the stereotaxic measurements of a single postmortem brain. This coordinate
system is ubiquitous in brain imaging research as it defines brain regions and
Brodmann’s areas in stereotaxic coordinates, allowing investigators to make
anatomical comparisons among different brains. Another commonly used coordinate
system is called the MNI template (MNI stands for Montreal Neurological
Institute, the institution that established this system). This template is a
probabilistic mapping system based on the averages of hundreds of individual
brain scans and scaled to match the landmarks within the Talairach atlas.
After an individual subject’s brain is adjusted to either the Talairach or MNI
templates, signal intensity is compared at different time points according to the
task paradigm. There are two general strategies of analyzing signal intensity in
the brain: voxelwise analysis or region-of-interest (ROI) analysis. Most fMRI
studies present data using voxelwise analysis, in which each voxel is analyzed
for significant differences in signal intensity in time between two experimental
conditions. In ROI analysis, the brain is divided into a set of discrete regions
assigned by the investigator. Rather than voxel-by-voxel comparisons, entire
brain regions are compared for significant differences in signal intensity. ROI
analysis can provide information about specific structures, but can initially
be more time-consuming, as each voxel has to be assigned to a specific ROI.
Sometimes an investigator will first analyze data using a voxelwise analysis and
then analyze discrete ROIs.
Each individual’s fMRI data are analyzed for significant differences, and
then these data are pooled together for all the individuals within a certain subject
pool. For a within-subjects study, the subject pool is the same and the investigator
determines if there is a significant difference in signal intensity between
two different stimuli or tasks. For a between-subjects study, the subject pools
are different and the investigator determines if there is a significant difference
in signal intensity between the two groups for a stimulus or task. Following this
data analysis, the statistically significant differences in the pooled subject data
are presented as figures for publication.
Preparation of Figures
The most common functional imaging figures presented in the literature take
the form of color-coded brain activation data superimposed on a structural
image of a brain slice (Figure 1.19). As mentioned previously, the brain slice
is almost always a T1-weighted image because these images present the greatest
contrast between brain regions. These brain slices can either be taken from
one of the subjects during the experiment, or from a stock set of images from
the Talairach or MNI templates. Sometimes, investigators superimpose imaging
data on an inflated brain in which the sulci and gyri of the brain are
expanded into a balloon-like shape to better show neural activity in the sulci
(Figure 1.20).
It is important to realize that these fMRI figures are statistical comparisons
in signal intensity over time for a grid of voxels, and that color-coded
34  Guide to Research Techniques in Neuroscience
activation does not just light up during a scanning session. The color-coded
voxels superimposed over the structural brain image are essentially a grid of
numbers (Figure 1.19). Each voxel is assigned a statistical value that represents
the difference in signal intensity for that region of brain space between
two conditions. The exact color scheme is chosen by the investigator to reflect
4
1
3
2
fMRI figure depicting an increase in neural
activity in the visual cortex
Increased magnification of the figure reveals
statistical analysis for each voxel
Degrees
of significance
FIGURE 1.19  Preparation of a functional magnetic resonance imaging (fMRI) figure for
publication. A typical fMRI figure contains a representation of the changes of blood oxygen
level-dependent signal intensity over time superimposed over a T1-weighted structural image. It
is important to remember that the colored representation of signal intensity is essentially a grid
of numbers, with each number representing a measure of statistical significance. (fMRI image
reprinted with permission of Springer Science + Business Media and Jens Frahm from Windhorst,
U. and Johansson, H. (eds.), 1999. Modern Techniques in Neuroscience Research, Chapter 38:
Magnetic Resonance Imaging of Human Brain Function, p. 1064, Figure 5.)
FIGURE 1.20  An inflated representation of the right hemisphere of the human brain used to
show activity within sulci. The dark gray regions represent the exposed sulci of the inflated brain,
while the light gray regions represent gyri. (Courtesy of Dr Rory Sayres.)
Whole-Brain Imaging Chapter | 1  35
the statistical magnitude of signal intensity: usually bright colors represent
high differences in intensity, while darker colors represent more subtle differences
in intensity.
CONCLUSION
The goal of this chapter was to provide a thorough introduction to the technologies
that currently exist to image the structure and function of the brain, as
well as the approaches an investigator may take when designing and analyzing
functional imaging experiments (Box 1.2). These technologies are more complicated
than the brief descriptions found here, and readers interested in more
detailed explanations should consult the Suggested Readings section at the end
of this chapter. However, this survey of techniques hopefully provides an appreciation
for the ingenuity of scientists and engineers in developing and improving
these techniques over the past 10–20 years. Imaging neural function can
be performed based on the electrical, magnetic, metabolic, and light-scattering
BOX 1.2  Walkthrough of a Functional Magnetic Resonance
Imaging (fMRI) Experiment
Let us consider a hypothetical situation: Suppose you work in a laboratory that uses
fMRI to study cognition in humans. Your research group becomes interested in the
neural basis of how a person recognizes and identifies familiar people. One way to
frame this question is “Are there specific brain regions that are active in response
to familiar individuals versus unfamiliar individuals?”
How would you go about designing an fMRI study to answer this question?
A good starting point would be to identify the independent and dependent
variables in this experiment. The independent variable, the variable that varies
from trial to trial, is a person who is familiar or unfamiliar to the subject. This
immediately leads to another decision to make as an investigator: the choice
of stimuli. Will the stimulus be a picture of an individual? The sound of the
individual’s voice? The individual’s name? In this example, let us make the
independent variable a person’s name that is visually displayed on a screen.
Therefore, in each trial, the subject will see a name on a screen. Because the
independent variable is the stimulus and not the identity of the subject, this is
a within-subjects study.
The dependent variable is the change in BOLD signal intensity in the brain.
We might hypothesize that a specific brain region will show a significant difference
in signal intensity between the two conditions, but in this experiment such
a specific hypothesis is unnecessary, as we can scan major regions of the brain (if
not the entire brain) to identify all regions that significantly differ between the two
stimulus conditions.
What will be the subject’s task during the experiment? If all we ask the subjects
to do is stare at the screen, they may become bored, distracted, and even
fall asleep. Therefore, we must provide the subjects with a specific task so that
Continued
36  Guide to Research Techniques in Neuroscience
they fully attend to the stimuli. In this example, a good task may be to indicate
whether the name on the screen is a man’s or a woman’s. Each subject could
be presented with a handheld device with two buttons, one for man and one
for woman. At the end of the imaging session, we can measure the accuracy
of the responses to ensure that the subject was fully attentive throughout the
experiment.
Obviously, we will need to generate a list of names for both the familiar and
unfamiliar categories of stimuli. Each list should contain a balanced list of males
and females, old and young, and so forth, to ensure that there are no confounding
variables in the study. Familiar names could include famous celebrities and
politicians, while the unfamiliar names could include combinations of first and
last names that are not well-established celebrities. How can we ensure that the
subject knows all of the familiar names and none of the unfamiliar names? This
is impossible to confirm before the experiment begins, but after the experiment
is over, each subject could complete a brief survey in which he or she indicates
the level of familiarity of each name, perhaps on a scale of 1–10. This survey can
then be utilized when analyzing the functional imaging data to ensure that each
stimulus is adequately categorized.
The next major decision will be the best task paradigm to present stimuli to
subjects. A blocked design will allow us to present multiple stimuli of the same
category (familiar or unfamiliar names) to a subject over a 1- or 2-min period,
a condition that may allow for maximal signal intensity in active brain regions.
However, we may wish to evaluate the degree of signal intensity for each name
presented to the subject. In this case, we may wish to pursue an event-related
design, which will allow us to correlate the degree of signal intensity with the
degree of familiarity with a famous name, as indicated by the subject on a survey
after the experiment is over.
Once the IRB approves the study and our proposal to use human subjects, we
are ready to reserve scanner time and start collecting data! We will need 10–20
subjects for this experiment, but we should recruit at least 20–40 because some of
the data will need to be discarded. For example, some subjects may fail to show
up, some may fall asleep in the scanner, or some may turn out to have medical
conditions or treatments that may preclude them from participation in the study. In
order to recruit subjects, our lab could offer $25/h for their time. After the imaging
session ends, we will provide them with the familiarity survey and then start to
analyze the data.
For each subject, we morph the structural coordinates from their brain scans
to match a normalized template system, such as the MNI template. Then we can
proceed with a voxelwise comparison in signal intensity between the familiar
name trials and the unfamiliar name trials. Individual brains can then be pooled
across all subjects for each condition. The final figure will be the difference in
signal intensity between the two conditions, voxel-for-voxel, superimposed on a
T1 image from the MNI template. Because the template contains coordinates for
discrete brain regions, we can interpret the specific regions activated in response
to familiar or unfamiliar names.
BOX 1.2  Walkthrough of a Functional Magnetic Resonance
Imaging (fMRI) Experiment—cont’d
Whole-Brain Imaging Chapter | 1  37
properties of distinct brain regions. Producing so many different methods of
imaging activity in the human brain is a remarkable achievement, and it will be
exciting to observe, either as fellow scientists or participants, how these methods
are combined and improved over the next 10–20 years.
SUGGESTED READING AND REFERENCES
Books
Frahm, J., Fransson, P., & Kruger, G. (1999). Magnetic resonance imaging of human brain function.
In U. Windhorst & H. Johansson (Eds.), Modern techniques in neuroscience research (pp.
1055–1082). Berlin: Springer.
Huettel, S. A., Song, A. W., & McCarthy, G. (2004). Functional magnetic resonance imaging. New
York: Sinauer.
Mori, S. (2007). Introduction to diffusion tensor imaging. Amsterdam: Elsevier.
Schild, H. H. (1990). MRI made easy... Well almost. Germany: Schering AG.
Review Articles
Aguirre, G. K. (2014). Functional neuroimaging: technical, logical, and social perspectives. Hastings
Center Report, 44, S8–S18.
Friston, K. (2002). Beyond phrenology: what can neuroimaging tell us about distributed circuitry?
Annual Review of Neuroscience, 25, 221–250.
Logothetis, N. K. (2008). What we can do and what we cannot do with fMRI. Nature, 453, 869–878.
Mezer, A., et al. (2013). Quantifying the local tissue volume and composition in individual brains
with magnetic resonance imaging. Natural Medicine, 19, 1667–1672.
Miller, G. (2008). Neuroimaging. Growing pains for fMRI. Science, 320, 1412–1414.
Mori, S., & Zhang, J. (2006). Principles of diffusion tensor imaging and its applications to basic
neuroscience research. Neuron, 51, 527–539.
Savoy, R. L. (2005). Experimental design in brain activation MRI: cautionary tales. Brain Research
Bullettin, 67, 361–367.
Turk-Browne, N. B. (2013). Functional Interactions as Big data in the human brain. Science, 342,
580–584.
Primary Research Articles—Interesting Examples from the Literature
Adleman, N. E., Fromm, S. J., Razdan, V., Kayser, R., Dickstein, D. P., Brotman, M. A., et al.
(2012). Cross-sectional and longitudinal abnormalities in brain structure in children with severe
mood dysregulation or bipolar disorder. Journal of Child Psychology and Psychiatry, 53(11),
1149–1156.
Dagher, A., Leyton, M., Gunn, R. N., Baker, G. B., Diksic, M., & Benkelfat, C. (2006). Modeling
sensitization to stimulants in humans: an [11C]raclopride/positron emission tomography study
in healthy men. Archives of General Psychiatry, 63, 1386–1395.
deCharms, R. C., Maeda, F., Glover, G. H., Ludlow, D., Pauly, J. M., Soneji, D., Gabrieli, J. D.,
et al. (2005). Control over brain activation and pain learned by using real-time functional MRI.
Proceedings of National Academic of Sciences of the USA, 102, 18626–18631.
Gil-da-Costa, R., Martin, A., Lopes, M. A., Muñoz, M., Fritz, J. B., & Braun, A. R. (2006).
­Species-specific calls activate homologs of Broca’s and Wernicke’s areas in the macaque.
Nature Neuroscience, 9, 1064–1070.
Glasser, M. F., & Rilling, J. K. (2008). DTI tractography of the human brain’s language pathways.
Cerebral Cortex, 18, 2471–2482.
38  Guide to Research Techniques in Neuroscience
Gonsalves, B. D., Kahn, I., Curran, T., Norman, K. A., & Wagner, A. D. (2005). Memory strength
and repetition suppression: multimodal imaging of medial temporal cortical contributions to
recognition. Neuron, 47, 751–761.
Hariri, A. R., Mattay, V. S., Tessitore, A., Kolachana, B., Fera, F., Goldman, D., et al. (2002).
­Serotonin transporter genetic variation and the response of the human amygdala. Science, 297,
400–403.
Honey, C. J., Sporns, O., Cammoun, L., Gigandet, X., Thiran, J. P., Meuli, R., et al. (2009). Predicting
human resting-state functional connectivity from structural connectivity. PNAS, 106(6),
2035–2040.
Kuhnen, C. M., & Knutson, B. (2005). The neural basis of financial risk taking. Neuron, 47,
763–770.
Logothetis, N. K., Pauls, J., Augath, M., Trinath, T., & Oeltermann, A. (2001). Neurophysiological
investigation of the basis of the fMRI signal. Nature, 412, 150–157.
McClure, S. M., Li, J., Tomlin, D., Cypert, K. S., Montague, L. M., & Montague, P. R. (2004).
Neural correlates of behavioral preference for culturally familiar drinks. Neuron, 44, 379–387.
Nishimoto, S., Vu, A. T., Naselaris, T., Benjamini, Y., Yu, B., & Gallant, J. L. (2011). Reconstructing
visual experiences from brain activity Evoked by Natural Movies. Curr Biology,
21, 1641–1646.
Pascual-Leone, A., Meyer, K., Treyer, V., & Fehr, E. (2006). Diminishing reciprocal fairness by
disrupting the right prefrontal cortex. Science, 314, 829–832.
Parks, N. A. (2013). Concurrent applications of TMS and near-infrared optical imaging:
­methodological considerations and potential artifacts. Frontiers in Human Neuroscience, 7, 592.
Thomason, M. E., Brown, J. A., Dassanayake, M. T., Shastri, R., Marusak, H. A., et al. (2014).
Intrinsic functional brain architecture Derived from Graph Theoretical analysis in the human
Fetus. PLoS ONE, 9(5), e94423.
Whalen, P. J., Kagan, J., Cook, R. G., Davis, F. C., Kim, H., Polis, S., et al. (2004). Human amygdala
responsivity to masked fearful eye whites. Science, 306, 2061.
Protocols
Ashburner, J., & Friston, K. J. (2000). Voxel-based morphometry—the methods. Neuroimage,
11(6 Pt 1), 805–821.
Ferrera, V., Grinband, J., Teichert, T., Pestilli, F., Dashnaw, S., & Hirsch, J. (2008). Functional
­imaging with reinforcement, eyetracking, and physiological monitoring. JoVE, 21 . http://
dx.doi.org/10.3791/992. http://www.jove.com/index/details.stp?id=992.
Haacke, E. M., & Lin, W. (2007). Current protocols in magnetic resonance imaging. In T. Downey
(Ed.), Current Protocols. New York: John Wiley & Sons.
Websites
fMRI 4 Newbies: http://fmri4newbies.com/
The Basics of MRI: http://www.cis.rit.edu/htbooks/mri/
Neuroimaging Informatics Tools and Resources Clearinghouse (NITRC): http://www.nitrc.org/
fMRI Methods Wiki: http://www.fmrimethods.org/
39
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00002-2
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 2
Animal Behavior
After Reading This Chapter, You Should be Able to:
	l	Compare and contrast the advantages and disadvantages of common model organisms
used to study animal behavior
	l	Discuss considerations for designing a behavioral assay
	l	Describe common behavioral assays used in rodents, invertebrates, and primates
Techniques Covered:
	l	Rodent motor assays: running wheel, homecage activity, open field test
	l	Rodent motor coordination assays: rotarod test, footprint pattern assay, hanging
wire assay, vertical pole test
	l	Rodent sensory assays: visual cliff assay, startle response, taste assays, olfactory
assays
	l	Rodent nociception assays: tail flick assay, Hargreaves assay, hot plate assay,
Von Frey assay, formalin assay
	l	Rodent spatial learning and memory assays: Morris water maze, Barnes maze,
radial arm maze
	l	Rodent nonspatial learning and memory assays: classical conditioning, operant
conditioning, novel object recognition, delayed match to sample and nonmatch to
sample assays
	l	Attention and impulsivity assays: go/no-go task, five choice serial reaction time task
	l	Rodent reward-related assays: self-administration, progressive ratio, conditioned
place preference/aversion
	l	Rodent social assays: resident–intruder assay, social approach/avoidance assay
	l	Rodent anxiety assays: open field test, elevated plus maze, defensive marble
burying, Geller–Seifter conflict test
	l	Rodent depression assays: forced swim test (Porsolt test), tail suspension test,
sucrose preference test
	l	Drosophila behavioral assays: locomotion, flight, sensory assays (vision, olfaction,
taste), learning and memory assays, courtship, aggression
	l	Caenorhabditis elegans behavioral assays: locomotion, sensory assays (mechanosensation,
thermosensation, chemosensation)
	l	Nonhuman primate behavioral paradigms
Humans are the only animals capable of verbally reporting what they feel, what
they perceive, and what they know. To gain insight into the emotional, perceptual,
and cognitive processes of other animals, a scientist can only observe their behavior.
40  Guide to Research Techniques in Neuroscience
Over the past several decades, scientists have developed and standardized a variety
of tests that probe animals for clues about their mental status, from their ability to
perceive a stimulus to whether or not they are emotionally depressed.There are now
dozens of assays commonly described in the literature, with novel assays continually
being developed to study a growing list of behaviors and psychiatric diseases.
The goal of the behavioral neuroscientist is not only to characterize an animal’s
behavior, but also to identify and describe the genetic, biochemical, and
cellular correlates of that behavior. Therefore, behavioral neuroscience does
not exist in isolation, but rather in combination with systems and molecular
techniques. To correlate electrical events in the brain with specific behavioral
outputs, a scientist can monitor neural activity during a behavioral task using
electrophysiology or optical methods (Chapters 4 and 7). A scientist can also
determine which genes, proteins, and/or neurons are necessary or sufficient for
a behavior to occur by investigating the effects of their perturbation (Chapters
3, 4, 8, 12, and 13). Thus, the behavioral paradigms described in this chapter are
used in conjunction with many other techniques described in this book.
The goal of this chapter is to describe the general strategies scientists use to
measure behavior in rodents, invertebrates, and primates. First, we will discuss
some of the issues a scientist must consider when choosing and performing
behavioral assays. Then we will survey the behavioral assays that are the most
commonly used in the literature.
CONSIDERATIONS FOR CHOOSING AND PERFORMING
A BEHAVIORAL ASSAY
There are many factors involved in the design of a behavioral assay. These
factors include choosing a model organism, choosing a behavioral paradigm,
reducing variability between individuals, and validating animal behavior as a
useful model for human behavior and disease.
Choosing an Appropriate Model Organism
A scientist should consider two major factors when choosing an animal model
to study behavior: the natural ethological capabilities of an animal and the
appropriateness of the animal for other experiments.
Ethology refers to the natural capability of an animal to perform specific behaviors.
Worms and flies can be good models for studying behaviors in which animals
interact with the environment, such as sensory transduction, food seeking, and
motor control. Flies have also been used to study addiction, learning and memory,
courtship, circadian rhythms, and other behaviors. Mice and rats are able to perform
more complicated cognitive tasks that underlie emotion, cognition, and social
behavior. Primates are capable of highly intelligent behaviors, such as complex
decision making, identification of faces, and use of tools. In addition to these standard
model organisms, scientists can choose to study other animals with specialized
adaptations that make them an optimal model for certain behavioral tasks (Box 2.1).
Animal Behavior Chapter | 2  41
A scientist must also consider the model organism in the context of other
experiments. If the goal of the study is to identify novel genes that regulate a
behavior, the best choice of animal model will be a genetically tractable organism,
such as a worm, fly, or mouse. If the goal is to record from several neurons in
the brain while the animal performs a behavior, a larger animal is necessary, such
as a large rodent or primate. Mice are often used in behavioral assays because
they are amenable to genetic, molecular, and electrophysiological approaches.
It is also important to consider the ethical and moral implications of using a
specific animal model in an experiment. The use of animals in research is both a
necessity and a privilege that a scientist should never fail to appreciate (Box 2.2).
Choosing an Appropriate Behavioral Paradigm
Whether investigating a commonly studied behavior or developing a new behavioral
paradigm from scratch, a scientist should consider many factors when preparing
to use an assay. First and foremost, the assay must be quantifiable, with
the ability to measure discrete, easily observable variables. For example, consider
an experiment in which a scientist wishes to measure drinking behavior
between two groups of rodents. The scientist has many potential variables to
measure: the total volume of liquid consumed, the rate of water consumption,
the number of licks applied to a liquid delivery system, etc. In the literature,
data are usually presented as bar graphs or scatter plots, with different graphs
representing different quantifiable aspects of each behavioral assay.
A scientist must also consider that animals, especially animals in which
an investigator has manipulated gene or protein expression or neural activity,
may exhibit abnormal behaviors for reasons the scientist may not expect. For
BOX 2.1  Neuroethology
Neuroethology is the study of the neural basis of an animal’s natural behaviors.
This term can be applied to traditional research animals, like rodents and primates.
However, it is more commonly used to describe nontraditional research animals
that have evolved unique adaptations to their ecological niches, making them particularly
good model organisms for specific research goals. For example:
	l	Echolocating bats have enlarged brainstem structures useful for studying the
auditory system.
	l	Various species of birds learn and practice song, allowing scientists to study
developmental learning, consolidation of learning, and language.
	l	Barn owls hunt in the dark and have an amazing capacity for sound localization,
as well as coordinating visual and auditory information.
	l	Some species of prairie voles exhibit monogamous behavior and an increased
tendency, compared with other rodents, to stay in stable male/female pairs.
Studying these animal models results in not only a greater understanding of the
neurobiological basis of ethologically relevant behaviors, but also a greater understanding
of human biology. For example, much of our understanding about the
human auditory system has come from research on echolocating bats.
42  Guide to Research Techniques in Neuroscience
example, animals injected with a pharmacological agent may take a statistically
longer time to complete a maze than control animals injected with saline. However,
this effect may not be due to problems in spatial navigation or memory,
but altered motor behavior that indirectly increases the amount of time spent in
the maze. Therefore, when choosing a behavioral assay, scientists must consider
alternate explanations for why experimental groups may differ, adding additional
behavioral experiments as necessary. It is also important to examine the
overall health of the animal (body weight, heart rate, etc.) to ensure that animals
do not perform abnormally because they are sick.
Variability in Individuals
There are many factors that can increase the variability of a model organism’s
performance on a behavioral task, making it difficult to achieve consistent
BOX 2.2  Ethical Considerations for Animal Use
Using animal models in biological research is necessary to make conclusions
about a fully functional, living organism. However, using animals is also a privilege,
and scientists deciding to use animal models must consider many ethical
considerations. These ethical concerns are not solely the responsibility of the
individual scientist; the use of animals must also be justified to animal oversight
committees that exist to maintain the welfare of research animals. Each institution
has an Institutional Animal Care and Use Committee (IACUC) that must approve
protocols involving animals and ensure that the research is justified. Furthermore,
granting agencies and many scientific journals now require that scientists state that
all animal protocols were approved by their institution’s IACUC.
When using animals in experiments, distress must be minimized, and the potential
benefits to humans must be maximized. Animal welfare guidelines throughout
the world are based on the principles proposed by Russell and Burch in The Principles
of Humane Experimental Research (1959), commonly referred to as the three Rs:
Replacement: If there is any way to do the research without using animals,
it should be done. This includes using computational modeling and cell culture
methods. In the case of behavioral research, modeling is obviously impossible
without actually performing the task. However, another component of replacement
is the use of less-sentient animal species: choosing to study invertebrates
rather than vertebrates, or mice rather than monkeys, etc.
Reduction: Use the minimum number of animals to obtain scientifically valid
data. If possible, use the same animals for multiple experiments. Also, investigators
should perform mathematical analyses to determine the minimum number of animals
required to provide enough statistical power for scientifically significant results.
Refinement: Procedures must minimize distress and pain and enhance animal
well-being. This is also important for the consistency and interpretability of experimental
results; stress has numerous effects on physiology that can confound results.
Following these guidelines is standard practice for developing animal protocols
and justifying research goals.
Animal Behavior Chapter | 2  43
results. One factor is an animal’s genetic background, which can have profound
effects on behavior. Consider mice as an example: there are over 10 strains
of mice commonly studied in research laboratories. Some strains behave very
differently than others. For example, C57/BL6 mice are known to be much
more aggressive than the FVB strain. An investigator performing social experiments
on C57/BL6 mice might, therefore, observe different results than another
investigator performing the same experiments using FVB mice. Strain-specific
differences are also present in other model organisms, such as worms, flies,
rats, and even primates. Therefore, it is important to always perform behavioral
assays in the same strain and, if possible, the same generation of animal. Comparisons
between genetically modified animals and wild-type animals should be
performed on individuals from the same litter.
Environmental variables can also impact behavior. These variables include
animal handling, diet, circadian rhythms, seasonal changes, social interactions,
etc. For example, an investigator may observe different behaviors on a Monday
compared to a Thursday if it turns out that an animal technician changes the
animal cages (and thus handles the animals) each Monday. Alternatively, an individual
animal may perform differently on a behavioral assay if it is housed with
other animals compared to if it is housed alone. These factors require a scientist to
foresee and minimize any aspects of an animal’s environment that may introduce
variability in behavior. Additionally, scientists may need to perform behavioral
assays on large numbers of animals to produce reliable measurements and results.
Using Animal Behavior as a Model for Human Behavior
One of the primary goals of biomedical research is to learn more about humans
by studying animal models. Therefore, it is worthwhile to ask the following:
Can scientists extrapolate and generalize the results of an animal behavioral
assay to people? Is it possible to model human neurological and psychiatric
disorders, such as Alzheimer’s disease and depression, using rodents?
The answers to these questions depend on the exact behavior and disease
studied, but scientists generally gauge the ability of an assay to provide information
about human behavior by examining the assay’s validity. There are
three components to the validity of an animal experiment as a model of human
behavior: face validity, construct validity, and predictive validity. A model has
face validity if the model’s behavior/phenotype is similar to the analogous
human behavior/phenotype. For example, mouse models of autism spend statistically
less time engaging in social behaviors than wild-type mice, just as many
humans diagnosed with autism have difficulty interacting socially with others.
Construct validity refers to a state in which the animal model and human model
have the same underlying genetic or cellular mechanism that may result in a certain
behavior, such as a transgenic mouse engineered to have the same mutation
that causes Huntington’s disease in humans. A model has predictive validity
if treatments used in human patients have the same effect on the animal model.
44  Guide to Research Techniques in Neuroscience
For example, some antidepressants prescribed to humans decrease behavioral
measures of depression in rodent models.
It is possible for animal models to exhibit one form of validity but not others.
The more forms of validity that an animal model exhibits, the greater its
relevance and value to humans. Note that validity is not something that can be
objectively measured, but a concept judged by scientists and their colleagues
within various subfields of neuroscience.
Now that we have discussed some general features of behavioral paradigms,
we describe many commonly used assays in modern neuroscience research.
RODENT BEHAVIORAL PARADIGMS
Rodents are perhaps the most commonly used mammalian model organisms in
behavioral neuroscience because they can be simultaneously used for molecular,
genetic, and electrophysiology experiments. The following assays represent
common approaches used by investigators to measure motor function, sensation,
learning and memory, reward-related behaviors, social interactions, and emotion.
Locomotor Activity
Locomotion assays are used to determine the net motor activity of an animal
over a given time period. These assays are useful for determining if two cohorts
of animals (e.g., knockout animals compared to wild-type littermates) have the
same baseline activity. If measurements are recorded continuously over days
and weeks, these assays are also useful for studying circadian rhythms—the
regular, roughly 24-h cycle of stereotyped biochemical, physiological, and
behavioral activity.
Running Wheel
A running wheel is the simplest way to measure the locomotor activity of an animal
over time. The wheel is coupled to a device that measures the total number
of revolutions and the speed of each revolution. If the animal is free to enter or
exit the running wheel voluntarily (as occurs in most running wheel assays), this
assay cannot measure locomotor activity when the animal is not on the wheel.
However, a running wheel exerts significantly more energy than simple movements
a rodent may perform around its cage, and therefore running wheels can
serve as reliable measurements of deliberate activity.
Home Cage Activity
Locomotor activity can be measured in specially designed cages that project
infrared beams from one side of the cage to the other. Each time an animal
moves around the cage, it breaks the beam, and a computer records the time and
position (Figure 2.1). Alternatively, if cages with infrared beams are not available,
a scientist can place a camera above the cage and record activity. Computer
Animal Behavior Chapter | 2  45
tracking programs can be used to statistically analyze the total locomotor activity
over time. Depending on the specific setup, a scientist can examine horizontal
activity, vertical activity, time spent in various regions of the cage, and total
distance traveled.
Open Field Locomotion Test
An open field test utilizes a large cubic box, usually measuring 1 m long × 1 m
wide × 1 m high (Figure 2.2). The top of the cube is typically left uncovered.
An animal is placed in the middle of the bottom surface, and its movements are
recorded over the course of minutes to hours as it moves around and explores its
environment. After the experiment is completed, computer tracking programs
analyze the movements of the animal over time. This assay can measure horizontal
activity, time spent in various regions of the open field, and the total distance
traveled. This assay can also be used to measure anxiety (described later
in assays of stress).
Motor Coordination and Balance
Assays for motor coordination and balance are used to test the strength of an
animal’s motor and vestibular systems. These tests can be used in isolation or
to exclude motor deficits as a reason for poor performance in other behavioral
experiments. For example, if pharmacological or genetic manipulation of an
animal results in abnormal performance on a learning and memory assay, a
scientist could ensure that this abnormal behavior was due to impairments other
than general motor deficits.
FIGURE 2.1  Locomotor activity can be monitored in a specially designed home cage.
Courtesy of the Stanford Behavioral and Functional Neuroscience Laboratory.
46  Guide to Research Techniques in Neuroscience
Rotarod
A rotarod is a device consisting of a cylindrical rod suspended above a padded
base (Figure 2.3). A scientist places a rodent on the rod and gradually accelerates
the rotation until the rodent falls to the bottom of the chamber. Most animals
are able to maintain balance for many minutes, but rodents with deficits
in motor coordination or balance fall more quickly. This assay can also be used
as a measure of motor learning, as most animals perform better on the rotarod
assay after consecutive training days.
Footprint Pattern Assay
A scientist performs a footprint pattern assay by dipping an animal’s paws in
inkandallowingittowalkdownapaper-linedtunnel.Thesefootprintscanreveal
gait abnormalities and ataxia—abnormal muscle coordination (Figure 2.4).
Dipping the forepaws and hind paws in different colors of ink allows a scientist
to measure a variety of gait parameters: distance between each stride,
changes in stride length, variability around a linear axis, width between left
and right hind paws, regularity of steps, and overlap between forepaws and
hind paws. Modern automated versions of this task can also track pressure
and walking speed. Ataxic gait is characterized by a highly variable stride
length and path.
Hanging Wire Assay
The hanging wire assay is used to measure neuromuscular deficits. An investigator
places a rodent on a cage lid made of a wire grid, shaking the lid, so the
FIGURE 2.2  Exploratory locomotor activity can be monitored in an open field testing chamber.
Courtesy of the Stanford Behavioral and Functional Neuroscience Laboratory.
Animal Behavior Chapter | 2  47
rodent grips the wire. The investigator then gently flips the lid, so the rodent
hangs upside down. To prevent falling, the rodent grasps the wire, requiring both
balance and grip strength. The investigator measures the time it takes before the
rodent falls to a padded surface. A normal rodent can hang upside down for
several minutes, but a 60-s cutoff is usually used for experimental purposes.
Vertical Pole Test
In a vertical pole test, a rodent is placed on the center of a horizontal pole
wrapped in cloth to provide traction. The pole is gradually lifted toward a
FIGURE 2.3  A rotarod tests balance and motor coordination.  Animals are placed on a rotating
rod (red arrow) until they eventually fall onto a padded floor (blue arrow). Multiple animals can be
tested at one time with a device that has individual lanes to record each animal’s latency to fall.
Courtesy of the Stanford Behavioral and Functional Neuroscience Laboratory.
48  Guide to Research Techniques in Neuroscience
vertical position, and the investigator records the amount of time and the angle
at which the animal falls off. Normally, rodents can move up and down the pole
without falling before the pole reaches a 45° angle.
Sensory Function
Abnormal sensory function is usually more difficult to observe than abnormal
motor function, as it is impossible to directly measure what any animal perceives.
In many sensory assays, a rodent uses motor output to report detection
of a sensory stimulus; thus, these assays are sensitive to confounding effects due
to deficits in motor function.
Visual Cliff Assay
A visual cliff assay evaluates the ability of a rodent to see the drop-off at the
edge of a horizontal surface. The edge of a box or table can serve as a horizontal
plane with a vertical drop to a lower horizontal plane, such as the floor. Each
surface is coated with a black-and-white checkerboard pattern to enhance the
detection of visual planes (Figure 2.5). A clear sheet of Plexiglas is placed over
the drop-off, extending across the cliff so an animal cannot actually fall. Normally,
rodents visually detect the drop-off and approach the edge cautiously,
stopping at the cliff. Blind rodents approach the cliff but continue walking,
unable to see the edge but able to feel the Plexiglas sheet. Rodents with normal
vision can also use their whiskers to feel the Plexiglas extension, so the whiskers
are often cut prior to an experiment.
FIGURE 2.4  Footprint pattern analysis can
reveal gait abnormalities. Footprint patterns of a
wild-type (+/+) mouse compared to a mutant (stg/
stg) showing a wider stance (proximity of blue forelimb
and black hindlimb footprints) and stumbling
(red arrow). Reprinted from Meng, H., et al. (2007).
BDNF transgene improves ataxic and motor behaviors
in stargazer mice. Brain Research, 1160, 47–57,
with permission from Elsevier.
+/+ stg/stg
Animal Behavior Chapter | 2  49
Startle Response Assay
In a startle response assay, a scientist exposes a rodent to an unexpected
and disruptive sensory stimulus and measures the degree to which the animal
responds, typically exhibited as an eye blink, body flinch, or overall muscle
contraction. These stimuli can be visual (a bright light), auditory (loud and disrupting
noise), or tactile (unexpected touch or air puff). Investigators can either
observe and score the number of times a startle response occurs or use specialized
sensors, such as an electromyogram, to more quantitatively measure the
degree of the response. This assay can be confounded by deficits in emotional
and motor components of the startle reflex circuitry. This assay can also be used
to measure animal learning by measuring extinction of the startle response over
successive trials (Box 2.3).
Taste
Investigating an animal’s ability to taste usually involves a choice assay, in
which different gustatory compounds are placed in different solutions in water
bottles. The scientist measures the volume of water in each bottle to determine
the animal’s taste preferences. It is possible to investigate whether an animal can
detect bitter tastes by placing quinine, an extremely bitter compound, in a water
solution and determining if the animal consumes less water than a solution with
no quinine. Likewise, it is possible to investigate whether an animal can detect
sweet tastes by placing sucrose or saccharine in a solution and determining if an
animal drinks more water than a solution with no sweet compounds.
FIGURE 2.5  Visual cliff assay. A checkerboard pattern enhances the perception of a drop-off.
Rodents that can see this drop-off will pause at the edge, while blind rodents will feel the Plexiglas
surface and continue walking without pause.
50  Guide to Research Techniques in Neuroscience
Olfaction
A scientist can test for normal olfactory processing by measuring how long it
takes for an animal to find a piece of familiar, palatable food (like a cookie,
cheese, or chocolate chip) hidden within the cage.
Nociception
Nociception is the ability to detect a noxious stimulus, usually perceived as
pain. Nociceptive assays rely on physical indicators of discomfort, such as withdrawal
reflexes, licking, and vocalizations. These assays are usually used to
investigate either the neural basis of pain or the therapeutic potential of analgesic
drugs. Genetically or pharmacologically manipulated animals that exhibit
decreased sensitivity to pain must be monitored closely during these assays to
prevent tissue damage induced by nociceptive stimuli.
Tail Flick Assay
In the tail flick assay, a high-intensity beam of light is aimed at a rodent’s tail.
In normal animals, the beam of light produces a painful heat sensation, causing
a reflex that moves the tail. The investigator measures the amount of time
it takes before the tail flicks to the side. Sex, age, and body weight can affect a
rodent’s response, so all animals used for these experiments (experimental and
control animals) must be similar to avoid confounding results.
Hargreaves Assay
The Hargreaves assay is similar to the tail flick assay, but the high-intensity
beam of light is aimed at the rodent’s hind paw rather than the tail. The investigator
measures the time it takes for the rodent to withdraw its paw.
Hot Plate Assay
In the hot plate assay, a scientist places a rodent on a heated surface and prevents
the animal from leaving by enclosing it within a tall cylinder (Figure 2.6).
BOX 2.3  Prepulse Inhibition
Interestingly, a startle response can be reduced if a nonthreatening stimulus is
presented immediately before the disruptive sensory stimulus. This phenomenon
is known as prepulse inhibition (PPI) and reflects the nervous system’s ability to
prepare for a strong sensory stimulus after a small warning (the prepulse). Human
patients with attention deficit disorder, Alzheimer’s disease, and schizophrenia
show deficits in PPI. Therefore, PPI can be a useful paradigm to test animal
models of psychiatric disease and establish face validity for animal experiments.
Furthermore, PPI has been a useful behavioral assay to test potential antipsychotic
medications that improve sensory processing.
Animal Behavior Chapter | 2  51
The surface is calibrated so that a normal animal will react within about 10 s of
exposure (usually 52–53 °C). The investigator records the latency and amount
of time an animal reacts to the heat stimuli. Individual reactions vary: licking a
rear paw is a reliable indicator of discomfort, though some animals will jump
or vocalize.
Von Frey Assay
A Von Frey assay is used to examine the sensitivity to pinch and mechanical
stimuli. Von Frey hairs are fine-gauge metal wires. The investigator pokes the
hind paw of an animal standing on an elevated mesh platform by inserting a
Von Frey hair through the mesh from below. Normal rodents usually react by
withdrawing or licking their paws and possibly vocalizing.
Formalin Assay
A formalin assay is used to examine an animal’s sensitivity to noxious chemical
stimuli.An investigator injects a small volume of formalin, a noxious chemical,
into a rodent’s hind paw and records the total amount of time the rodent
spends licking, biting, or withdrawing the affected limb. This activity is usually
assessed in two phases: one phase starting immediately after the injection and
lasting about 10 min and another phase starting 20 min after the injection and
lasting about an hour. The second phase is a response to tissue damage caused
by inflammation.
FIGURE 2.6  Hot plate test of pain perception. Courtesy of the Stanford Behavioral and
Functional Neuroscience Laboratory.
52  Guide to Research Techniques in Neuroscience
Spatial Learning and Memory
Spatial learning and memory are ethologically relevant behaviors to a mouse or
rat. In the wild, these animals spend most of their lives locating good nesting
spots, searching for food, and avoiding predation. Therefore, rodents are great
model species to assay for spatial navigation and memory.
Morris Water Maze
The Morris water maze is one of the most frequently used spatial learning and
memory paradigms. Rodents are placed in a large circular pool of opaque water
(Figure 2.7). Their natural dislike of water makes rodents highly motivated to
escape from the pool. In initial trials, a visible platform is placed within the
pool so that an animal can emerge from the water. A scientist places visual cues
around the pool so the animal can relate the spatial environment with the location
of the platform. In subsequent trials, the platform is hidden just below the
surface of the water, and a scientist measures the time required for the rodent to
swim to the platform to escape the water.
In the first few trials, the rodents become progressively better at swimming
directly toward the platform, demonstrating their ability to learn the
platform’s location. To test memory at the end of training, the investigator
completely removes the platform to measure the amount of time the animal
spends searching in each quadrant of the pool. This experiment measures the
ability of the animal to identify a spatial location based on the visual cues
placed around the chamber. Because water is stressful to rodents (in mice
FIGURE 2.7  The Morris water maze is one of the most common rodent tests for learning and
memory. Courtesy of the Stanford Behavioral and Functional Neuroscience Laboratory.
Animal Behavior Chapter | 2  53
more than rats), it is important for a scientist to account for stress-induced
performance effects.
Barnes Maze
The Barnes maze consists of a circular table with holes around the circumference,
placed in a room with visual cues in the periphery (Figure 2.8). Most of
these holes lead to an open drop to the floor, but a single hole leads to a drop
box, a dark box in which the animal can hide. A rodent is naturally motivated
to avoid open spaces and bright lights, and therefore attempts to find the drop
box. In initial trials, the scientist gently leads the animal to the drop box. In
subsequent trials, the animal is placed in the center of the table and must find
the drop box on its own. After a few trials, rodents typically remember which
hole contains the drop box and quickly proceed in a direct path toward the
hole. Investigators can measure the amount of time to find the correct hole,
the number of incorrect holes explored, and the length of the exploratory path.
The Barnes maze is considered to be less stressful than the Morris water maze.
Radial Arm Maze
The radial arm maze consists of an array of arms, usually eight or more, which
radiate from a central starting point (Figure 2.9). At the end of each arm is a cup
that may or may not contain a food reward. Animals are trained to recognize that
only one of the arms will contain food. Investigators measure the amount of time
it takes for animals to find the arm leading to food, as well as the number of times
it traverses an arm it has previously visited. Exploring a previously visited arm
FIGURE 2.8  The Barnes maze. Rodents learn which one of the holes contains a hidden
drop box they can use to escape from light. Courtesy of the Stanford Behavioral and Functional
Neuroscience Laboratory.
54  Guide to Research Techniques in Neuroscience
indicates that the animal did not remember previously choosing that spatial path.
This is a relatively difficult task for rodents, requiring several days or weeks to train
rats and many weeks to train mice. Unlike the Morris water maze and the Barnes
maze, the radial arm maze does not use distant visual cues to aid spatial learning.
Nonspatial Learning and Memory
The neural circuitry that mediates nonspatial learning and memory differs from
circuitry that mediates spatial learning and memory. Thus, multiple assays using
both nonspatial- and spatial-based tasks are used to evaluate learning and memory
as a whole. Nonspatial learning and memory assays include innate learning,
such as motor performance improvement over time (such as the rotarod assay),
novel object recognition, as well as learned associations through conditioning.
While classical (Box 2.4) and operant (Box 2.5) conditioning can be used to
directly assay an animal’s ability to learn, these paradigms are often used to train
and test animals on more sophisticated tasks. For example, the delayed match to
sample and nonmatch to sample assays use operant conditioning techniques to
examine an animal’s working memory. These techniques are described below.
Novel Object Recognition
The novel object recognition paradigm assesses an animal’s innate ability to
distinguish an old from a new object. Rodents are naturally curious and will
take time to explore novel objects. When presented with both a new object and
FIGURE 2.9  Radial arm maze. Courtesy of the Stanford Behavioral and Functional Neuroscience
Laboratory.
Animal Behavior Chapter | 2  55
a previously explored object, normal rodents remember the old object and spend
relatively more time exploring the new object. A scientist measures the amount
of time spent interacting with both objects.
Delayed Match to Sample/Nonmatch to Sample
Operant conditioning (Box 2.5) can be used to train an animal to make a
response such as poking its nose into a hole or pressing a lever for a reward in
response to a stimulus, often a light appearing above the lever. To test working
memory, the investigator trains the rodent to perform an operant response
under specific conditions—for example, pressing a specific lever when multiple
levers are present. In a match to sample task, a light appears above one lever,
BOX 2.4  Classical Conditioning
In classical conditioning (also called Pavlovian conditioning), the investigator couples
an initially neutral stimulus, such as a tone or light, with a salient stimulus,
such as food or an electric shock. The neutral stimulus is referred to as the conditioned
stimulus (CS), while the salient stimulus is referred to as the unconditioned
stimulus (US). The unconditioned stimulus elicits a reflexive response, such as salivation
or fear behaviors. By pairing the conditioned and unconditioned stimuli, the
conditioned stimulus alone is eventually able to elicit the reflexive response of the
unconditioned stimulus. Examples of Pavlovian conditioning are cued fear conditioning
and contextual fear conditioning. Fear conditioning often utilizes a foot
shock (US) to induce freezing behavior. By associating an auditory or visual cue
(CS) with the foot shock, animals will freeze in response to the sensory cue alone
(i.e., without any foot shock) demonstrating a learned association. Contextual fear
conditioning measures freezing behavior when the animal is returned to the training
chamber without foot shock—exhibiting a response without the unconditioned
stimulus shows that the animal has learned the association between a location and
the shock. Cued conditioning should be measured in a new testing chamber with
a very different context (shape of the box, visual cues, lighting, odors, textures)
but with the same auditory or visual cue from training to test the specific learned
association between the cue and the shock.
BOX 2.5  Operant Conditioning
In operant conditioning, a voluntary response (e.g., a lever press or a nose poke) is
induced by positive reinforcement or suppressed by punishment. Positive rewards
often include food or water (especially in food- or water-deprived animals) or drugs
of abuse. Punishing or aversive stimuli often include foot shocks or an unpleasant
sensory stimulus such as a loud tone. Several training sessions are often required for
animals to learn the association between performing a task and receiving reinforcement
or punishment. While operant conditioning is a form of learning, this paradigm
is often used to study other behaviors, such as reward, attention, and impulsivity.
56  Guide to Research Techniques in Neuroscience
and the animal must choose to press the lever beneath that light. In a nonmatch
to sample task, a light appears, and the animal must choose to press the opposite
lever. After learning these tasks, the scientist adds artificial delays, such as
withdrawing the lever, between the time when a cue appears and the time that an
animal responds. The longer the delay, the longer the animal must remember the
light cue that was presented, thus testing the animal’s working memory.
Attention and Impulsivity
Rodent models of attention and impulsivity test an animal’s ability to learn a
relatively difficult task in which success is dependent on sustained attention to
a cue and reduced desire to impulsively engage in behavioral responses. These
tasks are performed using rats more often than mice because often mice have
difficulty learning the task paradigms.
Go/No-Go Task
A go/no-go task is a form of operant conditioning (Box 2.5) in which a rodent is
trained to produce a response, such as a lever press, in the presence of a specific go
cue (such as a green light), yet withhold a response in the presence of a separate
no-go cue (such as a red light). Rodents are relatively successful on the go trials
but often have difficulty withholding responses on the no-go trials. These success
rates are exacerbated in animal models of impulsivity or low attention span.
Five Choice Serial Reaction Time Task
In the five choice serial reaction time task, an animal is placed in an operant
chamber (Box 2.5) with five lights coupled with five trays capable of delivering
a reward, such as a food pellet. At the beginning of a trial, one of the lights is
briefly illuminated, and the rodent must indicate via nose poke or lever press
which light was on. Controlling the length of time the light is on prior to requiring
the animal to produce an action can vary the difficulty of the task. Furthermore,
there is an intertrial interval during which an animal must withhold all
responses, demonstrating inhibitory control and reduced impulsivity.
Reward-Related Behaviors
Scientists can measure the rewarding aspects of a stimulus, such as an addictive
drug or food, by measuring how much an animal is willing to work to obtain the
reward or the degree to which the animal associates the stimulus as a positive
reinforcement. These assays are frequently used to study the neurobiology of
addiction, as well as motivational behaviors such as hunger and thirst.
Self-Administration
Self-administration is a form of operant conditioning (Box 2.5) in which animals
are trained to perform a particular action, such as pressing a lever, to
Animal Behavior Chapter | 2  57
receive a rewarding stimulus, such as an addictive drug. Animals are free to perform
the action freely and repetitively, demonstrating the intrinsically rewarding
properties of the stimulus. In a continuous reinforcement paradigm, an
animal performs a single action to receive a rewarding stimulus, whereas in a
fixed ratio paradigm, an animal performs a set amount of actions (such as five
lever presses). In a progressive ratio paradigm, the animal must perform an
action more and more to receive the rewarding stimulus (for example, pressing
a lever 1 time, then 2 times, then 4 times, then 8 times, etc.), and the scientist
measures the breakpoint at which the animal no longer works to receive the
reward.
Conditioned Place Preference
The conditioned place preference assay is a form of classical conditioning
(Box 2.4) in which a rodent associates a particular environment with a rewarding
experience. An animal is placed inside a test chamber (the conditioned stimulus)
and allowed access to an unconditioned stimulus (a drug, food, odor, etc.)
for minutes each day for multiple days. The animal is also placed in a control
chamber without any stimulus present for minutes each day for multiple days.
On the test day, the animal is allowed free access to both chambers, and the
scientist measures the amount of time the animal spends in one chamber relative
to the other. If animals spend significantly more time in the chamber with
the unconditioned stimulus, they are said to have developed a conditioned place
preference, associating that chamber with the reward. Alternatively, the unconditioned
stimulus could be relatively unpleasant for the animals and could lead
to conditioned place aversion.
Social Behaviors
Rodents display a variety of innate social behaviors. Mating, parenting, nesting,
and grooming are all innate behaviors that can be observed within an individual’s
cage. Rodents also exhibit specific behaviors based on social recognition
when multiple animals are exposed to one another. Exploratory sniffing allows
animals to decide on an appropriate social response, such as defending territory
or attempting copulation. Two standard assays are well represented in the literature:
the resident–intruder assay and social approach/avoidance assays.
Resident–Intruder Assay
The resident–intruder assay measures territorial behavior in males. A scientist
adds an intruder animal to the cage of a resident animal and measures specific
aggressive behaviors: tail rattling, quivering or thrashing of the tail, physical attacks
such as biting or clawing, wrestling, and chasing.The investigator can then compare
the amount of time each animal spends investigating the other (following, sniffing,
grooming) compared to the amount of time displaying aggressive behavior.
58  Guide to Research Techniques in Neuroscience
Social Approach/Avoidance
If a scientist presents a rodent with a choice between interacting with another
(unfamiliar) rodent or interacting with an inanimate object, most animals will
spend more time interacting with the other rodent, a phenomenon referred to as
social approach. Animals with abnormal social behaviors may spend relatively
little time with the other animal, referred to as social avoidance. For example,
in some mouse models of autism, there is no difference in the amount of time
spent exploring the object and exploring the other mouse. These behaviors are
thought to be analogous to the asocial behaviors exhibited by humans diagnosed
with autism.
Anxiety
Paradigms that measure anxiety generally involve placing rodents in naturally
stressful environments, such as brightly lit, open spaces. These assays are useful
for investigating brain regions thought to modulate anxiety, as well as testing
novel anxiolytic drugs hypothesized to reduce anxiety. In addition to the common
assays described here, investigators can also measure physiological correlates
of anxiety, such as heart rate, defecation and freezing behavior.
Open Field Test
The open field test (Figure 2.2), previously described as an assay of locomotor
activity, can also be used to measure anxiety. A rodent placed in a bare, open
chamber will initially stay near the walls and avoid the center (Figure 2.10).
Normal animals typically acclimate to the chamber and eventually explore the
center area. More anxious animals spend significantly less time in the open area
and more time closer to the walls. The investigator can monitor the animal’s
activity with a camera placed above the open field, and computer software measures
time spent in the center compared with the periphery. Anxiolytic drugs
increase the amount of time rodents spend in the open area.
(A) (B)
FIGURE 2.10  The open field test as a measure of anxiety.  (A) After being acclimated to the
chamber, rodents will normally explore the entire area, but (B) a rodent model of anxiety will stay
near the perimeter of the chamber.
Animal Behavior Chapter | 2  59
Elevated Plus Maze
An elevated plus maze is a four-armed platform resembling the shape of a plus
sign positioned two to three feet above the ground (Figure 2.11). Two arms of
the maze have sides, and two do not. A scientist places an animal in the center
of the maze and allows it to move freely in any of the arms. Normal animals avoid
the open arms, spending relatively more time in the protected, closed arms. Anxiolytic
drugs increase the proportion of time rodents spend in the exposed arms.
Defensive Marble Burying
Rodents tend to bury objects present in their environment, such as glass marbles,
within the bedding of their cages. Animals that are more anxious tend to bury
more objects in the 30–60 min after they are introduced. To perform a defensive
marble burying assay, a scientist places 10–20 marbles in an animal’s cage
and quantifies the number of marbles buried after 30 min. Anxiolytic drugs tend
to decrease the number of marbles buried over time.
Geller–Seifter Conflict Test
The Geller–Seifter conflict test is one of the oldest assays to study anxiety and
remains a robust test of anxiolytic drug effects. A scientist uses operant conditioning
(Box 2.5) to train an animal to press a lever for a food reward. During
the experiment, the investigator pairs a lever press with an unpleasant electrical
shock. Thus, the animal must decide whether to receive food while getting
FIGURE 2.11  The elevated plus maze. Courtesy of the Stanford Behavioral and Functional
Neuroscience Laboratory.
60  Guide to Research Techniques in Neuroscience
shocked or not receive food at all. Anxious animals tend to press the lever significantly
fewer times than nonanxious animals. Anxiolytic drugs (but not other
psychoactive drugs) increase the number of lever presses.
Depression
How is it possible to measure depression in a mouse or rat? Paradigms that
investigate depression in animal models assay for the feelings of hopelessness
and despair experienced by humans diagnosed with depression. Scientists can
induce depression in a rodent by applying conditions of learned helplessness
and chronic mild stress. The learned helplessness paradigm exposes an animal
to aversive stimuli at random intervals. Theoretically, this treatment creates
a condition in which the animal experiences a lack of control, exhibiting
symptoms of behavioral despair. Applying conditions of chronic mild stress
(leaving the lights on all the time, tilting the animal’s cage, wetting the animal’s
bedding, applying intermittent air puffs) over a period of days to weeks can also
theoretically lead to conditions of behavioral despair and helplessness. Both
learned helplessness and chronic mild stress cause differences in behavior on
the assays of depression described here. Importantly, antidepressants reverse
these behaviors, demonstrating the predictive validity of these animal models.
Forced Swim Test (Porsolt Test)
In the forced swim test (also called the Porsolt test), rodents are placed in a
small, confined space, such as a large graduated cylinder filled halfway with water
(Figure 2.12). Initially, there is a period of vigorous activity during which the animal
triestoescape.Eventually,theanimalceasesvigorousactivityandexhibitsacharac-
teristicimmobilityinwhichitonlymovestomaintainitsheadabovewater.Thisphysical
immobility is thought to be an indication of behavioral despair. Investigators
measure the amount of time between when the animal is placed in the chamber and
the onset of immobility. Rodent models of depression exhibit a decrease in the time
spent trying to escape, but this decrease is reversed with antidepressants.
Tail Suspension Assay
In the tail suspension assay, either a device or the investigator holds the rodent
in the air by its tail. The animal’s natural reaction is to move vigorously to
escape. Depressed animals give up more quickly than nondepressed animals.
Antidepressants increase the amount of time the animals struggle. An advantage
of this paradigm over the forced swim test is that tail suspension is less stressful
to the animal because there is no exposure to water.
Sucrose Preference Test
The sucrose preference test is hypothesized to model anhedonia—the loss
of desire to pursue an activity that was once enjoyable. Normally, rodents that
Animal Behavior Chapter | 2  61
are allowed to choose between drinking tap water or water with sucrose will
choose the water with sucrose. Rodent models of depression have a significantly
reduced preference for water with sucrose. Importantly, this effect is reversed
by administration of antidepressants.
DROSOPHILA BEHAVIORAL PARADIGMS
One of the powerful advantages of using Drosophila melanogaster, the fruit
fly, as a model organism is its genetic tractability. Drosophila has proven to
be very useful for studying the genetic basis of behavior due to the coupling of
many genetic tools with a variety of fly-specific behavioral assays. The lab of
Seymour Benzer famously used Drosophila to study genes necessary for learning,
memory, vision, circadian rhythms, nociception, and many other behaviors.
Many other labs have used Drosophila to study sleep, addiction, courtship, and
aggression.
As with any animal model used to study behavior, age, diet, and environmental
factors must be strictly controlled when performing behavioral experiments
to attain consistent results. If experiments are performed over multiple
days, the scientist should perform assays at the same time each day to avoid
FIGURE 2.12  Performance on the forced swim
(Porsolt) test is used as a test of behavioral despair to
model depression in rodents.
62  Guide to Research Techniques in Neuroscience
potentially confounding results due to circadian rhythms. Applying anesthesia
or handling the flies just prior to behavioral tests can also affect the outcome.
Locomotor Behavior
Locomotor behavior in flies can be studied similarly to locomotor behavior in
rodents. A scientist can project infrared beams of light through a housing chamber,
allowing flies to break the beams and thus indicate their movement over
time. Alternatively, a scientist can record a housing chamber with a video camera,
and tracking software can record the activity of many flies over time. Locomotor
behavior is useful when studying motor function, circadian rhythms, and
the effects of alcohol and other drugs.
Flight
Traditional locomotor assays measure movement in an enclosed container.
Another ethologically relevant movement for a fly is flight. Therefore,
scientists have developed specialized flight simulators that suspend a fly
from a thin pin and project visual stimuli to depict the external, visual environment
(Figure 2.13). These simulators contain torque meters to record turning
responses to various stimuli, allowing analysis of sensory and motor reflexes.
IR laser diode
Torque meter
Light source
Light guides
Diffusor
Electric motor
Yaw torque signal
FIGURE 2.13  A Drosophila flight simulator. This device can be used to measure free-flight
behaviors or flight responses to visual stimuli, as well as to train flies by using operant conditioning
methods that use a laser to generate heat reinforcement. Courtesy of Dr Björn Brembs.
Animal Behavior Chapter | 2  63
These flight simulators can also be used to study sensorimotor processing and
spatial learning and memory.
Sensory Function
Sensory modalities commonly studied in the fly include vision, olfaction, and
taste. These modalities are all ethologically relevant for a fly, which spends most
of its life navigating through space using visual and chemical cues in search of
food or responding to chemical signals produced by other flies.
Vision
Flies exhibit a natural tendency to move toward light, a phenomenon known
as phototaxis. To test proper visual function, scientists can use a device called
a countercurrent apparatus, made up of a series of test tubes (Figure 2.14).
Flies are placed in one test tube, which is gently tapped so that the flies fall to the
bottom. The test tube is then laid horizontally across from a second test tube. This
second tube is illuminated with a fluorescent light. The scientist waits approximately
15 s before preventing the flies from crossing between the two tubes. The
flies trapped within the second tube are considered to have demonstrated more
of a phototaxic response. This process is repeated several times, with flies demonstrating
a phototaxic response being used for the next trial. The end result is a
fractionated population of flies that vary in their phototaxic responses.
Vision can also be assayed using a device called a T-maze (Figure 2.15).
This apparatus consists of two arms oriented in opposite directions—a
“T” shape—with an additional loading arm. Flies are placed into the loading
arm and moved to the center of the other two arms of the T-maze, where they
can travel to either arm. Each arm contains a different visual stimulus—for
example, two different wavelengths of light, bright light versus darkness, flashing
light versus constant light, and so forth. Thus, a T-maze allows scientists to
understand the visual preferences of a fly, as well as investigate the genes necessary
and/or sufficient for these preferences to occur.
FIGURE 2.14  The countercurrent apparatus can be used to isolate a population of
phototaxing flies.
64  Guide to Research Techniques in Neuroscience
Olfaction
Olfaction assays can also take place in a T-maze. Each arm of the maze contains
a different odor, and flies decide which odor to move toward. Investigators can
use this assay to determine whether an odor is attractive, repulsive, or neutral.
In an olfactory trap assay, odors absorbed on a cotton swab or filter paper
are placed at the bottom of a chamber with a pipette tip that allows flies to get in
but not get out. By simply waiting a certain amount of time for flies to experience
the baited odor, an attraction index can be calculated from the ratio of flies
trapped in the odor vial compared to a water control. An attraction index of 1
indicates complete attraction to the odor, while an attraction index of 0 indicates
either no preference for an odor or anosmic flies.
There are many simple avoidance assays that assess behavioral responses to
olfactory cues. In the olfactory avoidance (also called dipstick) assay, a scientist
inserts a piece of filter paper or cotton swab containing a specific odor into
a fly vial, then measures the distance that the fly maintains from the offending
odorant. A similar assay is the chemosensory jump assay or olfactory jump
response, which exploits the tendency of flies to exhibit a startle response when
encountering a novel odor.
Taste
Hungry flies exhibit an unconditioned reflex called the proboscis extension
response. A scientist can elicit this response by applying taste ligands to
FIGURE 2.15  A Drosophila T-maze can be used to test fly sensory preferences or to train
and test flies on conditioning training.
Animal Behavior Chapter | 2  65
gustatory receptor neurons on the leg or proboscis. This reflex can be used to test
specific gustatory receptor responses and sensitivity to different taste ligands.
A scientist can also assay taste using a feeding acceptance assay. Starved
flies are provided a choice between appetitive, aversive, or neutral stimuli, each
dyed a different color. The amount of each stimulus ingested can be scored by
examining the color of the fly’s stomach.
Learning and Memory
Learning and memory assays can be performed in Drosophila using a T-maze
and classical conditioning. An odor is paired with an electric shock. This odor
is then presented in one arm of a T-maze. Flies that avoid this arm are considered
to have learned the association. This assay has been widely used in genetic
screens that assay both learning and memory, as well as olfaction.
Investigators can also assay learning and memory on individual flies in the
flight simulator through classical or operant conditioning. For example, flies can be
trained to navigate a virtual scene to find an appetitive sensory stimulus or reward.
Social Behaviors
There are many social behaviors that a scientist can study using Drosophila.
Many of these behaviors are innate (as opposed to learned), so they can be used
to investigate the specific genes necessary and/or sufficient for these behaviors
to occur. Perhaps the two most commonly studied social behaviors in the fly are
courtship and aggression.
Courtship
Flies exhibit a stereotyped courtship ritual composed of several distinct stages:
(1) orientation, (2) tapping, (3) wing song, (4) licking, (5) attempted copulation,
and (6) copulation. Because these behaviors are innate, scientists can use
them to study the genetic basis of a hard-wired behavior. Groups of male and/or
female flies can be placed into tiny chambers and recorded with a video camera.
Later, an investigator scores the behavior, measuring a variety of parameters
related to the timing, performance, and progression of these different stages.
Aggression
To study aggression, a scientist places two male flies in a chamber. The flies
naturally begin to fight, and the scientist can record many different parameters:
which fly initiated the fight, the outcome, the interfight interval, the order of
events of each fight, etc.
CAENORHABDITIS ELEGANS BEHAVIORAL PARADIGMS
The relatively simple and well-understood neural circuitry of Caenorhabditis
elegans, coupled with its genetic tractability, make this species a powerful
66  Guide to Research Techniques in Neuroscience
animal model to study the genetic basis of behavior. In fact, many genes in
the worm are named after the behavioral phenotypes that led to their discovery:
Unc (uncoordinated) mutants have disrupted locomotion, Egl (egg-laying)
mutants have disrupted egg-laying, and Mec (mechanosensation) mutants have
disrupted responses to touch. Most behavioral assays in C. elegans examine
motor and sensory functions.
Locomotor Behavior
Locomotor behavior in the worm can be automatically tracked using computer
software, such as Worm Tracker (Figure 2.16). This software allows for
a detailed analysis of the sinusoidal movement of worms, allowing scientists
to determine the genetic and cellular components that make these movements
possible.
Sensory Behavior
Caenorhabditis elegans has proved very useful for investigating sensory transduction,
as worms depend on detecting mechanical, thermal, and chemical stimuli
to stay alive and find food.
Mechanosensation
A gentle touch with a fine hair to the body, head, or tail of a worm results in different
behavioral responses. For example, if touched on the nose, worms move
Digital
video camera
To PC
Zoom lens
Worm arena
Transmitted-light
source
Time (s)
Speed(mm/s)
FIGURE 2.16  The Worm Tracker provides detailed information about the movements of
individual worms.  Representative tracks generated by the worm tracker can indicate starting position
(x), path length, speed, and turning events (circles). Reprinted from Ramot, D., et al. (2008).
The parallel worm tracker: a platform for measuring average speed and drug-induced paralysis in
nematodes. PLoS ONE, 3(5), e2208, under the Creative Commons Attribution License.
Animal Behavior Chapter | 2  67
backward. If touched on the body, worms immediately stop and sometimes
move away. Because the complete circuitry diagram of C. elegans is known, it is
possible to use these behavioral assays to map functional connections between
different neurons in different locations of the worm’s body.
Thermosensation
Worm behavior is strongly influenced by environmental conditions—temperature,
population density, and feeding status. Animals avoid temperatures at which
they have been starved, and accumulate at temperatures associated with food.
This requires thermosensation, as well as a memory of the associations. There
are various methods for creating thermal gradients on which to test worms: an
example of a simple method is to place frozen glacial acetic acid on one end
of a plate and an incubator on the other end. A more sophisticated method is
to use a thermoelectric device to maintain a steady heat gradient across a plate.
Investigators can then measure thermotactic migration by placing worms on the
plate and identifying the temperature where migration stops.
Chemosensation
Chemosensation can be thought of as a worm’s sense of taste or smell. A scientist
can assay attraction or avoidance of specific compounds by dissolving these
compounds in agar and placing them on opposite sides of a Petri dish. Worms
are placed at the intersection of the two chemosensory cues, and the scientist
measures the number of worms that migrate to each compound.
NONHUMAN PRIMATE BEHAVIORAL PARADIGMS
Nonhuman primates can be used to study the neural basis of complex motor
actions, sensory perception, and cognitive processes such as decision making,
attention, and complex learning. Experiments almost always couple electrophysiological
recordings with behavior so that scientists can correlate the firing
of neurons with distinct behavioral events. In addition to simply measuring
neural activity, it is possible to manipulate neural activity using electrical and
pharmacological methods (Chapter 8).
One of the greatest strengths of primates as research models is their genetic,
anatomical, and behavioral similarities to humans, allowing scientists to extend
results to theories about how the human brain works. Primates are also capable
of more complex behaviors and tasks than rodents and other animal models. In
fact, many of the specific tasks performed by nonhuman primates are the same
or adapted versions of tasks performed by humans in cognitive neuroscience or
psychology studies.
The most commonly used monkeys in research labs include rhesus macaques
(Macaca mulatta) and cynomolgus (also known as crab-eating or long-tailed)
monkeys (Macaca fascicularis). These animals can be very expensive to purchase
and maintain. Therefore, each scientist typically only uses one to three
68  Guide to Research Techniques in Neuroscience
monkeys over a 3- to 5-year period. While other behavioral paradigms described
in this chapter typically require large numbers of individuals to achieve statistical
significance, experiments involving nonhuman primates typically only need
to be performed on a minimum of two animals. This is primarily due to differences
in what the scientist measures: in rodent assays, scientists typically
measure quantifiable behaviors. In primate assays, scientists typically measure
response properties of neurons. Therefore, statistical power is achieved not by
the number of animals studied but by the number of neurons recorded.
Primate behavioral/electrophysiological experiments usually follow a specific
structure: an investigator designs a behavioral task, trains an animal to
perform the task, completes a surgical procedure on the animal to allow access
to the brain, conducts many experimental trials while recording from neurons in
the brain, and analyzes data outside of the experiment to correlate activity with
specific, behavioral events.
There are no standard behavioral tasks for primates as there are for rodents and
invertebrates. Each investigator designs tasks specific to his or her research question,
creating stimuli to maximize the response properties of the neurons to be
studied. However, there are many common elements to primate experiments: they
usually involve a single primate, working alone in a chamber for a juice reward,
staring at a visual stimulus on a screen (Figure 2.17). Sometimes animals are
required to make eye movements to follow moving dots on a screen; other experiments
require that monkeys follow a path with their fingers; while still others
require a monkey to press a lever in response to a learned cue. Like different levels
of a video game, each trial within a task is slightly different, with some trials more
difficult than others. Once the animal learns how to play the game, an investigator
can design dozens or hundreds of individual trials with different conditions.
Training a monkey to perform a task is often the most time-consuming
aspect of an experiment. Much of this time is dedicated to simply acclimating
the monkey to research life: sitting in a chair, learning how to accept a juice
reward, becoming comfortable working alone in a chamber, and so forth. Most
training involves operant conditioning (Box 2.5), in which successful completion
of a task is reinforced with a juice reward. Depending on the task, monkeys
are trained to perform at an 80–90% success rate. To increase success, monkeys
are often given restricted amounts of food and water before the task begins, so
they are motivated to work for juice.
Fixate Targets On Motion On Delay Period Go Cue Hold Target
FIGURE 2.17  A primate behavioral task.  Here, the monkey has been trained to make saccadic
eye movements in the direction indicated by overall movement of a random dot stimulus. The monkey
fixates at a point on the screen where the motion stimulus will appear. The fixation point disappears
indicating the monkey can saccade to the correct target to receive a reward. Courtesy of Rachel Kalmar.
Animal Behavior Chapter | 2  69
During the actual experiments, a monkey performs many repetitions (trials),
completing a task for juice rewards. The number of trials performed in a day is
primarily determined by the monkey, which will only work for a certain amount
of time. Like people, monkeys differ in the amount of work they are willing to
do in one sitting. After the completion of the daily experiments, scientists return
monkeys to their cages and analyze the data. Many investigators only record
from primates every other day to allow time for data analysis.
CONCLUSION
This chapter has provided a framework for thinking about behaviors in commonly
used animal models, surveying many specific assays frequently found
in the literature. These techniques are only a fraction of the assays that investigators
can use to elucidate the neural basis of behavior. Most published studies,
especially studies using rodents, utilize many different behavioral assays in
combination to confirm hypotheses and strengthen conclusions.
SUGGESTED READING AND REFERENCES
Books and Review Articles
Bailey, K. R., Rustay, N. R., & Crawley, J. N. (2006). Behavioral phenotyping of transgenic and
knockout mice: practical concerns and potential pitfalls. ILAR Journal, 47(2), 124–131.
Buccafusco, J. J. (Ed.). (2009). Methods of behavior analysis in neuroscience (2nd ed.) Boca Raton,
FL: CRC Press.
Conn, P. M. (1993). Paradigms for the study of behavior. San Diego: Academic Press.
Conn, P. M. (2008). Sourcebook of models for biomedical research. [Digital]. Totowa, NJ:
Humana Press.
Connolly, J. B., & Tully, T. (1998). Behaviour, learning, and memory. In D. B. Roberts (Ed.), Drosophila:
A practical approach (Chapter 9) (2nd ed.) (p. 389). Oxford: IRL Press at Oxford
University Press. pp. xxiv.
Crawley, J. N. (2007). What’s wrong with my mouse? Behavioral phenotyping of transgenic and
knockout mice (2nd ed.). Hoboken, NJ: Wiley-Interscience.
Russell, W. M. S., & Burch, R. L. (1959). The principles of humane experimental technique. [Print].
London: Methuen.
Silverman, J. L., Yang, M., Lord, C., & Crawley, J. N. (2010). Behavioural phenotyping assays for
mouse models of autism. Nature Reviews Neurology, 11, 490–502.
Whishaw, I. Q., & Kolb, B. (2005). The behavior of the laboratory rat: A handbook with tests. New
York: Oxford University Press.
Primary Research Articles—Papers First Describing Standard Behavioral
Assays
Barnes, C. A. (1979). Memory deficits associated with senescence: a neurophysiological and behavioral
study in the rat. Journal of Comparative Physiological Psychology, 93, 74–104.
Geller, I., & Seifter, J. (1960). The effect of meprobamate, barbiturates, d-amphetamine and promazine
on experimentally induced conflict in the rat. Psychopharmacologia, 1, 491–492.
Hargreaves, K., Dubner, R., Brown, F., Flores, C., & Joris, J. (1988).A new and sensitive method for
measuring thermal nociception in cutaneous hyperalgesia. Pain, 32, 77–88.
70  Guide to Research Techniques in Neuroscience
McKenna, M., Monte, P., Helfand, S. L., Woodard, C., & Carlson, J. (1989). A simple chemosensory
response in Drosophila and the isolation of acj mutants in which it is affected. Proceedings
of the National Academy of Sciences of the United States of America, 86, 8118–8122.
Morris, R. G. M. (1981). Spatial localisation does not depend on the presence of local cues. Learning
and Motivation, 12, 239–260.
Olton, D. S., & Samuelson, R. J. (1976). Remembrance of places passed: spatial memory in
rats. Journal of Experimental Psychology: Animal Behavior Processes, 2, 97–116.
Ramot, D., Johnson, B. E., Berry, T. L., Jr., Carnell, L., & Goodman, M. B. (2008). The parallel
worm tracker: a platform for measuring average speed and drug-induced paralysis in nematodes.
PLoS ONE, 3, e2208.
Wicks, S. R., de Vries, C. J., van Luenen, H. G., & Plasterk, R. H. (2000). CHE-3, a cytosolic dynein
heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans.
Developmental Biology, 221, 295–307.
Woodard, C., Huang, T., Sun, H., Helfand, S. L., & Carlson, J. (1989). Genetic analysis of olfactory
behavior in Drosophila: a new screen yields the ota mutants. Genetics, 123, 315–326.
Primary Research Articles—Interesting Examples from the Literature
de Bono, M., & Bargmann, C. I. (1998). Natural variation in a neuropeptide Y receptor homolog
modifies social behavior and food response in C. elegans. Cell, 94, 679–689.
Moretti, P., Bouwknecht, J. A., Teague, R., Paylor, R., & Zoghbi, H. Y. (2005). Abnormalities of
social interactions and home-cage behavior in a mouse model of Rett syndrome. Human Molecular
Genetics, 14, 205–220.
Moy, S. S., Nadler, J. J., Perez,A., Barbaro, R. P., Johns, J. M., Magnuson, T. R., et al. (2004). Sociability
and preference for social novelty in five inbred strains: an approach to assess autistic-like
behavior in mice. Genes Brain and Behavior, 3(5), 287–302.
Ramot, D., MacInnis, B. L., Lee, H. C., & Goodman, M. B. (2008). Thermotaxis is a robust mechanism
for thermoregulation in Caenorhabditis elegans nematodes. Journal of Neuroscience,
28, 12546–12557.
Richter, S. H., Garner, J. P., & Wurbel, H. (2009). Environmental standardization: cure or cause of
poor reproducibility in animal experiments? Nature Methods, 6, 257–261.
Rothenfluh, A., & Heberlein, U. (2002). Drugs, flies, and videotape: the effects of ethanol and
cocaine on Drosophila locomotion. Current Opinion in Neurobiology, 12, 639–645.
Shahbazian, M.,Young, J.,Yuva-Paylor, L., Spencer, C.,Antalffy, B., Noebels, J., et al. (2002). Mice
with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation
of histone H3. Neuron, 35, 243–254.
Protocols
Brembs, B. (2008). Operant learning of Drosophila at the torque meter. JoVE, 16. http://dx.doi.
org/10.3791/731. http://www.jove.com/index/details.stp?id=731.
Crawley, J. N. (2007). Short protocols in neuroscience: Systems and behavioral methods: A compendium
of methods from current protocols in neuroscience. Hoboken, NJ: John Wiley.
Cunningham, C. L., Gremel, C. M., & Groblewski, P. A. (2006). Drug-induced conditioned place
preference and aversion in mice. Nature Protocols, 1(4), 1662–1670.
Current Protocols in Neuroscience. (2005). Behavioral neuroscience. (Chapter 8). Hoboken, NJ:
John Wiley and Sons, Inc.
Current Protocols in Neuroscience. (2007). Preclinical models of neurologic and psychiatric disorders.
(Chapter 9). Hoboken, NJ: John Wiley and Sons, Inc.
Animal Behavior Chapter | 2  71
Duistermars, B. J., & Frye, M. (2008). A magnetic tether system to investigate visual and olfactory
mediated flight control in Drosophila. JoVE, 21. http://dx.doi.org/10.3791/1063. http://www.
jove.com/index/details.stp?id=1063.
Hart, A. C. (Ed.). (2006). WormBook. The C. elegans Research Community, WormBook. http://
dx.doi.org/10.1895/wormbook.1.87.1. http://www.wormbook.org.
Komada, M., Takao, K., & Miyakawa, T. (2008). Elevated plus maze for mice. JoVE, 22. http://
dx.doi.org/10.3791/1088. http://www.jove.com/index/details.stp?id=1088.
Mundiyanapurath, S., Certel, S., & Kravitz, E. A. (2007). Studying aggression in Drosophila (fruit
flies). JoVE, 2. http://dx.doi.org/10.3791/155. pii.155 http://www.jove.com/index/details.
stp?id=155.
Nunez, J. (2008). Morris water maze experiment. JoVE, 19. http://dx.doi.org/10.3791/897. http://
www.jove.com/index/details.stp?id=897.
Shiraiwa, T., & Carlson, J. R. (2007). Proboscis extension response (PER) assay in Drosophila.
JoVE, 3. http://dx.doi.org/10.3791/193. http://www.jove.com/index/details.stp?id=193.
Whishaw, I. Q., Li, K., Whishaw, P. A., Gorny, B., & Metz, G. A. (2008). Use of rotorod as a method
for the qualitative analysis of walking in rat. JoVE, 22. http://dx.doi.org/10.3791/1030. http://
www.jove.com/index/details.stp?id=1030.
Witt, R. M., Galligan, M. M., Despinoy, J. R., & Segal, R. (2009). Olfactory behavioral testing in
the adult mouse. JoVE, 23. http://dx.doi.org/10.3791/949. http://www.jove.com/index/details.
stp?id=949.
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Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00003-4
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 3
Stereotaxic Surgeries and In Vivo
Techniques
After Reading This Chapter, You Should be Able to:
	l	Describe the process of performing stereotaxic surgeries in rodents and primates,
from applying anesthetic agents to monitoring recovery
	l	Describe common implants that allow permanent access to the brain
	l	Discuss methods of measuring neural activity and neurochemistry in vivo
Techniques covered:
	l	Stereotaxic surgeries in animals: sterile fields, anesthesia, stereotaxic positioning
of animals, accessing the brain, implantation, recovery
	l	Implants that allow access to the brain: sealable chambers, cannulae, cranial
windows
	l	Measuring activity: electrophysiology, fluorescent activity indicators
	l	Measuring neurochemistry: microdialysis, voltammetry, amperometry
An in vivo experiment is any procedure that takes place in a whole, living
organism. This type of experiment is in contrast with an in vitro experiment
that takes place in a controlled environment outside a living organism, such
as a cell culture dish or test tube. There is a great trade-off between these
two conditions: although in vivo experiments accurately reflect the physiology
and behavior of a living animal, measuring or manipulating activity in
intact organs and animals can be relatively difficult. In an in vitro experiment,
a scientist has a high degree of control over the extracellular environment and
can perform accurate measurements of physiological activity or biochemical
expression; however, these results may not correspond to conditions inside a
living organism. Chapter 14 discusses in vitro methods in more detail, including
their advantages and limitations.
The purpose of this chapter is to describe the rationale for and process of
penetrating the brain in a living animal. Accessing the brain allows a scientist
to measure the activity (Chapter 4) and neurochemistry of cells in vivo, as
well as to manipulate their activity (Chapter 8) to determine effects on behavior.
To gain access to an animal’s brain, scientists must perform a stereotaxic
74  Guide to Research Techniques in Neuroscience
surgery, a survivable procedure in which a substance is injected into a discrete
brain region and/or a device is implanted into the brain or onto the skull. After
describing the process of performing these surgeries in vertebrate animals, we
will survey methods of measuring and manipulating physiological activity
in vivo.
STEREOTAXIC SURGERIES
A stereotaxic surgery is an invasive procedure used to precisely target specific
regions of the brain. The word stereotaxic is derived from the roots stereos,
meaning “three-dimensional,” and taxic, meaning “having an arrangement.”
Thus, a stereotaxic surgery places an animal brain within a three-dimensional
coordinate system so an investigator can place neurobiological probes and
reagents in discrete brain regions. These tools can be used to measure neural
activity, sample bioactive substances within the extracellular environment, or
manipulate neural function.
Just as explorers use maps and astronomical reference points to navigate
the globe, scientists use brain atlases and anatomical landmarks to navigate
the brain. Published atlases provide three-dimensional coordinates of brain
structures for a variety of animals, including rodents, primates, and even birds
and bats (Figure 3.1A). The coordinates of brain structures are defined in terms
of distances to anatomical landmarks visible as seams on the skull: bregma
and lambda. Bregma is defined as the intersection between the sagittal and
coronal sutures of the skull; lambda is defined as the intersection between
the lines of best fit through the sagittal and lambdoid sutures (Figure 3.1B).
To properly align an animal brain so that its structures are consistent with the
coordinates in a brain atlas, a scientist must correctly position the animal’s
FIGURE 3.1  Stereotaxic atlases and anatomical landmarks.  (A) A stereotaxic atlas presents
the locations of distinct brain structures in three-dimensional coordinates. To use these coordinates,
a scientist must properly align an animal onto a stereotaxic instrument and identify
landmarks on the skull. (B) Bregma and lambda are commonly used landmarks for stereotaxic
targeting of brain structures. (A: Reprinted from Paxinos, G. and Franklin, K. (2001). The Mouse
Brain in Stereotaxic Coordinates, 2nd ed. With permission from Academic Press/Elsevier: New
York.)
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  75
head on a stereotaxic instrument (Figure 3.2). This specialized equipment
holds an animal in place and uses a fine-scale micromanipulator to precisely
measure and target distances in 1-μm increments.
Brain atlases provide a useful three-dimensional guide for targeting discrete
brain regions. However, because different animal strains and individuals vary, scientists
must empirically determine the actual coordinates for a region of interest
before any experiments take place. Scientists can verify the correct targeting of
brain regions by examining histological sections of the brain after experiments have
ended. In larger animals such as primates, it is also possible to examine the position
of brain structures and implants using magnetic resonance imaging (Chapter 1).
As with any procedures involving vertebrate animals, an Institutional Animal
Care and Use Committee must approve procedures before any experiments
take place. This committee evaluates survival surgery procedures to
ensure that pain and distress are minimized. These considerations are important
for the well-being of the animals, as well as for the experiments themselves, as
pain and distress can alter physiology and behavior. See Box 2.2, Chapter 2 for
ethical considerations for animal use.
In the following section, we describe general procedures for performing stereotaxic
surgeries in animals. We first describe procedures in rodents, as the
FIGURE 3.2  A rodent stereotaxic instrument.  This equipment is used to precisely hold an animal’s
head in place and target specific brain structures according to three-dimensional coordinates.
76  Guide to Research Techniques in Neuroscience
majority of stereotaxic surgeries in neuroscience are performed on mice and
rats. Next, we describe surgeries in nonhuman primates. These procedures can
theoretically be adapted for use in any mammalian organism, as well as birds
and reptiles.
Stereotaxic Surgeries in Rodents
Scientists routinely perform stereotaxic surgeries on mice and rats because of
the variety of useful studies that can be performed using these model organisms.
The rodent nervous system is more similar to the human nervous system
in structure and function than the nervous system of invertebrates. Furthermore,
rodents can be used in larger sample sizes than larger animals (such as cats or
primates) to increase the statistical power of experimental results.
Creating a Sterile Environment
The first step in any invasive surgical procedure is to prepare sterile working
conditions. Sterile does not simply mean clean, but also aseptic—free from
microorganisms that can invade the brain and lead to subsequent infection.
Some aspects of the surgical environment can be made clean, but not sterile.
For example, a scientist’s lab coat, scrubs, face mask, and gloves can decrease
the possibility of contamination but can never be 100% sterile. Cleaning the
surgical environment with alcohols and specialized disinfectant solutions can
also decrease the possibility of contamination but is also not 100% sterile. The
only aspects of the surgical environment that must be kept completely sterile are
the instruments and probes that will come in contact with the brain, as well as a
dedicated surface to place these tools when they are not in use.
In general, there are two ways to sterilize implants and tools: the use of an
autoclave, or a tabletop heat sterilizer. An autoclave is a large appliance that
increases pressure to heat water above its natural boiling point (Figure 3.3A).
The high-temperature steam kills any microorganisms growing on tools and
reagents placed inside the autoclave chamber. A heat sterilizer is a smaller
device that can sterilize tools or fine instruments when an autoclave is not convenient,
such as sterilization between surgeries (Figure 3.3B). These sterilizers
are often filled with thousands of tiny glass beads that allow the heat to evenly
surround the instruments.
Just before a surgery takes place, a scientist cleans the surgical environment
and sterilizes tools and probes in an autoclave or tabletop sterilizer. Some
probes cannot handle the high heat of sterilization and can be soaked in 70%
ethanol or another disinfectant prior to use. It is usually necessary to create a
sterile field, a dedicated surface that can serve as a resting place for tools when
they are not in use. To create a sterile field, a scientist can purchase sterile
cloths/papers commercially or can autoclave a large strip of gauze. If the tips of
probes or instruments come in contact with gloves or other nonsterile surfaces,
they must be considered non-sterile.
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  77
Anesthesia
Once the environment is clean and ready for a surgery to take place, a
scientist can anesthetize a rodent with either pharmacological agents
or gas anesthetics. A widely used pharmacological agent is a cocktail of
ketamine/xylazine. Ketamine is an anesthetic that acts by inhibiting
N-methyl d-aspartate and hyperpolarization-activated cyclic nucleotidemodulated
ion channels. Xylazine is a sedative and analgesic that serves as
an agonist for β2 adrenergic receptors. A ketamine/xylazine cocktail typically
anesthetizes an animal for 1–2 h. Reinjection of the cocktail is not recommended,
as long-term exposure can lead to bradycardia (abnormally slow
heart rate), blindness, seizures, or death.
As an alternative to pharmacological agents, scientists can administer gas
anesthetics, which are especially useful for long surgeries. A common gas anesthetic
is isoflurane, an inhalable ether that likely works by disrupting synaptic
transmission. To anesthetize the animal, the scientist places the rodent in a
small, ventilated chamber. Gas is released into the chamber, and the scientist
waits 1–3 min until the rodent stops all movements. Then, the animal is quickly
placed on a bite bar or provided with a nose mask that receives a steady supply
of oxygen mixed with the gas anesthetic.
After an animal is anesthetized, a scientist should verify that the animal is
indeed unconscious by testing for muscle responses to stimuli. For example,
FIGURE 3.3  Equipment for sterilization. (A) An autoclave uses high-temperature steam to
sterilize contents. (B) A bead sterilizer uses high temperatures to sterilize small instruments and
surgical tools before and between surgeries.
78  Guide to Research Techniques in Neuroscience
pinching the animal’s tail produces a leg withdrawal response in an animal
that is not fully unconscious. Only after the animal shows no signs of motor
responses should the scientist begin a surgical procedure.
Investigators who wish to perform stereotaxic surgeries may consult the animal
facility at their institution for recommended anesthetic agents, doses, and
protocols.
Positioning a Rodent on a Stereotaxic Instrument
After cleaning the surgical area and properly anesthetizing an animal, the scientist
places the animal on a stereotaxic instrument. The apparatus is designed
to position the animal’s head in a precise orientation so that three-dimensional
stereotaxic coordinates remain consistent from animal to animal (Figure 3.2).
First, the scientist inserts ear bars into the animal’s ear canals. This placement
allows the head to be tilted up and down but not side to side. To restrict
movement up and down, the animal’s teeth are positioned on a bite bar and the
nose is held firmly in a nose brace. It is crucial that the head is precisely fixed
in place so that the scientist can accurately traverse the three-dimensional brain.
To ensure proper orientation of the brain, the scientist must place the head in
the flat skull position, such that the top surface of the skull is flat from bregma
to lambda in the rostral–caudal direction. To achieve this position, the skull is
exposed by a careful incision down the midline of the skin. The skin is gently
moved aside, and the skull is cleaned. A micromanipulator on the stereotax is
used to measure three-dimensional coordinates in the x, y, and z axes. The scientist
uses the micromanipulator to ensure the top surface of the skull is flat. If
the skull is not flat, the scientist can raise or lower the nose brace/bite bar until
the flat skull position is achieved.
Accessing the Brain
Once the animal’s head is in the flat skull position, the investigator can target
a discrete brain region using three-dimensional coordinates. Starting from
bregma or lambda, the scientist orients the micromanipulator over the appropriate
area of the skull. A dental drill is used to perform a craniotomy, a small hole
in the skull that exposes the brain. Sometimes bleeding occurs at this stage, and
the investigator must remove excess blood with a cotton swab or thin tissue. If a
scientist intends to implant a delicate probe, such as a glass electrode, it is often
necessary to also remove a section of dura, the thin meningeal layer of tissue
surrounding the brain.
Penetrating the Brain
Once the brain is exposed, the scientist can use the micromanipulator to deliver
a bioactive substance or neurobiological probe into the brain. For example, the
scientist can deliver a pharmacological or viral agent into a brain region, or
implant a device such as a cannula or optical window (described later in this
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  79
chapter). Permanent implants can be held in place with dental acrylic and/or
cement that is specifically designed to adhere small objects to the surface of
bone. If properly applied, implants can remain securely adhered to the skull
throughout the life of the animal.
Finishing the Surgery
After the adhesive has fully hardened, the final tasks are to seal the incision,
clean the affected area, and help the animal recover. Depending on the length of
the incision, a scientist can use sterile sutures or glues designed for tissues to
seal the skin around an implant. After the skin is sealed, the top of the animal’s
head can be lightly treated with an iodine or Betadine® solution to clean the
affected area and help prevent infection.
During surgery, an animal can lose much body heat due to blood loss and
low heart rate. Therefore, recovering animals should be placed in a recovery
cage on a heating pad or under a warm light. As the animal recovers, a scientist
should inject further analgesic and/or antibiotic agents and should always monitor
the animal until it becomes conscious before returning it to an animal storage
room. It will take the animal from a few days to a full week to recover from the
surgery. During this time, the animal should be inspected for any signs of pain
or discomfort, including little movement, hunched posture, and a lack of eating
or drinking. Further injections of an analgesic and/or antibiotic may be necessary
to aid in the recovery of the animal.
Stereotaxic Surgeries in Nonhuman Primates
Stereotaxic surgeries in primates are conceptually similar to surgeries in
rodents: the investigator anesthetizes the animal, exposes the skull, performs
a craniotomy, applies dental acrylic to implant probes, sutures the skin, and
carefully monitors the animal throughout the recovery process. The major difference
between primate and rodent surgeries is that primates are more limited
resources, and individual investigators may only use one to three monkeys over
the course of single studies lasting 4–6 years. Therefore, investigators have no
room for error and take extra steps to ensure correct targeting of brain regions.
For example, although primate brain atlases exist, scientists often produce
structural MR images (Chapter 1) for each individual weeks before the actual
surgery takes place so they can target specific regions more accurately.
The purpose of most primate brain surgeries is to implant devices for future
electrophysiology experiments. In these experiments, the scientist trains the
monkey to perform a task and then records neural activity in a specific part
of the brain. To perform these experiments, a scientist usually implants one of
three devices:
	l	Sealable chamber.After performing a craniotomy, a scientist implants a chamber
around the hole with a screw cap. This allows the surface of the brain to be
80  Guide to Research Techniques in Neuroscience
exposed at any time after the surgery for the insertion of a recording electrode,
but protected by the cap at other times.
	l	Head post. The purpose of this post is to keep the primate’s head fixed in place
during experiments. It is especially important to stabilize a monkey’s head for
the presentation of visual stimuli when the monkey must fix its gaze upon a
target and make decisions about complicated visual scenes.A head post can be
as simple as a bolt that attaches to the top of the apparatus where the monkey
sits during experiments.
	l	Eye coil. The eye coil is also used to ensure that the monkey correctly fixes
its gaze during the presentation of visual stimuli. A coil is basically a wire
loop that is implanted around the outer circumference of the eye. This coil
is usually hidden beneath the eyelids after the surgery. The wire is threaded
beneath the skin from the eye to the top of the head, where it can be attached
to additional cables to measure eye movements during experiments. An eye
coil is not necessary for experiments in which there is no need to monitor the
animal’s eye movements. Many laboratories implant and adjust these devices
over two or three surgical sessions so that their placement is accurate, firm,
and precise.
IMPLANTS FOR LONG-TERM ACCESS TO THE BRAIN
After a scientist completes a stereotaxic surgery, access to the brain is lost.
However, there are a variety of implantable devices that allow long-term access
to the brain for chronic injections or physiological recordings. The most common
implants include sealable chambers, cannulae, and cranial windows, each
described below.
Sealable Chambers
As described above, a sealable chamber is a round, hollow well with one end
affixed to the skull and the other end capped with a screw top. This implant
allows an investigator chronic access to a specific region of the brain for electrophysiology.
Care must be taken to keep the chamber clean and as sterile as
possible. Whenever not in use, the chamber must always be closed to prevent
outside contaminants from penetrating the brain.
Cannulae
As described above, a cannula is a common device used to access deep structures
within the brain. It is composed of a narrow, cylindrical tube that a scientist
can insert into the brain and permanently affix to the skull. Cannulae can be
made of plastic, glass, or steel of various sizes, though thinner cannulae are less
damaging to neural tissue. Once implanted, a scientist can deliver multiple substances
or probes: pharmacological agents, viral vectors, narrow electrodes, and
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  81
even optical probes to deliver light stimulation or image deep brain structures.
At the end of experiments, scientists verify the correct placement of cannulae by
histological examination of brain sections.
Cranial Windows
There are many neuroscience techniques that allow imaging the structure and
function of living cells (Chapters 6 and 7), such as labeling neurons with fluorescent
probes. In order to visualize these probes in vivo, it is necessary to create
an observation window through the skull. To create such a cranial window, a
scientist performs a craniotomy and replaces the missing section of the skull
with a glass coverslip. Alternatively, scientists can thin and polish an area of
the skull so that it becomes translucent. These cranial windows allow imaging
of the same brain region over long periods of time. It is usually necessary to
implant a head post in addition to a window so that an animal’s head can be
properly aligned, and a scientist can locate the same cells and structures over
weeks and months.
Now that we have described stereotaxic surgeries and implants allowing
continued access to the brain, we briefly survey various techniques that allow
scientists to measure and manipulate neural activity in vivo.
Measuring Neural Activity In vivo
Neural activity can be measured in vivo using electrophysiology or visualization
of fluorescent proteins. These methods are described in greater detail in
other chapters, but we mention them here in the context of in vivo approaches to
studying the brain after stereotaxic surgeries.
Electrophysiology
Electrophysiology techniques (Chapter 4) are used to record the electrical
activity of neurons. In brief, there are three basic types of electrophysiological
probes that can measure activity in vivo:
	l	Single electrodes.An electrode can be inserted through an implanted chamber
on the surface of an animal brain. These electrodes are often mounted onto
a microdrive, which itself can be mounted on the chamber on the skull. The
microdrive precisely raises or lowers the electrodes to specific depths in the
brain.
	l	Multielectrode array. A multielectrode array is a grid of dozens of electrodes
capable of recording the activity of multiple neurons on the surface
of the brain. Although extremely small, the size and mass of arrays are often
too large to be inserted into deep brain structures. Therefore, they are usually
implanted on the outer surface of the brain.
82  Guide to Research Techniques in Neuroscience
	l	Tetrodes. A tetrode is a type of multielectrode array consisting of four active
electrodes. Unlike a microelectrode array, a tetrode is narrow and can be easily
inserted into relatively deep brain structures.
Fluorescent Activity Indicators
Fluorescent dyes and genetically targeted activity sensors, such as calcium indicators,
can be used as indirect measures of neural activity. Cranial windows are
used to observe these indicators in vivo. We describe fluorescent indicators in
much more detail in Chapter 7.
Measuring Neural Chemistry In vivo
It is often desirable to know how specific neurochemicals, such as neurotransmitters,
hormones, or peptides, change in concentration over time. Two techniques
are commonly used to identify and measure neurochemicals in vivo:
microdialysis and voltammetry. These techniques can also be used in vitro, but
their use in living animals allows scientists the opportunity to correlate changes
in neurochemical expression in discrete brain regions with specific behaviors or
physiological functions.
Microdialysis
Microdialysis is a technique used to sample chemical substances from the
extracellular fluid in a specific location in the brain. Based on the principles
of diffusion, a microdialysis probe creates a concentration gradient
between the extracellular fluid in the probed brain region and a continuously
perfused physiological solution (Figure 3.4). The probe is made of a
semipermeable membrane connected to two thin cannulae, with perfusion
Perfusate pumped
into the brain
Dialysate pumped
out of the brain
FIGURE 3.4  In vivo microdialysis. (A) A microdialysis probe can be implanted through a
cannula into an awake, behaving rodent. Scientists perfuse a physiological solution through the
probe and collect the dialysate containing neurochemicals. (B) A microdialysis probe placed in a
specific brain region. Neurochemicals in the probed brain region pass through the semipermeable
membrane of the probe through diffusion.
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  83
solution flowing into and out of the probed brain region. Because of the
concentration gradient, substances present in the extracellular fluid will passively
diffuse through the probe’s membrane into the collected solution, the
dialysate. This dialysate is collected continuously into a vial for an increment
of 1–10 min for subsequent analysis using sensitive chemical methods,
such as high-performance liquid chromatography, radioimmunoassay, or
mass spectrometry.
Numerous factors affect the molecules that can be collected into the dialysis
solution: the characteristics of the neurochemicals (size, charge, solubility),
properties of the dialysis membrane itself (material, pore diameter), flow speed
of perfusion, composition of perfusion solution, and the density of the brain
tissue surrounding the microdialysis probe. These factors affect both the identity
and the amount of substance that can be collected. Thus, the total amount
of substance collected in the dialysate over a set amount of time, known as
absolute recovery, is not the true extracellular concentration. Instead, scientists
present the concentration as relative recovery, the relative concentration of the
substance in the dialysate from the probed brain region compared to the concentration
in the perfusion solution.
It is possible to determine the actual concentration of a substance using at
least two different methods: (1) measuring the relative recovery for different
flow rates and extrapolating the information to a zero-flow rate, where recovery
should theoretically be 100%; and (2) perfusing with known concentrations of
the substance to determine the equilibrium point at which the collected concentration
does not change due to the lack of a concentration gradient. Some investigators
calculate extracellular concentrations by calibrating probes in vitro,
immersing the probes in beakers with known concentrations of the substance.
However, because of the differences in the diffusion properties in a simple solution
compared to the complex environment of the brain, it is not possible to
equate in vitro with in vivo recovery to determine the true extracellular concentration
of substances.
There are a number of advantages to using microdialysis to measure neurochemicals
in the brain. Microdialysis collection of extracellular fluid can be
followed by sensitive chemical assays that clearly identify most types of small
molecules. In addition, microdialysis can be combined with simultaneous extracellular
electrophysiological recordings and stimulation, even in freely moving
animals. Another use of the microdialysis probe is reverse microdialysis: the
delivery of drugs through the perfusion fluid. This method of drug delivery
allows for greater control over the flow of the pharmacological agent compared
with pressure injection through a cannula.
The greatest disadvantage of microdialysis is low temporal resolution.
The concentration of neurochemicals that can be collected in the dialysate
is typically very low, requiring a high volume of fluid to be collected for
analysis. This low yield can require long sampling periods (1–10 min), which
is an adequate timescale for measuring long-term changes in neurochemical
84  Guide to Research Techniques in Neuroscience
concentrations but inadequate for detection of neurotransmitter release at a
physiological timescale.
Voltammetry and Amperometry
Voltammetry is a technique used to detect neurochemicals capable of undergoing
oxidation reactions. These neurochemicals include neurotransmitters
such as serotonin and the catecholamines (e.g., epinephrine, norepinephrine,
and dopamine). A scientist inserts a carbon fiber microelectrode into the brain
and applies a specific voltage. When the chemicals encounter the surface of the
electrode, they undergo an oxidation reaction, releasing electrons that produce
a measurable change in current. The magnitude of the current is proportional
to the number of molecules oxidized. Therefore, it is possible to detect the
presence and relative concentration of these neurochemicals at a physiological
timescale.
A commonly used form of voltammetry is fast-scan cyclic voltammetry
(FCV), which has a high temporal resolution (fractions of a second). In FCV,
the voltage of the electrode is shifted back and forth from a nonoxidizing
potential to an oxidizing potential within milliseconds. The scientist can then
plot the amount of measured current versus the applied voltage, producing a
cyclic voltammogram (Box 3.1). Because electroactive neurochemicals are
oxidized and reduced at different potentials, a cyclic voltammogram can be
used as a fingerprint to identify the specific neurotransmitter detected at the
electrode.
Another subtype of voltammetry is amperometry. Unlike FCV, amperometry
holds the electrode at a specific, constant voltage. All molecules that can
Box 3.1 The Cyclic Voltammogram
In cyclic voltammetry, the carbon fiber microelectrode measures current as it
cycles between oxidizing and nonoxidizing potentials (Figure 3.5A). A scientist
can use these data to plot the current (I) versus voltage (V), an I/V curve similar to
those found in some electrophysiology experiments (Figure 3.5B, Chapter 4, Box
4.2). The rapid, large changes in potential produce a high background current.
However, these background currents are stable over a period of minutes, and thus
can be subtracted from the experimental I/V curve (Figure 3.5C). Subtracting this
background current produces the cyclic voltammogram, the fingerprint that identifies
an electroactive substance.
Another way scientists present voltammetry data is to show current traces
over time to indicate changes in the substance’s concentration, demonstrating
the responses to electrical, pharmacological, or behavioral stimuli (Figure 3.5D).
Three-dimensional graphs can be used to display changes in both voltage and current
over time, with the current encoded in color (Figure 3.5E).
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  85
be oxidized at that potential will be detected. Thus, one limitation of amperometry
compared with FCV is that it is difficult to identify the specific compound
detected by the electrode solely based on the shape of the current. However,
in amperometry, measured currents can be averaged over longer time periods,
allowing more precise measurements of the relative concentrations of
neurochemicals.
Compared to microdialysis, voltammetric techniques offer a much higher
spatial and temporal resolution for measuring neurochemicals. The higher
spatial resolution is due to the small size of the carbon fiber microelectrode
compared with the larger size of the microdialysis membrane. The temporal
resolution of voltammetry (fractions of a second) is much higher than the standard
sampling rate of microdialysis, which occurs every 1–10 min. However,
voltammetric techniques can only be applied to electroactive chemicals and
cannot be used to assay other bioactive molecules present in the extracellular
environment.
-0.4 V
1.0 V
Current (nA)
Time (s)
Potential(V)
-0.8 nA 12 nA
-0.4 V
-0.4 V
+1.3 V
-5 0 5 10
5 s
2 nA
-0.4
0
1.0
125 nA
-125 nA
-0.4 V
1.0 V
8 nA
- 8 nATime (s)
Stimulus
Stimulus
Oxidation
Reduction
Oxidation
Reduction
Oxidation
Reduction
FIGURE 3.5  Analyzing and depicting cyclic voltammetry data.  (A) A voltage is applied to the
microelectrode that causes oxidation and reduction of nearby neurochemicals. (B) The current (I) is
recorded at these oxidizing and reducing potentials. Large background currents are generated from
the rapid changes in potential, but they can be subtracted to produce the (C) cyclic voltammogram
signature of a neurochemical. Notice the difference in scale between (B) and (C). Plotting current
over time (D) can reveal changes in the neurochemical’s concentration in response to a stimulus.
(E) Three-dimensional plots can be used to show changes in both potential and current over time.
86  Guide to Research Techniques in Neuroscience
MANIPULATING NEURAL ACTIVITY IN VIVO
The ability to penetrate the brain and stereotaxically target specific brain regions
provides scientists with many experimental opportunities for determining the role
of these regions in animal behavior. In a loss-of-function experiment, a scientist
can inactivate a brain region to determine if it is necessary for a behavior to occur.
Alternatively, a scientist can perform a gain-of-function experiment and stimulate
neural activity to determine whether activity in that region is sufficient to cause a
behavior. To perform these experiments, scientists have a diverse toolkit of physical,
pharmacological, and electrical methods, along with cutting-edge genetically
encoded neural actuators. These techniques are described in Chapter 8.
CONCLUSION
Stereotaxic surgeries provide the ability to target specific brain regions for
observation or manipulation. Following a surgery, it is possible to measure the
neural activity and neurochemistry of a small population of cells, as well as to
manipulate activity to determine the role of these cells in physiology and behavior.
The techniques described in this chapter greatly aid in the study of behavior
(Chapter 2), as well as the study of individual neurons and neural circuits, as
will be described in the next few chapters. These techniques are also complemented
by in vitro assays, described in Chapter 14.
SUGGESTED READING AND REFERENCES
Books
Boulton, A. A., Baker, G. B., & Bateson, A. N. (1998). In vivo neuromethods. Totowa, N.J.: Humana
Press.
Institute of Laboratory Animal Resources (U.S.). (1996). Guide for the care and use of laboratory
animals (7th ed.). Washington, D.C.: National Academy Press.
Paxinos, G., & Watson, C. (2009). The rat brain in stereotaxic coordinates (Compact 6th ed.).
Amsterdam; Burlington, MA: Elsevier/Academic Press.
Review Articles
Du, C., & Pan, Y. (2011). Optical detection of brain function: simultaneous imaging of cerebral
vascular response, tissue metabolism, and cellular activity in vivo. Reviews in the Neurosciences,
22, 695–709.
Fillenz, M. (2005). In vivo neurochemical monitoring and the study of behaviour. Neuroscience &
Biobehavioral Reviews, 29, 949–962.
Hutchinson, P. J., O’Connell, M. T., Kirkpatrick, P. J., & Pickard, J. D. (2002). How can we measure
substrate, metabolite and neurotransmitter concentrations in the human brain? Physiological
Measurement, 23, R75–R109.
Isshiki, M., & Okabe, S. (2014). Evaluation of cranial window types for in vivo two-photon imaging
of brain microstructures. Microscopy (Oxford, England), 63, 53–63.
Kim, S. A., & Jun, S. B. (2013). In-vivo optical measurement of neural activity in the brain. Experimental
Neurobiology, 22, 158–166.
Stereotaxic Surgeries and In Vivo Techniques Chapter | 3  87
van Rooy, I., Cakir-Tascioglu, S., Hennink, W. E., Storm, G., Schiffelers, R. M., & Mastrobattista, E.
(2011). In vivo methods to study uptake of nanoparticles into the brain. Pharmaceutical
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Wotjak, C. T., Landgraf, R., & Engelmann, M. (2008). Listening to neuropeptides by microdialysis:
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Wright, N. J. (2014). Evolution of the techniques used in studying associative olfactory learning
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Primary Research Articles—Interesting Examples from the Literature
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88  Guide to Research Techniques in Neuroscience
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89
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00004-6
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 4
Electrophysiology
After Reading This Chapter, You Should be Able to:
	l	Understand the fundamental electrical properties of neurons and the ionic basis of
membrane potentials
	l	Describe the basic components of an electrophysiology rig
	l	Compare different categories of electrophysiological recording techniques, including
which research questions each technique can best address
	l	Compare different types of tissue preparations used in electrophysiology
experiments
Techniques covered:
	l	Common electrophysiological approaches: extracellular recordings, intracellular
recordings, patch clamp techniques
	l	Common electrophysiological preparations: heterologous expression systems,
primary cultures, brain slice cultures, anesthetized or awake animals
Electrophysiology is the branch of neuroscience that explores the electrical
activity of living neurons and investigates the molecular and cellular processes
that govern their signaling. Neurons communicate using electrical and chemical
signals. Electrophysiology techniques listen in on these signals by measuring
electrical activity, allowing scientists to decode intercellular and intracellular
messages. These techniques can answer systems-level questions, such as the
role of a neuron in a neural circuit or behavior. Alternatively, they can be used to
investigate the specific ion channels, membrane potentials, and molecules that
give each neuron its physiological characteristics.
The three main categories of electrophysiology techniques are defined by
where the recording instrument, the electrode, is placed in the neural specimen.
In an extracellular recording experiment, the electrode is placed just outside
the neuron of interest. In an intracellular recording experiment, the electrode
is inserted into the neuron of interest. Finally, using patch clamp techniques,
the electrode is closely apposed to the neuronal membrane, forming a tight seal
with a patch of the membrane. These different recording techniques are used to
examine the electrical properties of neurons both in vitro and in vivo. In vitro
cell cultures and brain slices allow for detailed investigations of the molecules
90  Guide to Research Techniques in Neuroscience
responsible for electrical signals, while in vivo preparations can demonstrate the
role of these electrical signals in animal behavior.
The purpose of this chapter is to differentiate between the major categories
of electrophysiological techniques and preparations, comparing the relative
advantages, disadvantages, and common uses of each. We will start with a brief
review of the physical principles that provide neurons their electrical characteristics.
To explain how these techniques are performed, we will survey the instruments
that make up an electrophysiology setup (the rig). We will also examine
some common methods of data analysis and presentation used in the literature.
A BRIEF REVIEW OF THE ELECTRICAL PROPERTIES
OF NEURONS
The electrical activity of a neuron is based on the relative concentration gradients
and electrostatic gradients of ions within the cell and in the extracellular
fluid, as well as the types of ion channels present within the neuron. The difference
in charge between the intracellular and extracellular sides of the membrane
creates an electrical potential, measured in units of Voltage (V). A neuron membrane
at resting potential is about −70 mV. This is due to differences in the permeability
of various inorganic ions, particularly sodium (Na+), potassium (K+),
and chloride (Cl−), as well as to the active contributions of a sodium–potassium
pump (Figure 4.1). Ions moving across the membrane generate a measurable
current (I), the movement of charge over time. The movement of ions across
the membrane is limited by the membrane resistance (R). This resistance is
generated by properties of the membrane, such as how many channels are open
or closed. The relationship among the membrane potential, the current flow, and
the membrane resistance is described by Ohm’s law: V = I × R. This relationship
is the fundamental basis of electrophysiological techniques.
Neurons communicate by causing changes in membrane potential in other
neurons. For example, a presynaptic neuron releases neurotransmitters that bind
to receptors on the postsynaptic neuron. These receptors are often ligand-gated
ion channels that open to allow more ions to flow through the membrane. Relative
to the membrane’s resting potential, this current flow can make the membrane
potential more positive, an effect called depolarization. Alternatively,
current flow can make the membrane potential more negative, an effect called
hyperpolarization. Whether a depolarization or hyperpolarization effect occurs
depends on the charge of the flowing ions. This local voltage change is called
a graded potential or localized potential, and its magnitude is proportional to
the strength of the stimulus. A local voltage change that makes the membrane
potential more positive is called an excitatory postsynaptic potential (EPSP),
while a local voltage change that makes the membrane potential more negative
is called an inhibitory postsynaptic potential (IPSP). Different EPSP and
IPSP events combine to form an overall signal in the postsynaptic neuron. These
localized potentials can add up in space (called spatial summation) and time
(called temporal summation). If enough localized potentials sum to depolarize
Electrophysiology Chapter | 4  91
the membrane to a threshold point, usually around −55 mV, an action potential
will occur.
An action potential, also referred to as a spike, is an all-or-none, rapid, transient
depolarization of the neuron’s membrane. A local depolarization to the
threshold potential opens voltage-gated sodium channels, and the rapid influx of
sodium ions brings the membrane potential to a positive value (Figure 4.2). The
membrane potential is restored to its normal resting value by the delayed opening
of voltage-gated potassium channels and by the closing of the sodium channels.
A refractory period follows an action potential, corresponding to the period when
the voltage-gated sodium channels are inactivated. The all-or-none generation of
an action potential initiates a wave of depolarization that preserves the amplitude
of the voltage change as it propagates down the axon’s membrane.
In a chemical synapse, depolarization stimulates the fusion of synaptic vesicles
with the presynaptic membrane and the release of neurotransmitter molecules
into the synaptic cleft. The neurotransmitters bind to receptor proteins
associated with particular ion channels on the postsynaptic membrane. These
ion channels then generate EPSP and IPSP events in the postsynaptic neuron,
which can, in turn, sum to generate an action potential in that neuron.
Sodium–
potassium
pump
90 mV
pressure from
concentration
gradient
70 mV
pressure from
concentration
gradient
50 mV
pressure from
concentration
gradient
Na+
Na+ Na+
Na+
Na+
Na+
Na+
Na+Na+
K+
K+
K+
K+
K+
K+K+
K+
K+
K+
K+
Cl- Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Cl-
Na+
Na+
Na+
K+K+
70 mV
pressure from
electrostatic
gradient
70 mV
pressure from
electrostatic
gradient
70 mV
pressure from
electrostatic
gradient
FIGURE 4.1  The ionic basis of the resting potential. The resting potential of neurons, about
−70 mV, is caused by the permeability of various inorganic ions. These ions experience pressure
to move in or out of the cell based on concentration gradients (differences in concentration of the
ion per unit distance in the local environment) and electrostatic gradients (differences in electrical
charge per distance in the local environment). In addition to passive diffusion, a sodium–potassium
pump continually pumps sodium ions out of the cell and potassium ions into the cell.
92  Guide to Research Techniques in Neuroscience
Many more details on the electrical properties of neurons can be found elsewhere.
What is important in the context of discussing electrophysiology methods
is to understand that scientists can study these properties at many different
levels of investigation. For example, an investigator may want to know the frequency
of action potentials in a specific neuron over time to decipher how a
neuron encodes a particular stimulus or action. This kind of experiment can
be performed using an extracellular recording, either in vitro or in an awake,
behaving animal. Alternatively, an investigator may want to know how the presence
of a drug in the extracellular fluid affects the ability of a specific ion channel
to pass current. This experiment could be performed using a patch clamp
technique in a heterologous expression system. Whether in the context of circuit
analysis or the molecular basis of the membrane potential, nearly any aspect of
neuronal physiology can be investigated with the electrophysiology methods
described in this chapter. Before we describe these methods, let us examine the
tools and equipment necessary to perform an electrophysiological recording.
THE ELECTROPHYSIOLOGY RIG
Each electrophysiology lab setup is different, reflecting the questions being
addressed, the requirements of the experiment, and the personal preferences
of the investigators. There is, however, a standard set of equipment necessary
and desirable to record electrical signals from neurons (Figure 4.3). In general,
a signal is detected by a microelectrode, which transmits that signal to an
-70
-50
-30
-10
10
30
50
70
1 2 3 4
MembranePotential(mV)
Time (ms)
Sodium channels open
Potassium channels open
Sodium channels close
Potassium channels close
FIGURE 4.2  The ionic basis of an action potential. Localized potentials within the neuron sum
to bring the membrane voltage to its threshold potential—around −55 mV. This potential causes
voltage-gated sodium channels to open, further depolarizing the membrane. Potassium channels
open as the membrane potential becomes more positive. At about 25 mV, sodium channels close,
and the membrane potential decreases until it becomes hyperpolarized. Finally, potassium channels
close, and the membrane potential returns to a resting state.
Electrophysiology Chapter | 4  93
amplifier, an oscilloscope, and a computer. The oscilloscope presents a visual
display of the membrane potential over time, which can also be heard using a
loudspeaker system. In modern electrophysiology rigs, the oscilloscope is now
completely digital and part of a software program on the computer. An electrophysiology
setup also includes equipment to stabilize the microelectrode and
correctly position it to record from a neuron of interest. A microscope is used
to ensure proper placement of the electrode for in vitro recordings, while stereotaxic
equipment (Chapter 3) is used to position electrodes for in vivo recordings.
Box 4.1 details various components of a standard electrophysiology rig.
Now that we have reviewed the electrical properties of neurons and surveyed
the equipment needed to study these properties, we describe various electrophysiology
techniques and how they are used.
CATEGORIES OF ELECTROPHYSIOLOGY RECORDINGS
Electrophysiological recordings can be categorized into three main types
based on the placement of the electrode in relation to the cell: (1) extracellular
recordings; (2) intracellular recordings; and (3) patch clamp techniques (Figure
4.4). Each technique can be used to address specific questions concerning
Ground electrode Amplifier
Oscilloscope (can be digitized
and displayed on the computer)
Computer
10x Microscope
Microelectrode
Headstage/
Micromanipulator/
Microdrive
FIGURE 4.3  Some of the fundamental components of an electrophysiology rig. In general,
an electrical signal is recorded by a microelectrode and passed along to an amplifier. The amplifier
compares the recording to a ground electrode, then transmits the signal to an oscilloscope and/
or computer. Various other equipment is necessary and desirable depending on the nature of the
experiment.
94  Guide to Research Techniques in Neuroscience
BOX 4.1 The Electrophysiology Rig
The following is a brief description of some of the equipment that may or may not
be part of a scientist’s electrophysiology rig. Some of this equipment is mandatory,
such as a microelectrode and amplifier. Some equipment is not, such as a loudspeaker
system.
Microelectrode
There are two main types of electrodes: (1) glass micropipettes filled with an electrolyte
solution (2 or 3 M sodium chloride or potassium chloride); and (2) metal
electrodes (usually tungsten, steel, or platinum–iridium). An important characteristic
of both kinds of electrodes is their electrical resistance, which is related to the
exposed tip size. Smaller tips have higher resistances, and they restrict the area
from which potentials can be recorded, thus permitting the isolation of the activity
of either a fiber or a cell. Large tips and low resistances pick up the activity from
a number of neurons and are of limited use in efforts to identify the functional
properties of single cells. Tips with very high resistances are also of little use, as
they cannot record neural activity unless they are very close to the cell membrane
or actually inside a cell. Electrodes often also have significant capacitances at the
metal/fluid interface. Usually, the impedance (a measure of electrical resistance
plus capacitance) of the most successful electrodes is in the range of 5–20 MΩ
when measured by an AC current at 50 Hz. Electrodes with impedances of less
than 3 or 4 MΩ tend to record from more than one cell simultaneously. The spikes
are smaller, and usually only a multiunit recording can be made.
Glass micropipettes are necessary for patch clamp recordings because of the
way the pipette must make a tight seal with the cell membrane (as discussed
later in the chapter). For intracellular or extracellular electrophysiology experiments,
metal electrodes are more commonly used. These metal electrodes not
only provide more stable isolation of single units than micropipettes, but they
tend to sample from a larger morphological variety of cells and also help in better
localization of electrode tracks to identify where recordings took place in whole
brains. The main advantage of using a glass electrode in extracellular or intracellular
experiments is that the pipette can be filled with a dye or other materials that
can subsequently be injected into the cell or cellular environment for staining and/
or pharmacological experiments.
A pipette puller is used to create glass electrodes. A glass capillary is loaded
into the machine, which heats the glass and pulls it apart to end in a fine tip. For
glass micropipettes that will be used in patch clamp recordings, the micropipette
is also fire-polished, rounding and smoothing the electrode tip. Glass capillaries
and pipette pullers come in many varieties and are commercially available. Metal
electrodes are generally purchased ready-for-use.
It is important to note that two electrodes are necessary for any electrophysiological
recording: the recording electrode itself and a reference electrode (also
called a “ground” electrode) placed outside the cell of interest. A reference electrode
is necessary because an electrophysiological measurement is a comparison—
for example, a comparison of the potential difference across the membrane of a
neuron. In extracellular electrophysiology, both electrodes are located outside the
neuron but are placed in different locations in the extracellular environment.
Electrophysiology Chapter | 4  95
Headstage
The headstage is the central hub that connects the electronic equipment to the tissue
preparation. It contains an electrode holder that stabilizes the microelectrode
during recordings and also directly connects the microelectrode to the first stage of
amplifier electronics needed to detect the electrical signals. The headstage passes
the signal to the main amplifier for the main signal processing. The headstage is
carefully positioned by the micromanipulator and is also attached to the microdrive.
Micromanipulator
The micromanipulator is a device that allows fine movements in the X, Y, and Z
axes, permitting precise positioning of the microelectrode in tissue. Good micromanipulators
have fine-scale units of measurement (usually μm) and can be used
to stereotaxically place the microelectrode in specific regions of brain or tissue.
Microdrive
A microdrive is used to lower or raise the microelectrode to a specific depth in tissue
in very fine steps. It is usually preferable to use remote-controlled microdrive
systems to eliminate hand vibration. Thus, the headstage (and consequently the
electrode) can be set into place by hand using the micromanipulator and then
finely adjusted in and out of tissue using a microdrive for the final approach to
the cell.
Amplifier
The signal is passed from the microelectrode on the headstage to the main amplifier
where amplification of the signal takes place (100–1000×). A scientist needs
an amplifier to enhance the relatively weak electrical signal derived from a microelectrode,
just as a radio listener must amplify the signal from an FM antenna so
it can drive the speakers on a radio. The amplifier also receives signal from the
reference electrode, and it is here that the signals from the two electrodes are compared.
The amplifier then transmits the signal to an oscilloscope and/or computer.
Oscilloscope
An oscilloscope receives the electrical signal from the amplifier and displays the
membrane voltage over time. This is the major source of data output in electrophysiology
experiments. While many computer programs now contain virtual
oscilloscopes that also receive the signal from the amplifier, a physical oscilloscope
is often used to detect subtle dynamics that can get accidentally filtered out
by a computer.
Loudspeaker System
Changes in voltage over time are visualized using an oscilloscope or digital oscilloscope,
but they can also be heard by connecting the output of the amplifier to a
loudspeaker. Action potentials make a distinctive popping sound, so recording the
activity of an active neuron can sound like popcorn popping. Loudspeakers can
be helpful when trying to locate a neuron of interest, because different types of
neurons have distinctive firing patterns. An investigator lowering an electrode into
neural tissue using a microdrive can be alerted to a specific type of cell or group of
nuclei by the characteristic sounds of neurons near the electrode tip.
BOX 4.1 The Electrophysiology Rig—cont’d
Continued
96  Guide to Research Techniques in Neuroscience
Microscope
A microscope is almost always necessary for all kinds of physiological recordings. For
extracellular recordings, a low-power dissecting microscope is usually adequate to
see laminae or gross morphological features of the tissue and brain. For intracellular
physiology or patch clamp techniques, a microscope with high enough magnification
power to see individual cells (300–400×) is necessary. These microscopes are usually
equipped with optical contrast enhancement to view cells in unstained preparations.
An inverted microscope is usually preferable for two reasons: (1) it allows easier
electrode access to the sample, as the objective lens is below the chamber; and (2)
it provides a larger, more solid platform upon which to bolt the micromanipulator.
Computer
Computers have greatly aided electrophysiological studies by automating stimulus
delivery and electrical signal recording. Computer software makes it easy to
write programs that can reproducibly introduce sensory or electrical stimuli to
animals or tissue preparations and record neural responses. Computers can easily
manipulate many parameters during recordings, such as the recording thresholds
and stimulus delivery timing. Computers also allow simple real-time data analysis,
displaying the results of an experiment, even while the experiment is occurring.
Vibration Isolation System
A vibration isolation system, usually an air table, is used to absorb tiny changes in
vibration that can disturb the placement of the microelectrode. Antivibration tables
are usually composed of a heavy surface on compressed-air supports.
Faraday Cage
A Faraday cage can be a simple enclosure made from conductive material that
blocks external electrical interference. This is needed to eliminate noise from sensitive
electrical recordings that can detect electrical activity from outside sources.
Other equipment may be necessary, depending on the exact nature of the
experiment. For example, in experiments that study auditory physiology, a sound
booth is necessary to block environmental sounds that could influence recordings.
Pharmacological injection equipment is often included in the rig to inject or perfuse
drugs and other substances into neural tissue. Special stimulus delivery systems
may be necessary, depending on the stimuli needed for the recordings. Each
scientist’s rig is often a highly specific setup, with all of the equipment designed
and calibrated for the particular needs of the experiments being performed.
BOX 4.1 The Electrophysiology Rig—cont’d
the electrical properties of neurons. For example, questions regarding signals
from neurons in vivo are most easily addressed using extracellular methods.
Questions regarding the open and closed states of a specific ion channel in the
presence of neuropeptide activators are best addressed using patch clamp techniques.
Table 4.1 compares some of the questions that can be addressed using
various categories of electrophysiology recordings.
Electrophysiologists using intracellular or patch clamp methods have the
benefit of being able to make use of closed electrical systems to explore the
Electrophysiology Chapter | 4  97
Extracellular
Recording
Patch Clamp
Recording
Intracellular
Recording
FIGURE 4.4  The three categories of electrophysiological recordings. Each type of recording
is defined by where the scientist places the recording electrode: outside the neuron (extracellular
recording), inside the neuron (intracellular recording), or adjacent to the membrane (patch clamp
recording).
TABLE 4.1  Questions That Can Be Addressed by Different
Electrophysiological Techniques
Extracellular
recordings:
Recording from
the extracellular
environment near
a cell (or cells) of
interest
How does a neuron encode information in action potentials?
What is the role of a neuron in a given sensory, motor, or
cognitive behavior?
How does the activity (or inactivity) of one neuron affect
the activity of another neuron?
How do pharmacological agents, neurotransmitters, and
neuromodulators affect the firing of a neuron?
How is the spiking activity of a group of neurons coordinated?
Intracellular recordings:
Recording from inside
a cell of interest,
piercing the membrane
with an electrode
How does the activity (or inactivity) of one neuron affect the
local potentials and action potentials of another neuron?
How do pharmacological agents, neurotransmitters, and
neuromodulators affect the local potentials and action
potentials of a neuron?
Patch clamp recordings:
Recording from the
membrane of a cell of
interest
How do an ion channel’s open and closed times depend
on the membrane potential?
How do the concentrations of ions, pharmacological
agents, neurotransmitters, and neuromodulators affect the
current flowing into an ion channel or cell?
How much current does a single ion channel carry?
What contributions does a single channel provide to an
entire neuron?
98  Guide to Research Techniques in Neuroscience
relationship between membrane potential and current flow: voltage clamp and
current clamp. Using a voltage clamp, a scientist can measure currents generated
by ions moving across the membrane by holding the membrane potential
at a set voltage, the holding potential. Deviations between the membrane
potential and the holding potential are corrected through a feedback system
that injects current to maintain the membrane voltage at the holding potential.
In this way, membrane potential is clamped to a set voltage. Data collected
using a voltage clamp can be plotted in I/V curves (Box 4.2) to examine the
relationship between current and voltage or to measure fluctuations in current
BOX 4.2  Data Analysis: I/V Curves
The I/V curve (current/voltage curve) is one of the most commonly used methods
of data analysis in electrophysiology. It is simply a plot of the voltage across a
neuronal membrane and the associated ionic current flow through channels in the
membrane. Voltage (V) is measured in units of volts, and current (I) is measured in
units of amperes. I/V curves can be produced for an entire neuron (with current
typically in the range of nanoamps, nA) or for an individual ion channel or class of
channels within the neuron (with current typically in the range of picoamps, pA).
To produce an I/V curve, an investigator uses the voltage clamp technique to
hold the voltage of a neuron at a specific value. Current can be recorded using
one of the patch clamp methods. The I/V curve is the curve of best fit for each data
point of current for a given voltage. By convention, currents with a negative value
are referred to as inward current, while those with a positive value are known as
outward current. An inward current is the result of positively charged ions crossing
a cell membrane from the outside to the inside, or a negatively charged ion crossing
from the inside to the outside. An outward current is the result of positively
charged ions crossing a cell membrane from the inside to the outside, or a negatively
charged ion crossing from the outside to the inside.
Electrophysiology Chapter | 4  99
The relationship between current and voltage is described by Ohm’s law:
V IR V egatlov
I tnerruc
R ecnatsiser
R/1 ecnatcudnoc
The slope of the I/V curve is the conductance of the membrane/channel for all
ions that can pass through. If the investigator is interested in the conductance of
a specific ion, pharmacological agents are used to isolate specific channels. The
conductance depends on an ion channel’s permeability for a specific ion (how
easily the channel allows the ion to pass through), as well as the concentration of
the ion in the extracellular/intracellular solution.
In the preceding example, the I/V curves could represent the current/voltage
relationships for particular channels within a membrane. Note that the I/V curve
for the channel on the right is linear, meaning it acts like a simple resistor, and
the conductance of the channel is independent of voltage. An electrophysiologist
would say there is no voltage-dependent gating of this ion channel. In contrast,
the I/V curve on the left is not linear. At a value of about −20 mV, there is a strong
inward current. If the ion that passes through this channel is positively charged,
it will flow into the cell when the membrane potential is −40–160 mV. This is an
indication that the channel is voltage gated. To determine the type of ion that can
flow through this channel, the ionic concentrations in the bath solution can be
adjusted so that the investigator can determine which ionic species are required
for current to flow through the channel.
BOX 4.2  Data Analysis: I/V Curves—cont’d
at a particular voltage over time (Box 4.3). In current clamp mode, membrane
potential is free to vary, and the investigator records whatever voltage the cell
generates on its own or as a result of stimulation.
Extracellular Recording
The dramatic changes in membrane voltage that occur during an action potential
generate local, temporary differences in potential on the outer surface of an
active neuron. Therefore, action potentials can be detected in the extracellular
space near the membrane of an active neuron by measuring the potential difference
between the tip of a recording electrode and a ground electrode placed in a
distant extracellular position. In the absence of neural activity, there is no difference
in potential between the extracellular recording electrode and the ground
electrode. However, when an action potential arrives at the recording position,
positive charges flow away from the recording electrode into the neuron. Then,
as the action potential passes by, positive charges flow out across the membrane
BOX 4.3  Data Analysis: Current Over Time
Many questions in neurophysiology involve knowing how much current flows
through a particular ion channel for a particular voltage or other environmental
conditions. An I/V curve displays the relationship between the current that passes
through a channel or membrane and the voltage across the membrane. For questions
involving the ability of a cell or channel to pass current under conditions
other than changes in voltage (such as the presence of a compound in the bath
or the temperature of the bath), data are often presented as a plot of changes in
current over time. These measurements are typically taken under voltage clamp to
eliminate contributions to current dynamics due to changes in voltage.
The size of detected current changes depends on the patch clamp technique
used to collect the data. For example, in whole-cell mode, the glass pipette is
continuous with the cytoplasm, and a large current trace can be obtained. Current
data in whole-cell mode are typically presented in nanoamps and can be applied
to timescales ranging from 1 ms to even minutes in length. In the following wholecell
recording, an agonist for a ligand-gated ion channel is applied to the bath, as
indicated by the blue bar.
Current data obtained from other patch clamp modes are typically smaller in
value and presented in picoamps.This is because the current flowing through single
channels rather than an entire cell generates a smaller signal. The timescale of these
current traces is also much smaller, in the range of milliseconds instead of seconds,
due to the rapid and transient events of a single channel opening and closing.
In these patch clamp modes, data are often presented as a long, continuous
trace. For example, in the following experiment, data are obtained in a cellattached
mode from a single channel before and after application of a pharmacological
agent:
Notice how the scale is different from the whole-cell recording. This experiment
shows that the presence of the pharmacological agent reduces the probability
that the channel will be open and conduct current.
Electrophysiology Chapter | 4  101
toward the recording electrode. Thus, the extracellular action potential is characterized
by a brief, alternating voltage difference between the recording and
ground electrodes (Figure 4.5). The results of an extracellular experiment can
be displayed and analyzed in a variety of ways (Box 4.4).
A major advantage to performing extracellular recordings is their relative
ease and simplicity. They do not require the precise and delicate electrode
positioning that is necessary in intracellular or patch clamp recordings.
-20 µV
0 µV
20 µV
40 µV
-40 µV
-60 µV
-80 µV
-20 mV
0 mV
20 mV
40 mV
-40 mV
-60 mV
-80 mV
Extracellular Electrode Intracellular Electrode
FIGURE 4.5  Comparison of recordings from an extracellular and intracellular electrode.
Each action potential exhibits a different waveform due to its placement relative to a neuron. Note
the differences in the units of measurement.
BOX 4.4  Data Analysis: Spikes
The goal of many electrophysiology experiments is to quantify the number of
action potentials, or spikes, that a neuron fires in response to a specific stimulus.
Usually, these experiments use extracellular recording electrodes, though intracellular
electrodes can also detect action potentials. Data displaying changes in the
number of spikes that occur over time following the presentation of a stimulus are
commonly presented in several ways.
For example, consider the data collected during an extracellular electrophysiology
experiment in which an electrode is placed in the auditory cortex of a mouse.
The investigator presents tones to the mouse and records from a neuron close to
the tip of the electrode to determine whether this neuron responds to tones of a
particular frequency. The simplest way to present data from this experiment is to
show a plot of voltage over time:
Stimulus
Recording
Note that changes in voltage in this plot are in the magnitude of microvolts; an
intracellular electrode would record changes in voltage on the scale of millivolts.
102  Guide to Research Techniques in Neuroscience
This plot clearly shows that the stimulus leads to specific changes in voltage over
time. However, a simple voltage over time plot does not present the effects of
multiple types of stimuli—in this case multiple frequencies of sound. To show the
effects of a continuous distribution of stimuli on spike counts, there are other, more
efficient methods of data analysis and presentation that can combine the results
of multiple experiments with different stimuli. For example, the results of multiple
frequencies can be presented as a raster plot.
Time (ms)
Frequency(kHz)
In this type of plot, each dot represents a spike as recorded by the electrode.
The Y-axis shows the stimulus frequency, while the X-axis shows the time when the
spike was recorded. Stimulus onset and duration are indicated by the vertical gray
bar. The data are arranged so that the spikes resulting from a stimulus are aligned
with the onset of the stimulus. A raster plot provides a strong visual depiction of
the kinds of stimuli that give rise to the greatest number of spikes, as well as an
indication of the background noise during an experiment.
Data in a raster plot can also be presented as a peri-stimulus time histogram
(PSTH) (also referred to as a poststimulus time histogram). In this type of plot, spikes
are quantified and sorted into bins of a defined size. In the following plot, the number
of spikes (X-axis) is presented for bins that are each 2 kHz in size, with the
number of spikes plotted on the Y-axis. A PSTH provides an easy visualization of
the stimuli that cause the greatest proportion of firing events. Different mathematical
analysis tools, such as a Gaussian distribution curve, can be used to characterize the
most effective stimuli and determine the tuning properties of an individual neuron.
Sound Frequency (kHz)
NumberofSpikes
BOX 4.4  Data Analysis: Spikes—cont’d
Electrophysiology Chapter | 4  103
Finally, it is possible to analyze the effects of multiple stimulus variables using a
three-dimensional heat map plot. For example, if the investigator presented tones
of different intensity in addition to tones of different frequency, the data could be
presented using color to indicate the number of spikes.
5
10
15
20
10 20 30 40 50
Intensity (db)
Frequency(kHz)
0%
100%
Efficiency
(%ofstimulithat
evokeaspike)
This plot shows that the neuron being investigated responds to a specific frequency
of tones that produce a maximum firing rate but that the neuron can be
made to produce more spikes if the intensity of the stimulus is increased.
Remember that the raw number of spikes that the investigator includes as data
is determined by the settings on the electrophysiology rig and computer. For example,
changing the threshold of voltage change that determines what is considered
to be a spike will alter the number of spikes included in the data analysis.
BOX 4.4  Data Analysis: Spikes—cont’d
A disadvantage to extracellular recordings is that it is impossible to measure
localized potentials. Therefore, extracellular recordings cannot report
detailed information about subthreshold potential changes, such as EPSPs
or IPSPs. However, the summed synaptic activity of neurons in the general
vicinity of the extracellular recording electrode produces measurable local
field potentials, the sum of all postsynaptic activity within a volume of
neural tissue.
By using multiple electrodes, the activity of multiple cells can be recorded at
the same time. A popular configuration is the use of tetrodes, a bundle of four
individual electrodes carefully arranged into a single implant (Figure 4.6). Grids
of individual electrodes or groups of tetrodes can be arranged into a single device
called a multielectrode array (MEA) (Figure 4.6). This allows for stimulation
and extracellular recording from several neighboring sites at once. The number of
microelectrodes in an array varies, but can be anywhere from four (a single tetrode)
to over 100.
When recording from a single electrode, it can be difficult to determine
whether the neural activity that is recorded is produced by a single neuron
or multiple neurons in the vicinity of the electrode tip. A single unit spike
will depend on the cell’s unique shape, size, and distance from the recording
104  Guide to Research Techniques in Neuroscience
electrode. Identifying individual cells based on their spiking activity and assigning
the waveforms to particular cells is known as spike sorting. A major advantage
to using tetrode recordings over single electrode recordings is that it is
easier to compare recordings made by each individual tetrode microelectrode.
Signals are processed through triangulation methods based on the position of
the electrodes and the strength and characteristic shape of a recording. These
comparisons result in specific and reproducible waveforms for each individual
neuron recorded, enabling single unit data to be obtained by recording from
multiple electrodes at once.
By investigating the activity of dozens of neurons at a time, it is possible to
answer questions regarding connectivity and timing within a neural network. A
scientist can also manipulate specific neurons within the network and monitor
the effect on other neurons recorded by the array. Ultimately, using MEAs to
study the simultaneous response of neural circuits provides pivotal information
on neuronal interactions, as well as spatiotemporal information about neural
networks.
MEAs can be used to record from multiple neurons in vitro or in vivo. In
in vitro preparations, a brain slice can be placed over a grid of many microelectrodes
for extracellular recordings. In vivo tetrodes or MEAs can be implanted
in the intact brains of live animals for multiunit recordings. Multielectrode
technology has also been used in nonhuman primates for long-term recording.
For example, recordings of network activity from the premotor cortex of monkeys
performing reaching movements is helping to develop neural prosthetics
that allow animals (including humans) to move prosthetic devices using conscious
thought alone.
Tetrode
Electrodes
Guide tube
Plug
(A)
Multielectrode Array
Electrodes
Base
(B)
FIGURE 4.6  Two specialized types of electrodes. (A) An individual tetrode is composed of four
microelectrode wires. A scientist implants a tetrode into the brain of an animal and connects the
top plug to a cable attached to an amplifier. (B) A multielectrode array is composed of 25 or more
electrodes (sometimes over 100) and is used to record from neurons on the surface of the brain.
Multielectrode arrays can also be used to record from slices in vitro.
Electrophysiology Chapter | 4  105
Intracellular Recording
While extracellular recordings detect action potentials, intracellular recordings
detect the small, graded changes in local membrane potential caused by synaptic
events. Intracellular recording requires impaling a neuron or axon with a
microelectrode to measure the potential difference between the tip of the intracellular
electrode and the reference electrode positioned outside the cell.
Note that the measurement of an action potential is different for extracellular
and intracellular recordings (Figure 4.5). From the point of view of an
extracellular electrode, the difference in potential between an electrode and
ground initially decreases, then increases, and then returns to baseline. From
the point of view of an intracellular electrode, the potential difference initially
increases and then returns to baseline. Also notice that the units of measurement
are different between the two techniques. Intracellular electrodes measure
potential differences in units of millivolts (mV). Extracellular electrodes
measure much smaller potential differences and are often expressed in units
of microvolts (μV).
In the early days of electrophysiology, intracellular recordings were used
to examine the ionic basis of membrane potential. In fact, Alan Hodgkin and
Andrew Huxley won the Nobel Prize in 1963 for using intracellular methods to
determine the ionic basis of action potentials. However, most experiments that
once used intracellular electrodes are now usually performed using patch clamp
techniques due to higher signal-to-noise ratios and the ability to ask questions
about the nature of single ion channels. Indeed, after developing patch clamp
techniques and using them to study the function of single ion channels, Erwin
Neher and Bert Sakmann were awarded the Nobel Prize in 1991.
Patch Clamp Techniques
In patch clamp methods, a glass micropipette is used to make tight contact with a
tiny area, or patch, of neuronal membrane. After applying a small amount of suction
to the back of the pipette, the seal between pipette and membrane becomes
so tight that ions can no longer flow between the pipette and the membrane. Thus,
all of the ions that flow when a single ion channel opens in the patched membrane
must flow into the pipette. The resulting electrical current, though small, can be
measured with a sensitive amplifier. This arrangement is usually referred to as the
cell-attached mode. Resistance of the seal to flow between the pipette and the
cell membrane must be very high to ensure that all current to be recorded flows
through the pipette and does not leak out through other regions of the membrane.
In practice, seal resistances in excess of 1 GΩ can be obtained by a cell-attached
patch, often referred to as a gigaseal.
The cell-attached method allows the investigator to study single ion channels.
Minor technical modifications provide other recording possibilities. There
are three other variations of the patch clamp technique: whole-cell, inside-out,
and outside-out recordings (Figure 4.7).
106  Guide to Research Techniques in Neuroscience
If the membrane patch within the pipette is disrupted by briefly applying
strong suction, the interior of the pipette becomes continuous with the cytoplasm
of the cell. This arrangement allows measurements of electrical potentials and
currents from the entire cell and is, therefore, called the whole-cell recording
Cell-attached mode
Whole-cell mode
Inside-out mode
Outside-out mode
Careful retraction of the pipette
causes the ends of the broken
plasma membrane to anneal
Gentle suction is applied to the glass
pipette
Tight contact between the pipette
and membrane forms a “gigaseal”
Strong pulse of suction
opens up the
membrane, making
the cytoplasm continuous
with the pipette
Careful retraction
of the pipette
removes patch
of membrane
Extracellular
Intracellular
FIGURE 4.7  The patch clamp techniques. The four fundamental patch clamp techniques
include the cell-attached mode, whole-cell mode, inside-out mode, and outside-out mode.
Electrophysiology Chapter | 4  107
method. The whole-cell configuration also allows diffusional exchange between
the pipette and the cytoplasm, producing a convenient way to inject substances
into the interior of a patched cell.
Two other variants of the patch clamp technique originate from the finding
that once a tight seal has formed between the membrane and the glass pipette,
small pieces of membrane can be pulled away from the cell without disrupting the
seal. Simply retracting a pipette that is in the cell-attached configuration causes
a small vesicle of membrane to remain attached to the pipette. By exposing the
tip of the pipette to air, the intracellular surface of a small patch of membrane is
exposed. This arrangement, called the inside-out recording configuration, allows
the measurement of single-channel currents with the added benefit of being able
to change the medium exposed to the intracellular surface of the membrane. Thus,
the inside-out configuration is particularly valuable when studying the influences
of intracellular (cytoplasmic) molecules on ion channel function.
Alternatively, if the pipette is retracted while it is in the whole-cell configuration,
a membrane patch is produced that reseals to expose its extracellular
surface to the bath solution. This arrangement, called the outside-out recording
configuration, is optimal for studying how channel activity is influenced
by extracellular chemical signals, such as neurotransmitters, because the bath
solution can be easily manipulated during recordings.
Table 4.2 compares the relative advantages and disadvantages of the four
variations of the patch clamp techniques. Sometimes electrophysiologists
TABLE 4.2  Relative Advantages and Disadvantages of Different Patch
Clamp Configurations
Advantages Disadvantages
Cell attached Single ion channels can be
recorded, and channel properties
are not changed.
Exact potential of the patch is
unknown.
Physiologically relevant
because cell is intact.
Not easy to control
environment of single ion
channels.
Easiest patch clamp configuration
to obtain.
Whole cell Ability to manipulate the cell
cytoplasm pharmacologically. The
relatively large size of the current
makes it easier to measure.
The cell cytoplasm is
continuous with the recording
electrode so cell contents may
be diluted or leak out.
Excised patch
(inside-out or
outside-out)
Recordings can be taken from
individual ion channels. Excellent
environmental control over both
the intracellular and extracellular
sides of single ion channels.
There is a risk that channel
properties are changed
because the cellular
environment is severely
altered.
108  Guide to Research Techniques in Neuroscience
use a fifth method: the perforated patch. The purpose of this technique is to
make the glass pipette continuous with the cell, as in the whole-cell mode, but
without the disadvantage of cytoplasmic contents potentially leaking into the
pipette. Instead of applying suction in the cell-attached mode to transition into
the whole-cell mode, a chemical (often an antibiotic, such as amphothericin-B
or nystatin) is applied from within the glass pipette, causing small perforations
in the membrane. Thus, the perforated patch technique can be thought of as an
intermediate between the cell-attached mode and whole-cell mode; this configuration
prevents the leaking of cytoplasmic contents but generates a smaller
signal than whole-cell mode.
ELECTROPHYSIOLOGY TISSUE PREPARATIONS
Electrophysiology experiments can be categorized by the type of tissue
preparation in addition to the type of recording technique. Physiological
experiments in vitro usually fall into one of three main categories: recording
from heterologous expression systems, primary cultured cells, or brain
slices. While in vitro preparations allow probing of cell physiology in a
controlled environment (Chapter 14), conditions in the cell’s natural environment
in vivo may be different. It is possible to perform experiments
in vivo using extracellular techniques (and more rarely, intracellular and
patch clamp techniques), either in an anesthetized animal or in an awake,
behaving animal.
In Vitro Recordings
In vitro culture preparations provide unparalleled physical and visual access
to individual cells, allowing detailed studies of the molecules and proteins that
affect neuronal physiology. Access to individual ion channels, functional subcellular
regions, and small circuits can be gained using various in vitro preparations:
heterologous expression systems, isolated primary cells, and brain slices.
Control over the cellular environment through the culture or bathing
media aids the ability to finely dissect cellular and subcellular physiological
events. For in vitro electrophysiology experiments, the neural specimen is
incubated in a well-regulated bath solution. The composition of the solutions
used during the experiments is critical because the basis of neuronal
electrical properties depends on the concentration gradient of ions inside
and outside the cell. When deciding what ingredients should make up the
bath solution, the guiding principle is to maintain an environment that
allows the observation of physiologically relevant electrical activity and
channel function. In patch clamp techniques, the solution inside the microelectrode
should mimic the intracellular milieu. This solution is typically
composed of salts, ionic buffering agents, and a pH buffer that can be maintained
at biologically relevant temperatures to provide cells and tissue with
Electrophysiology Chapter | 4  109
a simulated in vivo environment. One of the greatest advantages of in vitro
recordings is the ability to study the electrophysiological properties of neurons
by altering components of the bath. For example, to isolate the contribution
of specific channels or receptors to the electrical signal, investigators
frequently add pharmacological agents that block certain receptors, such as
CNQX (to block AMPA receptors) or AP5 (to block NMDA receptors), to
the bath solution.
Heterologous Expression Systems
To study the physiological properties of individual ion channels or investigate
the role of structure on a channel’s function, scientists often utilize
heterologous expression systems. A heterologous expression system is a
type of cell culture system that can be easily transfected with a foreign gene
(see Chapter 11 for more about transfection), such as a gene that encodes
a specific ion channel. The ion channel is efficiently expressed within the
cells, and its function can be studied with methods such as the patch clamp
technique. By expressing mutant forms of a channel, scientists can observe
the effect of molecular changes on a channel’s electrical properties, such
as the role of a specific amino acid on a gating mechanism. Heterologous
expression systems are also useful for isolating the role of individual channels
because of the absence of endogenous channels that may otherwise
alter overall cell physiology.
Common expression systems are Xenopus laevis oocytes, Chinese hamster
ovary (CHO) cells, and human embryonic kidney (HEK 293T) cells. These
cells are usually easy to culture, readily available, have few endogenous channels,
and can easily take up and express foreign DNA. The ability to combine
molecular and physiological methods in a single cell system has made heterologous
expression systems a powerful experimental tool for understanding the
structure–function relationships of ion channels (Box 4.5).
Primary Cultures
Primary cultures of cells isolated from a region of interest in the nervous system
can reveal properties close to those found in vivo while still providing excellent
access to and control of individual channels. For example, if an investigator is
interested in the physiological properties of spinal cord neurons, he or she can
culture these neurons for electrophysiology experiments. Such cultures are useful
for comparing the electrophysiological properties of different populations of
neurons, as well as the effects of different bath solutions. For example, an investigator
may want to know what happens to neuronal physiology when the extracellular
concentration of a particular ion or neuropeptide increases or decreases.
An investigator can also compare the physiological properties of neurons from
different populations of animals—for instance, to examine changes in neuronal
physiology between a genetic knockout animal and a wild-type animal.
110  Guide to Research Techniques in Neuroscience
BOX 4.5  Walkthrough of an In Vitro Electrophysiology Experiment
Let’s say that you are interested in examining the properties of a newly discovered
ion channel. Your research group has cloned the channel and created an
expression construct (Chapter 10) and introduced this channel into Xenopus laevis
oocytes (Chapter 11). We can now use this heterologous expression system, the
large (∼1 mm) Xenopus oocytes expressing our channel of interest, to answer questions
about the electrical properties of the channel. You might be interested in a
number of properties: What ions pass through the channel? How is the channel
gated, and what stimuli open the channel to let ions pass? You might first look at
the similarity of the amino acids to known channels. This can provide hypotheses
about the properties of the channel, such as whether the channel is selective for
specific ions or what types of compounds may affect gating properties. To examine
single-channel properties, patch clamp recordings will be the most appropriate
technique.
Assuming the lab has established rigs on which to work, the first step in running
the experiment will typically be to make the appropriate solutions and prepare
fresh glass micropipettes. The composition of both the bath solution and the solution
that will fill the micropipette are important because ionic concentration gradients
affect the electrical properties of the membrane. The solution’s osmolarity and
pH will also affect pressure on the cell membrane, which can cause a cell to shrink
or swell and burst if not properly regulated. In a whole-cell recording, the pipettefilling
solution will be continuous with the inside of the cell, so it should closely
match the cell cytoplasm. The bath solution can contain pharmacological agents
that block the activity of known channels so the signal from the channel under
investigation is clearer. The currents of endogenous Xenopus ion channels, however,
are typically much smaller than those of heterologously expressed channels;
thus, it is not always necessary to add blocking agents to the bath solution. For
cell-attached recordings, activity of the predominant endogenous Ca2+-activated
Cl− channels can be reduced by keeping the Ca2+ concentration low in the bath
solution, by adding chelators such as EGTA and using impermeable ions instead of
Cl−, or by adding a pharmacological blocker.
Patch pipettes are generally used immediately after they are made to ensure that
the tip of the micropipette is not contaminated, which can cause a poor seal to
form between the membrane and the pipette. A pulled glass pipette should be firepolished
to round and smooth the tip. Now it is ready to be filled with solution and
positioned in the electrode holder. Using the microscope and a micromanipulator,
you can bring the electrode tip in a targeted cell into the field of view. By applying
gentle, positive pressure to the electrode through a tube to prevent dirtying the
electrode tip, you can insert the tip into the bath solution. By applying a test voltage
pulse, the patch electrode will generate a current, and you can calculate the
electrode’s resistance. Observing the current response to the test pulse can help
guide the electrode position. As the tip nears the cell, resistance should increase.
You can now gently touch the electrode tip against the surface of the cell,
checking the computer output of data to see that resistance has increased, and
then release the positive pressure. This release can be observed as a small dimple
against the cell surface as the tip is pressed against the cell. You can now apply
Electrophysiology Chapter | 4  111
gentle suction to the patch pipette, using resistance and microscopic appearance
as evidence that contact has been made between the cell surface and the patch
electrode tip. If you have a gigaseal, you should see the resistance rise to at least
1 GΩ. Establishing the gigaseal is one of the trickiest parts of patch clamping—perhaps
the step that makes this technique an art.
At this point, you are in cell-attached mode and can switch the amplifier to
voltage clamp. Cell-attached mode can provide single-channel resolution, and you
may be able to see characteristic steps in the current that demonstrate transitions
between the opened and closed state of a channel. Now you can test whether
changes in voltage can activate the patched channel (or channels) by applying
voltage steps. Alternatively, you can test whether the addition of ligands affects the
patched channel by adding various agonists or antagonists to the solution filling
the micropipette (as this is the extracellular environment of the patched channel).
Monitoring the effect of experimental manipulations on current will reveal
open/closed times and current amplitude. Current amplitude can be analyzed to
determine the conductivity and ion specificity of the channel. I/V curves (Box 4.2)
reveal characteristics of voltage-activated conductance, including the reversal
potential, the voltage at which there is no overall flow of ions across the membrane.
The reversal potential can be used to identify the ion species that pass
through the channel.
Analyzing current flow through individual channels in this heterologous
expression system exposes many of the channel’s properties. Further studies can
use mutant versions of the channel to dissect the requirements of specific aspects
of the protein’s structure on its function. This knowledge can then be used to investigate
the contributions of this channel of interest in intact or natural preparations:
isolated neurons, brain slices, and whole animals.
BOX 4.5  Walkthrough of an In Vitro Electrophysiology Experiment—cont’d
Slice Cultures
The controlled in vitro environment in cell culture conditions can be substantially
different from the in vivo environment. Brain slices, which preserve some
endogenous connections while still providing the level of access available
through cell culture conditions, can more closely mimic the in vivo environment.
While it is possible to culture tissue slices for extended periods of time to
capture conditions closest to those in vivo, most electrophysiological recordings
from slices are from acute preparations cut the same day. At the beginning of
the experiment, the brain is removed and sliced into 200–500-μm-thick sections
using a vibratome. Many neurons remain healthy despite the mechanical shock
and damage of slicing, though physiological responses may be slightly altered.
The brain slice is placed in a chamber that is continuously bathed in a solution
containing the proper proportion of inorganic ions, nutrients, and gases to allow
the neurons to survive.
There are several compelling advantages for recording from a brain slice
rather than an intact brain. First, it is much easier to make intracellular recordings
112  Guide to Research Techniques in Neuroscience
from neurons if the tissue is not subject to the periodic pulsing of blood caused by
a beating heart. Second, it is also easier to study neurons from a particular region
of the brain if an electrode does not have to penetrate several millimeters of cells
before it can get to that region. Brain slices provide much easier access to internal
brain structures. Third, it is possible to study the pharmacology of known synapses
because specific drugs or other pharmacological agents can easily be applied to
a brain slice. In addition, the specific role of individual neurons in circuits can be
studied because it is possible to record from both pre- and post synaptic neurons
in a known synaptic circuit, if they can be accessed in the same section.
In Vivo Recordings
Though in vitro recordings allow control of the environment and access to the
brain, they may not accurately reflect neural activity in an intact organism. Most
electrophysiology experiments performed in vivo submerge an electrode into the
brain and record extracellularly due to the fact that it is difficult to visualize the
cellular environment and extremely difficult to correctly position an electrode for
intracellular or patch clamp recordings. Therefore, most in vivo recordings are
extracellular, performed using electrodes, tetrodes, or multielectrode arrays.
However, it is possible to perform intracellular or even patch clamp recordings
with specialized, dedicated equipment. To perform intracellular recordings,
a scientist can use a very sharp pipette to impale cells for sharp electrode
recordings. To perform in vivo patch clamp recordings, a scientist can use twophoton
microscopy (see Chapter 5) in conjunction with an animal expressing
a fluorescent protein in a cell type of interest to visualize the placement of the
patch pipette on the surface of the membrane. A scientist can also perform patch
clamp recordings blind, but this approach is quite difficult.
Acute versus Chronic Recordings
While most in vitro recordings are performed during a single session, in vivo
recordings can be performed either acutely (over hours) or chronically (over multiple
days). An acute recording is an experiment that uses an animal only once. To
perform a chronic recording, cannulae or electrodes can be surgically implanted
into an animal’s brain, permitting long-term access to a recording site. Chronic
recordings from implanted multielectrode arrays permit the monitoring of neural
circuit activity during complex behavioral tasks over long periods of time or
during a variety of different conditions and multiple tasks. Sealable chambers
implanted on the skull allow an investigator to place new electrodes within the
brain over periods of months in rodents or years in nonhuman primates.
Anesthetized versus Awake Animals
In vivo electrophysiology experiments often involve animals stabilized under
anesthesia. Anesthetized animals are immobile, allowing scientists to record
neuronal responses to passive stimuli and characterize tuning properties of
Electrophysiology Chapter | 4  113
single neurons. Anesthesia affects the excitability and neurotransmission of
normal neural activity, with varied effects depending on the anesthetic agent
or neuron population of focus. Thus, it is important for an investigator to
know the specific effects of the anesthetic used to be able to properly interpret
the data. For some experiments, the animals are also paralyzed to eliminate
motion from breathing—in these instances, artificial, controlled ventilation
is provided.
Recording from awake, behaving animals make it possible to correlate
specific behaviors with the activity of individual cells. An animal is first
trained on one or more specific behavioral tasks. After a certain level of
performance has been reached, a surgical operation is performed to create
a small opening in the skull over the brain area of interest. This area is then
sealed by implanting a chamber with a screwable cap (Chapter 3). After the
animal has recovered from the operation, electrophysiological recordings can
be performed for a few hours each day with an electrode mounted on a lightweight
microdrive. Single or multiunit recording during a behavioral task
allows scientists to examine the neural basis of various cognitive and behavioral
phenomena.
MANIPULATING NEURAL ACTIVITY DURING
ELECTROPHYSIOLOGY EXPERIMENTS
During neurophysiological recordings, an investigator may wish to inhibit or
stimulate certain neurons for a loss of function or gain of function experiment.
For example, if an investigator hypothesizes that neuron A stimulates neuron
B, a way to test this hypothesis would be to stimulate neuron A while recording
from neuron B. Alternatively, an investigator may want to know the contribution
of certain types of neuron (e.g., GABAergic, cholinergic, glycinergic) to a
physiological process; in this instance, the investigator could inhibit only those
neurons while examining the physiological properties of a neuron of interest.
Methods of perturbing neural activity during electrophysiology experiments
are covered in detail in Chapter 8: Manipulating Neural Activity.
An increasingly popular technique called dynamic clamp allows scientists
to simulate artificial membrane conductances or synaptic connections in living
cells, even creating hybrid circuits between real and simulated neurons.
In a dynamic clamp experiment, a cell’s voltage is monitored, typically using
whole-cell patch clamp, and fed into a computational model, either in the computer
software or implemented through hardware as an analog chip. The model
calculates and then sends back a current to inject into the cell, creating a realtime
feedback loop. This applied current creates an experimental condition in
which a channel with the model’s properties seems as if it is embedded in the
cell’s membrane, creating an artificial conductance. A dynamic clamp can be
used to simulate specific channel knockins, artificial synaptic inputs, or virtual
pharmacology experiments.
114  Guide to Research Techniques in Neuroscience
CONCLUSION
Electrophysiology remains the technique of choice for analyzing neural activity
and the physiological properties that give rise to this activity. A wide range of
techniques and tissue preparations make it possible to record the activity of neurons
in a dish, a slice, or an awake, behaving animal. Many scientists, even nonelectrophysiologists,
consider electrophysiology techniques to be the backbone
of neuroscience research—they are the primary methods to precisely investigate
the activity of neurons that produce cognition and behavior, the ultimate output
of the nervous system.
SUGGESTED READING AND REFERENCES
Books
The axon guide: A guide to electrophysiology & biophysics laboratory techniques (3rd ed.). (2008).
Sunnyvale, CA: Molecular Devices/MDS Analytical Technologies.
Boulton, A. A., Baker, G. B., & Vanderwolf, C. H. (1990). Neurophysiological techniques: Applications
to neural systems. Clifton, NJ: Humana Press.
Hille, B. (2001). Ion channels of excitable membranes (3rd ed.). Sunderland, MA: SinauerAssociates.
Molleman, A. (2003). Patch clamping: an introductory guide to patch clamp electrophysiology.
NY: Wiley.
Nicholls, J. G., Wallace, B. G., Martin, A. R., & Fuchs, P. A. (2001). From neuron to brain (4th ed.).
Sunderland, MA: Sinauer Associates.
Nicolelis, M. A. L. (2008). Methods for neural ensemble recordings (2nd ed.). Boca Raton,
FL: CRC Press.
Review Articles
Buzsaki, G. (2004). Large-scale recording of neuronal ensembles. Nature Neuroscience, 7, 446–4451.
Cunningham, J. P., &Yu, B. M. (2014). Dimensionality reduction for large-scale neural recordings.
Nature Neuroscience, 17, 1500–1509.
Economo, M. N., Fernandez, F. R., & White, J. A. (2010). Dynamic clamp: alteration of response
properties and creation of virual realities in neurophysiology. Journal of Neuroscience, 30,
2407–2413.
Miller, E. K., & Wilson, M. A. (2008). All my circuits: using multiple electrodes to understand
functioning neural networks. Neuron, 60, 483–488.
Quian Quiroga, R., & Panzeri, S. (2009). Extracting information from neuronal populations: information
theory and decoding approaches. Nature Reviews Neuroscience, 10, 173–185.
Super, H., & Roelfsema, P. R. (2005). Chronic multiunit recordings in behaving animals: advantages
and limitations. Progress in Brain Research, 147, 263–282.
Windels, F. (2006). Neuronal activity: from in vitro preparation to behaving animals. Molecular
Neurobiology, 34, 1–26.
Primary Research Articles—Interesting Examples from the Literature
Bennett, C., Arroyo, S., & Hestrin, S. (2013). Subthreshold mechanisms underlying state-dependent
modulation of visual responses. Neuron, 80, 350–357.
Bliss, T. V., & Lomo, T. (1973). Long-lasting potentiation of synaptic transmission in the dentate
area of the anaesthetized rabbit following stimulation of the perforant path. Journal of
Physiology, 232, 331–356.
Electrophysiology Chapter | 4  115
Evarts, E. V. (1960). Effects of sleep and waking on spontaneous and evoked discharge of single
units in visual cortex. Federation Proceedings, 19, 828–837.
Evarts, E. V. (1968). A technique for recording activity of subcortical neurons in moving animals.
Electroencephalography and Clinical Neurophysiology, 24, 83–86.
Foster, D. J., & Wilson, M. A. (2006). Reverse replay of behavioural sequences in hippocampal
place cells during the awake state. Nature, 440, 603–680.
Kaufman, M. T., Churchland, M. M., Ryu, S. I., & Shenoy, K. V. (2014). Cortical activity in the null
space: permitting preparation without movement. Nature Neuroscience, 17, 440–448.
Lin, D., Boyle, M. P., Dollar, P., Lee, H., Lein, E. S., Perona, P., et al. (2011). Functional identification
of an aggression locus in the mouse hypothalamus. Nature, 470, 221–226.
Mante, V., Sussillo, D., Shenoy, K. V., & Newsome, W. T. (2013). Context-dependent computation
by recurrent dynamics in prefrontal cortex. Nature, 503, 78–84.
Sharp, A. A., O’Neil, M. B., Abbott, L. F., & Marder, E. (1993). Dynamic clamp: computergenerated
conductances in real neurons. Journal of Neurophysiology, 69, 992–995.
Protocols
Brown, A. L., Johnson, B. E., & Goodman, M. B. (2008). Patch clamp recording of ion channels
expressed in Xenopus oocytes. Journal of Visualized Experiments, 20. http://dx.doi.
org/10.3791/936. http://www.jove.com/index/details.stp?id=936.
Current protocols in neuroscience, Chapter 6: neurophysiology. (2007). John Wiley & Sons, Inc.
Kodandaramaiah, S. B., Franzesi, G. T., Chow, B.Y., Boyden, E. S., & Forest, C. R. (2012).Automated
whole-cell patch-clamp electrophysiology of neurons. In Vivo Nature Methods, 9, 585–587.
Martina, M., & Taverna, S. (Eds.). (2014). Patch-clamp methods and protocols (methods in molecular
biology) (2nd ed.). Totowa, NJ: Humana Press.
Murthy, M., & Turner, G. (2013). Whole-cell in vivo patch-clamp recordings in the Drosophila
brain. Cold Spring Harbour Protocals, 2013(2), 140–148.
Nicolelis, M. A., Dimitrov, D., Carmena, J. M., Crist, R., Lehew, G., Kralik, J. D., et al. (2003).
Chronic, multisite, multielectrode recordings in macaque monkeys. Proceedings of the National
Academy of Sciences of the United States of America, 100, 11041–11046.
Perkins, K. L. (2006). Cell-attached voltage-clamp and current-clamp recording and stimulation
techniques in brain slices. Journal of Neuroscience Methods, 154, 1–18.
Tammaro, P., Shimomura, K., & Proks, P. (2008). Xenopus oocytes as a heterologous expression
system for studying ion channels with the patch-clamp technique. Methods in Molecular Biology,
491, 127–139.
117
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00005-8
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 5
Microscopy
After Reading This Chapter, You Should be Able to:
	l	Define magnification and resolving power, as well as the parts of a microscope
that affect these parameters
	l	Describe the fundamental parts of a standard light microscope and how they
manipulate light to magnify an image
	l	Compare the relative strengths and limitations of common forms of microscopy
	l	Discuss issues related to image preparation and analysis
Techniques Covered:
	l	Light microscopy: brightfield, phase contrast, darkfield, differential interference
contrast (DIC/Nomarski)
	l	Fluorescence microscopy: epifluorescence, confocal, two-photon laser scanning
microscopy, total internal reflection fluorescence (TIRF) microscopy
	l	Electron microscopy: transmission electron microscopy (TEM), scanning electron
microscopy (SEM), electron tomography (ET)
	l	Microscopy data: image processing and interpretation
The first microscopes were used in the seventeenth century to expose the
microscopic world of cells and single-celled organisms. Scientific pioneers
such as Robert Hooke and Anton von Leeuwenhoek, often called the
fathers of microscopy, used homemade microscopes to study cell types
from a variety of living organisms. In the late nineteenth and early twentieth
centuries, Santiago Ramón y Cajal used a microscope in combination
with histology to produce highly detailed, seminal studies of nervous system
structure.
The microscope is now an indispensable tool in neuroscience. Because
organelles, glial cells, neurons, and even populations of neurons cannot be seen
by the naked eye, a microscope is essential for examining the nervous system
at the cellular level. Light microscopes can enlarge the image of an object up to
1000 times greater than normal, providing access to the structure of a cell and its
local environment. Fluorescent microscopes provide an even greater ability to
highlight individual subcellular structures. Electron microscopes can theoretically
enlarge an image 1 million times greater than normal, providing unparalleled
insight into the smallest neuronal structures including synapses, surface
receptors, and even individual proteins.
118  Guide to Research Techniques in Neuroscience
The goal of this chapter is to provide a fundamental understanding of common
forms of microscopy. First, we define the fundamental parameters and parts
of a light microscope. Next, we survey the different forms of microscopy and
why an investigator might choose one form over another. Finally, we examine
issues related to processing and interpreting microscopy data. The information
in this chapter will complement information in many other chapters, especially
methods in Chapters 6 and 7 used to investigate neural structure and function.
ESSENTIAL PRINCIPLES OF MICROSCOPY
While modern microscopes are undoubtedly more advanced than those used a
few centuries ago, the fundamental parts of a microscope and the theory behind
how they work together are essentially the same. Before surveying different
forms of microscopy, we review the fundamental parameters and parts of standard
light microscopes.
Fundamental Parameters in Microscopy
There are two important values to consider in microscopy: magnification and
resolution. Magnification refers to how much larger the sample appears compared
to its actual size. Figure 5.1 provides a sense of the relative size of nervous
system components, from an entire brain down to individual atoms. The naked
eye is able to perceive objects about 0.2 mm in size and larger. Therefore, it
is possible to view entire brains and large neural structures, but not individual
neurons or axons without additional magnification.
Resolution (or resolving power) refers to the minimum distance by which
two points can be separated yet still be distinguished as two separate points. For
example, imagine looking at the side of a building with a mosaic image made up
of thousands of colored tiles. If you were standing a block away from the building,
you would see the entire image but would be unlikely to make out the individual
tiles themselves because the resolution would be too low to see that level of detail.
If you move closer, the resolution becomes better and you can see the distinct tiles
making up the image. Likewise, in microscopy, it can be difficult to perceive two
structures as separate from one another—only by increasing the resolving power
is it possible to make out distinct points in a specimen. Figure 5.1 characterizes
the resolution of commonly studied neural structures, demonstrating the range of
objects that can be resolved by the naked eye and the increased resolving power
provided by light and electron microscopy (EM).
Though it may seem that the resolving power of a microscope is based on
the ability of the microscope to magnify an object, this is not the case. Magnifying
an object does not necessarily improve the clarity or resolution of the final
image. The resolution of a magnified object depends on two factors: numerical
aperture and the wavelength of the light source. The numerical aperture (NA) is
a measure of the light-collecting ability of the microscope objective, the lens that
gathers and focuses light from the specimen. An objective with a higher NA will
Microscopy Chapter | 5  119
collect more light rays, leading to better resolving power. The NA of an objective
depends on the angle that light enters into the objective and the index of refraction
of the medium in which the objective is working (Figure 5.2). The index of
refraction (or refractive index) indicates changes in the speed of light traveling
Mouse brain
Cell body
Dendrites
Brain nuclei
µ
µ
µ
Dendritic
spines
Synapses
Synaptic
vesicles
Ion
channels
DNA
Ions
Atoms
FIGURE 5.1  Resolving power in microscopy.  The resolution required for distinguishing neuroscience
structures, from a rodent brain to individual atoms.
120  Guide to Research Techniques in Neuroscience
through a particular medium (1.0 for air, 1.33 for water, and up to 1.55 for oils).
The greater the index of refraction, the greater the NA. As light travels from one
medium (e.g., air) to a medium with a higher index of refraction (e.g., oil), the
angle of the light changes, allowing an objective to collect more light. This is the
same principle that causes a straw in a glass of water to appear bent at the surface
of the water. An objective lens immersed in oil has a higher NA than an objective
in air, and thus the oil-immersed objective has a greater resolving power.
The other factor that determines the resolution of a magnified object is the
wavelength of light either illuminating or emanating from the specimen. In light
microscopy, light illuminates the specimen; in fluorescent microscopy, light
emanates from the specimen. Wavelength is the distance between two repeating
units of a light wave (Figure 5.3A). Light rays with different wavelengths
appear as different colors to the human eye (Figure 5.3B). The wavelength of
light affects how broad a single point of light appears. Shorter wavelengths of
light (UV through green) appear sharper than longer wavelengths (red through
infrared), which tend to appear more spread out, decreasing resolving power.
Therefore, the smallest object that a microscope can display is a function of
the magnification and resolving power used to image a specimen. Interestingly,
while the ability of a microscope to magnify an object is essentially unlimited,
the resolution is finite. A light microscope cannot resolve details of a specimen
finer than 0.2 μm—about the size of a neural synapse—no matter what the
magnification. This limitation is due to the minimum wavelength of visible light
(Box 5.1); wavelengths shorter than the UV to green portion of the spectrum are
invisible to the human eye. This limitation causes the resolving power of any
light microscope to have a maximal value. The unaided human eye is limited to
seeing details about 0.2 mm in size. A light microscope can only resolve details
of about 0.2 μm, so magnifying those details more than 1000× (1000 × 0.2 μm = 
200 μm = 0.2 mm) would not be useful. Increasing the magnification higher
α1 α2
1 2
FIGURE 5.2  Numerical aperture (NA) and the angle of light entering the objective. An
objective with a higher NA collects more light rays, leading to a higher resolving power. The NA
depends on the angle that light enters the objective, α, as well as the refractive index of the medium,
n. When light travels through material with a different n, such as between glass and air (n1), it
refracts more and yields a smaller α. Immersion, such as water or oil (n2), reduces the difference
and allows for a greater α.
Microscopy Chapter | 5  121
than 1000× is like using the zoom feature on a computer to enlarge a digital
picture of a fixed size: you won’t get any finer details with a digital zoom—you
simply make the pixels bigger. However, technologies that do not use visible
light to image a specimen, such as EM, can have much higher resolving powers
and can image much smaller structures than light microscopes.
Now that we have discussed magnification and resolution, letus examine the
fundamental parts and design of two categories of microscopes: the compound
microscope and the stereomicroscope.
The Design of a Compound Microscope
The simplest form of microscopy, one that has been used for centuries, is the
magnifying glass. A magnifying glass is a convex lens that enlarges nearby
objects (Figure 5.4). It works by refracting the light scattered by an object to a
focal point on the other side of the glass. What appears is a larger image of the
object behind the lens.
A single lens has limited magnification ability, but multiple lenses can be
arranged, one behind the other to multiply the magnification of each lens. This
is the basis of the compound microscope. The term compound refers to the two
or more lenses used in concert to magnify an object.
λ
λ
FIGURE 5.3  The visible spectrum of light. (A) The wavelength of light, λ, is the distance
between two repeating units of a propagating light wave. (B) Light rays of different wavelengths
appear as different colors to the human eye. Shorter wavelengths appear violet-blue while longer
wavelengths appear redder. (C) Approximate peak excitation (black) and emission (blue) wavelengths
of commonly used fluorophores.
122  Guide to Research Techniques in Neuroscience
Most light microscopes use at least two lenses. The first lens is called the
objective lens and is placed adjacent to the specimen (Figure 5.5). Investigators
can choose from multiple objective lenses for a desired degree of magnification,
usually 4×, 10×, 20×, 40×, or 100×. The second lens is located in the
eyepiece and is referred to as the ocular lens. This lens is often set at 10×. The
total magnification of the microscope is multiplicative, so if the objective lens
is set at 40× and the ocular lens is set at 10×, the total magnification achieved
is 400×.
A condenser focuses light from a light source onto a specimen (Figure 5.6).
Light transmitted through the specimen is then collected and magnified by the
objective lens. This magnified image is focused onto the ocular lens, which
essentially acts like a magnifying glass to further enlarge the image. Although
each manufacturer designs light microscopes with unique features, the conceptual
design of a compound microscope remains the same.
BOX 5.1 The Diffraction Limit of Resolution and Ways around It
Why does the wavelength of visible light ultimately set the limit of resolution
for light microscopes? The reason is due to a phenomenon known as diffraction.
Diffraction can be thought of as the tendency for a wave to spread out and bend
around obstacles as it propagates. Light behaves like a wave, so it too can bend
around obstacles, just like water ripples moving past a boulder in a river. When a
single point of light passes through the opening of a microscope objective, it does
not appear as an infinitely small single point of light; it has a finite size that looks
more like a bright point with concentric rings of decreasing brightness, like the
rings of ripples you see in a puddle when a raindrop hits. This ring of light ripples
is known as an Airy disk. Because each point of light is really a ring of light ripples,
the distance between two points needs to be large enough so the ripples do not
interfere with each other. Diffraction is what causes the points of light to create
these ripples. Because the ripples are the factor that limit how close together two
points of light can be and still be seen as two distinct points of light, this is known
as the diffraction limit of resolution. The minimum wavelength for light visible to
the human eye (∼400 nm) sets this diffraction limit.
New forms of superresolution microscopy attempt to get beyond the diffraction
limit of resolution to image nanoscale molecular interactions. In fact, work on superresolution
fluorescent microscopy received the Nobel Prize in 2014. Stimulated
emission depletion (STED), ground-state depletion, and saturated structuredillumination
microscopy break the diffraction limit by controlling the spatial and/
or temporal transition between a fluorophore’s molecular states (e.g., dark vs. bright
state).This is done by modifying the excitation light pattern to effectively shrink the size
of the Airy disk. Demonstrating its importance in cellular and molecular neuroscience,
STED was used to produce video-rate images of synaptic vesicles in live hippocampal
neurons at a resolution of 60 nm. Photoactivated localization microscopy, fluorescence
photoactivation localization microscopy, and stochastic optical reconstruction
microscopy break the diffraction limit by detecting the positions of single molecules.
By using photo-switchable fluorescent probes, overlapping individual fluorophores
can be separated in time to construct a superresolution image.
Microscopy Chapter | 5  123
FIGURE 5.4  The magnifying glass. A magnifying glass is a convex lens that refracts light to
make an object appear larger.
Eyepieces
(ocular lens)
Camera
Objective
Lenses
Excitation and Emission
Filters (inside)
Shutter
Stage
Mercury lamp source
Transmitted light source
Focus knob
Condenser
FIGURE 5.5  Components of a compound microscope. In brightfield microscopy, the light
comes from the transmitted light source. For fluorescent microscopy, the light comes from a mercury
lamp (green arrow). Light is transmitted through the specimen or excited from the specimen
and travels to the eyepieces or camera (red arrow).
124  Guide to Research Techniques in Neuroscience
Compound microscopes are classified as being either upright or inverted. In
an upright microscope, the specimen is placed just below the objective
(Figure 5.7A). This type of microscope is ideal for examining a specimen mounted
on a glass slide. However, there is little space between the objective and specimen,
Ocular lens
Objective lens
Condenser lens
Specimen
Magnified
Specimen
FIGURE 5.6  Compound microscopes use multiple lenses to magnify a specimen. A condenser
lens focuses light onto a specimen, which then transmits (or emits) light through the objective
and ocular lenses that magnify the image that reaches the eye.
Microscopy Chapter | 5  125
making it impractical for manipulating brain sections for electrophysiology or for
viewing live cells in a thick cell culture dish. In such situations, it is better to use an
inverted microscope, which positions the objective below the specimen with the
light source and condenser located relatively far above the sample (Figure 5.7B).
The other parts of a basic compound microscope (Figure 5.5) allow the
investigator to adjust the magnification, focus, and light level of the microscope,
as well as adjust the precise placement of the specimen.
The Design of a Stereomicroscope
A stereomicroscope, also called a dissecting microscope, serves a different purpose
than a compound microscope and works in a different way (Figure 5.8). The
main purpose of a stereomicroscope in neuroscience is to examine the surface of
brains, tissue slices, or large neural structures. It is especially useful for the fine
manipulation needed during dissections, surgeries, or the fabrication of small tools
such as electrodes or implants. Rather than passing through a single objective and
ocular lens system, light in a stereomicroscope goes through two separate lens systems.
While a compound microscope directs light to both eyes from a single lightpath,
a stereomicroscope directs light to each eye from two independent lightpaths.
Because light from a single point on the specimen travels independently through
two different paths to reach each eye, the specimen appears three dimensional.
In addition to the appearance of the specimen, there are at least two other
major differences between a stereomicroscope and a compound microscope.
First, stereomicroscopes typically use light that is reflected off a specimen,
while compound microscopes often use light transmitted through a specimen.
This makes stereomicroscopes useful for looking at specimens too thick for
FIGURE 5.7  Upright versus inverted microscopes.  (A) In an upright microscope, the specimen
is placed just below the objective. (B) In an inverted microscope, the specimen is placed just above
the objective, allowing for larger cell culture plates and access from above.
126  Guide to Research Techniques in Neuroscience
light to pass through. Second, the magnification power of a stereomicroscope is
often less than that of a compound microscope. The ocular lenses of a stereomicroscope
are usually fixed at 10× and the objective lenses typically range from
0.1 to 8×. Therefore, a stereomicroscope is useful for examining gross neural
structures, while a compound microscope is better for examining microscopic
structures such as single neurons or fiber tracts.
Now that we have examined the basic concepts of microscopy, we survey the
various categories of light microscopy and why each may be particularly useful
for a given experiment.
LIGHT MICROSCOPY
A light microscope is any microscope that uses visible light to illuminate and
image a specimen. This includes white light composed of all wavelengths, as
well as the light of a specific wavelength used in fluorescent microscopy. Usually
when people refer to light microscopy, they refer to nonfluorescent microscopy,
even though fluorescent microscopy does, of course, use light.
The most common and general form of light microscopy is brightfield
microscopy, in which light passes directly through or is reflected off a specimen.
Most cells and tissues are transparent due to their high water content.
Unless naturally pigmented or artificially stained, distinct structures are
FIGURE 5.8  A standard stereomicroscope.
Microscopy Chapter | 5  127
difficult to differentiate using brightfield microscopy. Thus, a variety of histological
procedures have been developed to preserve and stain specimens
to enhance contrast among different microscopic structures (Chapter 6).
However, most of these procedures result in the death of cells to preserve the
specimen.
For many experiments, working with preserved cells is not a problem. However,
when an investigator wishes to magnify living, unpreserved cells or tissues,
as in cell culture experiments, it is necessary to provide contrast without
killing the specimen. Therefore, methods of manipulating light to enhance contrast
have been developed to make resolvable details stand out to the eye without
requiring the application of special dyes.
Variations in density within cells cause tiny differences in the way different
regions scatter light; therefore, different cellular structures have different indices
of refraction. Phase-contrast microscopy takes advantage of these slight differences,
amplifying them into larger intensity differences with high contrast that
can be easily seen. Because this form of microscopy does not require special
treatment of the specimen to see many details, phase contrast is often used to
examine cultured cells. Darkfield (or darkground) microscopy illuminates a
specimen using oblique rays of light directed from the side so that only scattered
light enters the objective lens. Most regions of the specimen (e.g., cytoplasm)
do not scatter much light so they appear dark, while highly refractive organelles
that scatter large amounts of light will appear bright. Differential interference
contrast (DIC) microscopy, also known as Nomarski microscopy, exaggerates
changes in the light-scattering properties of cellular structures, highlighting
the edges around structures to create a textured, three-dimensional appearance.
Figure 5.9 demonstrates the different appearances of a single cell when viewed
using these various forms of light microscopy.
(A) (B)
(C) (D)
FIGURE 5.9  Four types of light microscopy used to analyze the same cell. Various methods of
light microscopy are used to enhance contrast and visualize details in unstained tissue preparations. (A)
Brightfield microscopy. (B) Phase-contrast microscopy. (C) Differential interference contrast (DIC/
Nomarski) microscopy. (D) Darkfield microscopy. Reprinted from Alberts, B., et al. (2002). Molecular
Biology of the Cell, (4th ed.). Fig. 9–8, with permission from Garland Science: NewYork, NY.
128  Guide to Research Techniques in Neuroscience
FLUORESCENCE MICROSCOPY
Fluorescence microscopy takes advantage of specialized molecules called fluorophores
that have the property of absorbing light at a specific wavelength and
then emitting light at a different (typically longer) wavelength. Fluorophores that
absorb blue light typically emit green light; fluorophores that absorb green light
emit red; fluorophores that absorb red emit infrared, and so on. There are dozens of
commercially available and commonly used fluorophore molecules each with their
own characteristic absorption (excitation) and emission wavelengths (Figure 5.3C).
These molecules can be linked to antibodies or other molecular probes to indicate
the presence of specific proteins, organelles, or other structures within a cell.
The principal use of fluorescence microscopy is to examine specimens
labeled with these fluorescent reagents or expressing genetically encoded fluorescent
proteins (Chapter 6). Light from an extremely bright source passes
through an excitation filter that allows only light in a specific range of wavelengths
through the objective to illuminate the specimen. That light is absorbed
by and excites the fluorophores in the specimen, which react by emitting a longer
wavelength of light. The emitted light is collected back through the objective
and passed through a second filter, an emission filter. The emission filter
blocks extraneous wavelengths of light, including the light used to illuminate
the specimen, but allows emitted light wavelengths to pass through to a detector
(i.e., your eye or a camera). The optical filters make it appear as if structures
tagged with the fluorophores light up against a dark background. Because the
background is dark, even a tiny amount of the glowing fluorescent reagent is
visible, making fluorescence microscopy a very sensitive technique.
A major appeal of fluorescent microscopy over conventional light microscopy
is that structures and proteins within a cell can be labeled with a wide
range of fluorophores. Because each fluorophore has its own characteristic excitation
and emission spectra (the wavelengths of light they absorb and emit), it
is possible to label different structures in the same sample and investigate their
relative locations. For example, an investigator can use a fluorophore that emits
light in the green range of the spectrum to label one protein, and a fluorophore
that emits light in the red range of the spectrum to label another. Because the
excitation and emission spectra of the green and red fluorophores do not overlap,
they distinctly mark the locations of these different proteins within the same
sample. This technique can even be used in living specimens to observe the
dynamic interactions of multiple fluorescently labeled proteins and molecules.
Despite the advantages of fluorescence microscopy, there are some disadvantages.
One major limitation is that fluorescent reagents cannot be illuminated
indefinitely. The intensity of the light emitted from a fluorophore will decrease
over time as it is continuously exposed to light, a process called photobleaching.
This makes it necessary to limit the light exposure of fluorescently tagged specimens
and capture images of them before fluorescence becomes too dim.Another
limitation in live cell imaging is phototoxicity, in which illumination leads
Microscopy Chapter | 5  129
to the death of cells expressing the fluorophore due to free-radical generation.
Finally, while fluorescently labeled structures can be detected easily, in practice,
background noise can mask the actual signal of interest. Background noise
is nonspecific fluorescence that does not indicate true specific signal. One form
of background can be autofluorescence, in which certain structures, chemicals,
and organisms naturally fluoresce without the addition of a fluorophore label. If
this fluorescence has the same emission wavelength as the fluorescent label, it
can be difficult to determine true signal from noise.
Fluorophores have advanced every area of neuroscience. Fluorescent molecules
have made breakthroughs possible in studying neural structure and function,
identifying the spatial relationships of proteins within a cell, examining
the fine branching of neural structures, and detailing the time course of a cell’s
functional response to chemical or electrical stimulation. Various forms of
microscopy attempt to maximize the advantages and minimize the disadvantages
of fluorescent reagents. We now survey the main categories of fluorescence
microscopy.
Epifluorescent Microscopy
The most elementary form of fluorescent microscopy is epifluorescent microscopy,
also known as wide-field fluorescent microscopy. Epifluorescent microscopes
work as just described: specimens labeled with fluorescent probes are
illuminated by light of the excitation wavelength (Figure 5.10A). The specimen
is then viewed using a second filter that is opaque to the excitation wavelength
but transmits the longer wavelength of the emitted light. Thus, the only light
transmitted through the eyepiece is the light emitted by the specimen.
Epifluorescence is an excellent tool, but has one major disadvantage: light
excites fluorophores throughout the entire depth of the specimen. Thus, fluorescence
signals are collected not only from the plane of focus but also from areas
above and below this plane. Such background fluorescence can lead to hazy,
out-of-focus images that appear blurry and lack contrast. Thus, the value of epifluorescence
in relatively thick specimens (>15–30 μm) is limited. Alternative
forms of fluorescent microscopy attempt to limit out-of-focus fluorescence to
produce sharper, clearer fluorescent images.
Confocal Microscopy
Confocal microscopes produce clear images of structures within relatively thick
specimens by selectively collecting light from thin (<1 μm) regions of the specimen.
It is the tool of choice for examining fluorescently stained neurons in brain
slices or small, intact organisms such as Drosophila or embryonic zebrafish.
With epifluorescence, the entire specimen is illuminated at once so all the fluorophores
emit light at the same time, making it difficult to observe signals from
a specific focal plane within a thick specimen. In a confocal system, light from
130  Guide to Research Techniques in Neuroscience
FIGURE 5.10  Comparison of different forms of fluorescence microscopy: epifluorescence,
confocal, and two-photon.  (A) Basic strategy of an epifluorescent microscope. Light from a bright
lamp passes through a filter that only allows light of the excitation wavelength through to the specimen.
Light emanates from the specimen and passes through a second filter that is opaque to the
excitation wavelength of light but that transmits the longer wavelength of the emitted light. (B)
Basic strategy of a confocal microscope. Light is focused from a pinhole aperture onto a single
point on the specimen. Fluorescent light emanating from the specimen is transmitted through a
second pinhole aperture before hitting a detector. The two apertures are confocal with each other,
so out-of-focus fluorescence is avoided by the detector pinhole. (C) Basic strategy of a two-photon
microscope. Pulsed laser light illuminates the sample with light about twice as long as the wavelength
required for excitation. When focused on a specific plane of section, two photons can arrive
near-simultaneously at one fluorophore in that plane and deliver enough energy to excite the fluorophore.
Above and below the plane in focus, light is too low-energy to excite fluorescence, eliminating
out-of-focus fluorescence.
Microscopy Chapter | 5  131
a laser passes through a pinhole aperture that focuses the light at a specific depth in
the specimen (Figure 5.10B).Although this pinhole aperture restricts focused illumination
to one plane, other regions receive dispersed, less intense illumination
and emit out-of-focus fluorescence. However, all the emitted light passes through
a second pinhole aperture before reaching a detector. The key aspect of confocal
microscopy is that the pinhole aperture in front of the detector is at a position
that is confocal with the illuminating pinhole—that is, the only light to reach the
detector comes from the same plane in the specimen where the illuminating light
comes to a focus, minimizing background fluorescence and maintaining sharp
focus on a single plane. The detected light is digitized and sent to a computer for
display and storage.
To generate a two-dimensional image, data from each point in the plane of
focus are collected sequentially by scanning across the field. Confocal microscopes
can focus the laser beam at precise intervals throughout the thickness
of the specimen, producing optical sections at multiple focal planes. Computer
image analysis can then be used to stack these sections to reconstruct a threedimensional
volume with optimal contrast and resolution.
A major advantage of confocal microscopy to standard epifluorescence is
the ability to produce sharp images of cells and cellular structures without background
fluorescence (Figure 5.11). However, there are still limitations. Confocal
lasers scan specimens point by point to form a complete image of the specimen.
Unfortunately, this scanning requires that confocal microscopes expose samples
to intense light for a longer time period than epifluorescent microscopes, making
photobleaching and phototoxicity more problematic. The longer time necessary
to capture an image also makes it less desirable for live cell imaging of extremely
fast events. In addition, though it is a great advantage to be able to create threedimensional
reconstructions by stacking multiple optical sections, the lateral
X-Y resolution is finer than resolution in the Z (depth) dimension, so threedimensional
reconstructions may appear warped. Despite these disadvantages,
(A) (B)
FIGURE 5.11  Comparison of images from epifluorescence and confocal microscopy.  (A) A
standard epifluorescent microscope captures fluorescence emitted from all planes, not just those
in focus, leading to a hazy background. (B) A confocal microscopy image of the same specimen
appears sharper and in focus.
132  Guide to Research Techniques in Neuroscience
confocal microscopy is the gold standard for imaging, especially for examining
brain sections or small invertebrate organisms.
Two-Photon Microscopy
Two-photon microscopy, also referred to as two-photon laser scanning
microscopy, is a further refinement of precision fluorescence microscopy. In
epifluorescent or confocal microscopy, a single photon of light at a specific
wavelength excites a fluorophore to emit light. In two-photon microscopy, a fluorophore
can be excited by effectively absorbing two photons simultaneously. In
this case, each photon by itself has a wavelength that is twice the usual excitation
wavelength, and thus has half the energy level per photon. When a fluorophore
absorbs the two lower-energy photons at the same time, each contributes half the
needed excitation energy to interact with the fluorophore as if a single photon
with more energy and a shorter wavelength had struck it (Figure 5.10C).
The main advantage of this method is that the near-simultaneous arrival of
two photons is an extremely rare event, so fluorescence excitation is restricted
to a narrow plane of focus within the specimen. This provides clearer images
than traditional confocal microscopy, even in cases where relatively few fluorophores
are present, because photons are only emitted from the excited fluorophores
located at the focal plane. Thus, all emitted light can be collected. There
is not enough energy to excite fluorophores located above and below the focal
plane to produce any background fluorescence.
Two-photon microscopy can penetrate deeper into tissue (500 μm−1 mm)
than confocal or epifluorescence microscopy because the excitation wavelength
is about twice as long as the usual wavelengths used. Light with longer wavelengths
scatters less, allowing deeper penetration into tissue. Because excitation
is restricted to a focal point, it also causes less photobleaching and phototoxicity,
making two-photon microscopy particularly useful for extended observation
of cells in undissected, living brain tissue in whole animals or fine-scale neural
structures in brain slices. The primary limitation of this method is that a twophoton
microscope setup requires expensive, specialized lasers and equipment.
Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescent (TIRF) microscopy is another method
used to eliminate hazy images with out-of-focus fluorescence by restricting the
excitation and detection of fluorophores to a thin section of the specimen. In
TIRF microscopy, the thin section of detection is fixed at the interface between
the specimen and the surface on which the specimen is mounted, usually a
glass coverslip (Figure 5.12). TIRF microscopes excite fluorophores with an
evanescent wave of light exclusively at the interface between the specimen and
its adjacent surface. The evanescent wave exponentially decays with distance
and is typically lost at a depth of 100 nm into the sample. Therefore, it only
Microscopy Chapter | 5  133
effectively excites the plasma membrane (∼7.5 nm thick) and the immediately
adjacent cytoplasmic zone.
This method is useful for investigating molecules and biochemical events
that occur at the cell surface. For example, a fluorophore-tagged molecule of
interest may be located on the plasma membrane as well as in the cytoplasm
of the cell. Using an epifluorescent microscope, fluorescence emitted from the
membrane can be overwhelmed by the fluorescence coming from the much
larger population of molecules in the cytoplasm. A TIRF microscope allows an
investigator to selectively excite and image the membrane-bound population
of this molecule. This kind of microscopy is often used to selectively visualize
processes that occur at the plasma membrane in living cells with high
resolution.
ELECTRON MICROSCOPY
Although there is no theoretical limit to the magnification power of a light
microscope, resolving power has a maximal value of about 200 nm due to the
fixed minimal wavelength of visible light (Box 5.1). However, an electron
has a much shorter wavelength than a photon. By focusing a beam of electrons
rather than a beam of photons to image a specimen, electron microscopy
(EM) can increase the resolving power as much as 1000-fold to 0.2 nm,
about the radius of a glutamate molecule. EM is essential for examining
small neural structures such as synapses, synaptic vesicles, and ion channels.
The major limitation to using EM is that specimens must be fixed, dehydrated,
and chemically treated. Therefore, EM cannot be applied to living samples.
Furthermore, artifacts could appear that do not exist in the living cell. To
reduce these artifacts, investigators can rapidly freeze specimens so that water
and other components in cells do not have time to rearrange themselves or even
crystallize into ice.
There are two major categories of EM: transmission electron microscopy
(TEM) and scanning electron microscopy (SEM).
FIGURE 5.12  The basic strategy of TIRF microscopy. Reflected laser light causes an evanescent
wave of excitation at the interface between a specimen and a glass coverslip. The light
penetrates only 100 nm, ensuring that all fluorescent excitation and emission occurs only at the
surface of the specimen, usually the plasma membrane.
134  Guide to Research Techniques in Neuroscience
Transmission Electron Microscopy
In transmission electron microscopy (TEM), a beam of electrons is aimed through
a thin section of a specimen that has been chemically treated to enhance contrast.
Extremely thin (<100 nm) sections of preserved tissue are stained with atoms of
heavy metals, which are preferentially attracted to certain cellular components.
These heavy metal stains are electron dense, so when the electron beam hits the
heavy metal atoms in the specimen, the electrons are absorbed or scattered, making
electron-dense areas appear dark. Electromagnets focus and magnify the image by
bending charged electron trajectories. Since we cannot see electrons, the variations
in electron intensity are converted into photons by projecting the electrons into a
special detector or onto a screen that fluoresces at intensities relative to the amount
of beam electrons hitting it. TEM produces two-dimensional images of thin tissue
sections that are useful for studying the internal structure of cells (Figure 5.13).
TEM is frequently used to define synapses, as the postsynaptic density appears
clearly as an electron-dense region at close contact with a presynaptic membrane
containing neurotransmitter vesicles.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) is useful for detailed study of a specimen’s
surface.A high-energy electron beam scans across the surface of a specimen, usually
coated with a thin film of gold or platinum to improve contrast and the signal-to-noise
µ µ
FIGURE 5.13  Comparison of images taken by different electron microscope techniques. (A)
A differential interference contrast image of stereocilia projecting from a hair cell in the inner ear.
(B) An image of the same structure taken with a transmission electron microscope and (C) a scanning
electron microscope Courtesy of Dr. A.J. Hudspeth and Dr. R. Jacobs.
Microscopy Chapter | 5  135
ratio.As the beam scans across the sample’s surface, interactions between the sample
and the electron beam result in different types of electron signals emitted at or near the
specimen surface. These electronic signals are collected, processed, and eventually
translated as pixels on a monitor to form an image of the specimen’s surface topography
that appears three dimensional (Figure 5.13). Low-energy secondary electrons
excited on the sample’s surface are the most common signal detected. High-energy
backscattered electrons and X-rays are emitted from below the specimen surface,
providing information on specimen composition.
Electron Tomography
Electron tomography (ET), also known as electron microscope tomography,
is similar to computerized tomography described in Chapter 1. In a CT
scan, the imaging equipment is moved around a patient to generate different
images of a slice of the brain. In ET, the specimen is tilted within the electron
microscope to produce TEM images from many different perspectives to
reconstruct a three-dimensional view. With a resolution of 2–20 nm, ET can be
used for determining both molecular structures and the three-dimensional ultrastructure
of organelles. ET has been especially informative for elucidating the
organization of presynaptic vesicles in axon terminals (Figure 5.14).
FIGURE 5.14  Example of an electron tomography image: synaptic vesicles docking at
release sites in the neuromuscular junction.  (A) Two-dimensional TEM images used in the ET
volume reconstruction of an active zone at a frog neuromuscular junction. AZM, active zone material,
denoted by arrows; sv, docked synaptic vesicles; pre, presynaptic membrane. Scale bar, 50 nm.
(B) Three-dimensional surface-rendered AZM, docked vesicles, and presynaptic membrane that
allow the shape, size, and associations of different components to be examined. Reprinted by permission
from Harlow, M.L., et al. (2001). The architecture of active zone material at the frog’s
neuromuscular junction. Nature: Macmillan Publishers Ltd.
136  Guide to Research Techniques in Neuroscience
PREPARING AND INTERPRETING MICROSCOPY DATA
Microscopy data are probably the most frequently presented form of data in
neuroscience, and possibly all life sciences. They may also be the most subjective
form of data to interpret. Therefore, it is important to understand standard
practices used to prepare and display data to know what to look for in published
results.
Image Processing
The digital images taken at the microscope are generally not the exact same
images that appear in publications. Raw images are usually processed by computer
software accompanying the microscope or by secondary programs such
as Adobe Photoshop® or NIH ImageJ. Image processing is appropriate and
sometimes necessary to extract meaningful information: to facilitate quantitative
measurements and accurate analysis, to highlight meaningful aspects of an
image, and to improve aesthetic appearance when it does not interfere with the
results. The key is to faithfully represent reproducible results and not alter the
inherent information contained in the image.
The most common form of processing is creating pseudocolor representations
of fluorescent microscopy images taken in different channels (different
emission wavelengths) to overlay them in a single image. Most microscope
cameras capture light intensity in grayscale rather than color. If multiple
fluorophores are imaged in a single specimen, different channels can be
selected by changing the excitation and emission filters on the microscope.
This process allows different fluorophores to be imaged independently, but
they will all appear in grayscale. The investigator can then assign a different
color to each signal and merge the separate images of each fluorophore into
a single image with multiple colors. This helps visualize relative intensity
differences and spatial relationships for each fluorescently labeled molecule
(Figure 5.15).
Other common forms of image processing include digitally enhancing
contrast or removing background fluorescence. Although this processing can
aid in elucidating useful information from an image, it must not introduce
false elements into the image to provide support for what an investigator
wishes to see. Many journals provide guidelines for the types of image processing
allowed and require that all processing be reported in the Materials
and Methods section.
In general, there are two important guidelines to follow when processing
microscopy images for analysis and publication: (1) Any processing applied to
one image of a set should be applied to all images within the set. For example,
when removing background fluorescence from one image, it is important that
all remaining images receive the exact same digital enhancements. This is especially
important when comparing images between groups. (2) An investigator
should process and analyze images blind to the identity of the samples between
Microscopy Chapter | 5  137
experimental and control groups. Even with the best of intentions, scientists
can subconsciously make mistakes in analysis or introduce subconscious bias.
When working with microscopy data, as with most data, blind processing and
scoring is critical to prevent potential bias. It is important to properly represent
reproducible features contained in the data itself rather than in interpretations
of the data.
Interpreting Images
Some general guidelines are useful when evaluating your own or published
microscope images. When evaluating images, it can be helpful to ask some critical
questions:
	l	Does it look right? Familiarity with other work in the field can provide an
intuition and expectation for standard images. If the image is supposed to be of
neurons, do the structures look like neurons? If the image is supposed to represent
a specific brain region, can you identify that region based on the image
presented? Is the image taken at an appropriate scale to make a judgment? If
the author claims that expression of a gene, protein, or lesion is localized to a
specific structure, does the image show that structure in the context of neighboring
regions?
	l	Does the signal-to-noise ratio allow for proper examination of the data?
Nonspecific background staining can potentially mask a specific signal. Is the
contrast between the background and the signal substantial enough to produce
a consistent, observable result?
	l	Are the exposure and brightness optimal? An overexposed image (Figure
5.16A) can mask details, enhance nonspecific background, or improperly
FIGURE 5.15  Merging images of different fluorophores into a single image. A sample is
viewed for fluorescence using filters for (from left to right) rhodamine (red), FITC (green), and
DAPI (blue). Because these spectra do not overlap, it is possible to overlay these images, assigning
each channel a distinct color, to compare the spatial relationship of fluorescent signals.
138  Guide to Research Techniques in Neuroscience
suggest a high concentration of the measured signal. An underexposed image
(Figure 5.16B) can make it appear as if the measured signal is not present
when it is. An investigator can control the proper exposure of an image during
both the capture of an image and the secondary image processing.When evaluating
microscopy data, ensure that the conclusions are not drawn from images
with inappropriate exposure conditions or inappropriate image processing.
	l	If this is the most convincing example, what do the other samples look like?
For obvious reasons, authors tend to publish images that best represent and
support their conclusions. Usually, these images represent one of many individual
data points. Therefore, a critical review of the data should assume
that the other data points are of an equal or lesser quality than the images
presented in a publication. If the image is suboptimal or difficult to interpret,
the validity of conclusions reached from unpublished images should be
questioned.
	l	Is there quantitative analysis of the images? Though a picture is worth a
thousand words, subjective interpretation of data is not as strong as objective,
quantifiable results. Therefore, the strongest evidence to support a conclusion
presents images alongside complementary quantification of the data
that demonstrates the reproducibility of the results from image to image.
Common types of quantitative information calculated from images include
relative cell counts, size and shape distributions, variation in fluorescence
intensity to indicate relative concentrations of the measured signal, and
colocalization of independently labeled proteins.
	l	Does the illumination look uniform? Improper microscope alignment can cause
differences in signal intensity that might lead to inappropriate conclusions. If
FIGURE 5.16  Over- and underexposure of an image.  (A) An overexposed image of neurons.
(B) The same cells underexposed and (C) with the proper level of exposure. The yellow arrows point
to a neural process that is lost when underexposed and difficult to see under a properly balanced
exposure level. It can be difficult to find the proper exposure to see both intensely and weakly fluorescent
structures in the same field of view.
Microscopy Chapter | 5  139
the entire field of view is not evenly illuminated, important information and
resolution can be lost.
	l	Could fluorescent images represent poor quality control issues, such
as photobleaching or autofluorescence? Photobleaching could lead an
investigator to believe there is no signal in a sample when the signal
has actually faded. Alternatively, autofluorescent substances in a sample
might lead an investigator to believe there is signal where there is
none. Investigators can avoid inappropriate conclusions caused by photobleaching
or autofluorescence by processing all samples in the same
way and by performing the appropriate positive and negative control
experiments.
	l	Could fluorescent images represent bleed-through of different emission spectra?
Investigators usually assume that signals from fluorophores of different
wavelengths are independent of each other. However, some fluorophores
exhibit overlapping excitation or emission spectra and may be detected when
not expected. Bleed-through, also known as cross-talk, occurs when the signal
for one fluorophore is present when examining the signal for a different
fluorophore(i.e. the signal of one fluorophore has bled through the filters that
are set for the other fluorophore). To control for this situation, specimens can
be labeled with a single fluorophore and the signal recorded in each channel.
Bleed-through of fluorophores becomes especially pertinent when an investigator
claims that two signals overlap, as when two proteins colocalize. It is
rare that the signal from two different fluorophores will be exactly identical,
no matter how strong the colocalization may be. Therefore, if two different
channels appear exactly the same, despite use of different detection agents, the
image could indicate an artifact.
CONCLUSION
Whether using photons or electrons, microscopes provide the ability to explore
structures much smaller than can be viewed by the naked eye. Although microscopes
are one of the most frequently used tools in neuroscience, many people
do not understand the essential concepts necessary to produce quality, scientifically
meaningful images. This chapter has served as an introduction to
microscopy and surveyed multiple techniques (Table 5.1). Most microscope
manufacturers (Nikon, Leica, Zeiss) provide detailed instructions in the proper
use and theory behind their products. These guides can provide further information
about the specific microscopes used in your laboratory. While optical methods
can be used to provide enhanced contrast to make out individual structures
within cells, histological preparations are utilized to examine specific processes
and structures. The next two chapters present methods on preparing samples to
visualize neural structure and function that depend on good microscopes and,
just as important, good microscopy skills.
140 GuidetoResearchTechniquesinNeuroscience
TABLE 5.1 Comparison of Different Forms of Microscopy
Description Advantages Disadvantages
Common Use in
Neuroscience
Brightfield Light transmitted through
specimens.
Contrast generated by
natural pigmentation or
added dyes.
Simple and inexpensive.
Different colored dyes can
be used.
Most cells and tissues are
transparent, so they can
be difficult to see.
Adding dyes to visualize
structures usually requires
fixation and sectioning.
Fixed samples processed
using detection reagents that
produce colored byproducts
(see Chapter 6).
Phase-contrast Contrast generated by
changes in index of
refraction because of
variation in organelle
densities.
Can be used on live cells.
Does not require added
chemicals or tissue
processing.
Out-of-focus signals. Tissue culture cells.
Darkfield Specimen illuminated
from the side at an
oblique angle.
Only scattered light is
imaged.
Can be used on live cells.
Sensitive.
High signal-to-noise ratio.
No added chemicals
necessary.
Cannot see structures that
do not scatter light.
Samples processed
for radioactive in situ
hybridization
(see Chapter 6).
DIC/Nomarski Optical methods used
to exaggerate changes
in index of refraction,
enhancing contrast at the
edges of objects.
Has a 3-D appearance.
Can be used on live cells.
Can create thin optical
sections.
Can create 3-D
reconstructions from thin
optical sections.
Can only image a single,
thin focal plane at a time.
Tissue culture cells
and tissues.
Microscopy Chapter|5 141
Epifluorescent Specimen is illuminated
by excitation wavelengths
and emits light from
excited fluorophores
throughout the entire
thickness.
(Advantages of all forms of
fluorescent microscopy)
Can be used to detect
specifically labeled
fluorescent molecules.
Multiple fluorophores can
be imaged in same sample.
High signal-to-noise ratio.
Sensitive.
(Advantage of
epifluorescence over
other forms)
Simpler and faster image
collection methods than
other forms of fluorescent
microscopy (confocal,
two-photon).
Out-of-focus fluorescence
can cause blurry pictures,
making structures difficult to
resolve.
Thin physical sections of
fluorescently stained tissue
or cells.
Time-lapse fluorescence
imaging in cells.
Confocal Out-of-focus illumination
is blocked by the use of
pinhole apertures, so only
in-focus emitted light is
collected.
Optical sectioning ability
creates sharp, in-focus
images.
Intense laser illumination
can cause photobleaching
and phototoxicity.
Long scan time.
Detecting fluorescence
in thick tissues or small
organisms (e.g., Drosophila).
Time-lapse fluorescence
imaging in thicker tissues
(e.g., slice cultures) or
for small structures
(e.g., synaptic vesicles).
Two-photon Fluorophores in a thin
focal plane are selectively
excited by effectively
absorbing the combined
energy of two photons that
cannot excite fluorophores
on their own.
Longer wavelengths of laser
illumination allow deeper
penetration of fluorescence
excitation.
Reduced photobleaching
and phototoxicity.
Expensive equipment. In vivo imaging of intact
organisms.
Long-term fluorescence
imaging.
Continued
142 GuidetoResearchTechniquesinNeuroscience
Total internal
reflection
fluorescence
(TIRF)
An evanescent wave
of illumination rapidly
decays after passing
the interface between a
cell membrane and the
surface it is on.
Images a thin optical section.
Eliminates out-of-focus
fluorescence from regions
more than 100 nm away
from the surface.
High resolution.
Only fluorophores at the
interface between the
membrane surface and
the glass coverslip are
illuminated; cannot image
an arbitrary region within
the cell.
Imaging protein dynamics
at the plasma membrane in
live cells.
Transmission
electron
microscopy
(TEM)
Electron beams
transmitted through
ultrathin sections.
Nanometer resolution. Cannot be used on live cells.
Harsh processing conditions
can cause artifacts.
Requires specialized
equipment.
Ultrastructure of cells.
Synapse structure.
Scanning
electron
microscopy
Detect secondary
electrons scattered off the
surface of the sample.
Provides 3-D
topological information.
Cannot be used on live cells.
Harsh processing conditions
can cause artifacts.
Requires specialized
equipment.
Topography of cells
and tissues.
Electron
tomography
Rotate specimen to take
TEM images from multiple
perspectives and create
3-D reconstruction.
Provides 3-D
organization information.
Cannot be used on live cells.
Requires intensive
computation to reconstruct
TEM views.
3-D cellular ultrastructure
and organization.
TABLE 5.1  Comparison of Different Forms of Microscopy—cont’d
Description Advantages Disadvantages
Common Use in
Neuroscience
Microscopy Chapter | 5  143
SUGGESTED READING AND REFERENCES
Books
Bradbury, S., Bracegirdle, B., & Royal Microscopical Society (Great Britain). (1998). Introduction
to light microscopy. Oxford: Bios Scientific Publishers, Springer.
Chandler, D. E., & Roberson, R. W. (2009). Bioimaging: Current concepts in light and electron
microscopy. Sudbury, MA: Jones and Bartlett Publishers.
Herman, B. (1998). Fluorescence microscopy (2nd ed.). Abingdon, UK; New York: Taylor &
Francis in association with the Royal Microscopical Society.
Murphy, D. B. (2012). Fundamentals of light microscopy (2nd ed.). Hoboken, NJ: John Wiley &
Sons, Inc.
Pawley, J. B. (2006). Handbook of biological confocal microscopy (3rd ed.). NY: Springer.
Russ, J. C. (2011). The image processing handbook (6th ed.). Boca Raton, FL: CRC Press.
Review Articles
Brown, C. M. (2007). Fluorescence microscopy—avoiding the pitfalls. Journal of Cell Science,
120, 1703–1705.
Kapitza, H. G. (1994). Microscopy from the very beginning (2nd revised ed.). Oberkochen,
Germany: Carl Zeiss.
McEwen, B. F., & Marko, M. (2001). The emergence of electron tomography as an important
tool for investigating cellular ultrastructure. Journal of Histochemistry and Cytochemistry,
49, 553–564.
Mancuso, J. J., Chen, Y., Li, X., Xue, Z., & Wong, S. T. (2013). Methods of dendritic spine detection:
from Golgi to high-resolution optical imaging. Neuroscience, 251, 129–140.
Osten, P., & Margrie, T. W. (2013). Mapping brain circuitry with a light microscope. Nature
Methods, 10, 515–523.
Pearson, H. (2007). The good, the bad and the ugly. Nature, 447, 138–140.
Saibil, H. R. (2007). How to read papers on three-dimensional structure determination by electron
microscopy. In D. Zuk (Ed.), Evaluating techniques in biochemical research. Cambridge, MA:
Cell Press.
Schneckenburger, H. (2005). Total internal reflection fluorescence microscopy: technical innovations
and novel applications. Current Opinion in Biotechnology, 16, 13–18.
Waters, J. C., & Swedlow, J. R. (2007). Interpreting fluorescence microscopy images and measurements.
In D. Zuk (Ed.), Evaluating techniques in biochemical research. Cambridge, MA: Cell
Press. http://www.cellpress.com/misc/page?page5ETBR.
Willig, K. I., & Barrantes, F. J. (2014). Recent applications of superresolution microscopy in neurobiology.
Current Opinion in Chemical Biology, 20, 16–21.
Primary Research Articles—Interesting Examples from the Literature
Ahrens, M. B., Orger, M. B., Robson, D. N., Li, J. M., & Keller, P. J. (2013). Whole-brain
functional imaging at cellular resolution using light-sheet microscopy. Nature Methods,
10, 413–420.
Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M., & McMahan, U. J. (2001). The
architecture of active zone material at the frog’s neuromuscular junction. Nature, 409,
479–484.
Jung, J. C., Mehta, A. D., Aksay, E., Stepnoski, R., & Schnitzer, M. J. (2004). In vivo mammalian
brain imaging using one- and two-photon fluorescence microendoscopy. Journal of Neurophysiology,
92, 3121–3133.
144  Guide to Research Techniques in Neuroscience
Lu, H. E., MacGillavry, H. D., Frost, N. A., & Blanpied, T. A. (2014). Multiple spatial and kinetic
subpopulations of CaMKII in spines and dendrites as resolved by single-molecule tracking
PALM. Journal of Neuroscience, 34, 7600–7610.
Micheva, K. D., Busse, B., Weiler, N. C., O’Rourke, N., & Smith, S. J. (2010). Single-synapse
analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron,
68, 639–653.
Westphal, V., Rizzoli, S. O., Lauterbach, M. A., Kamin, D., Jahn, R., & Hell, S. W. (2008).
Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science, 320,
246–249.
Protocols
Coling, D., & Kachar, B. (2001). Theory and application of fluorescence microscopy. Current
Protocols in Neuroscience (Chapter 2), Unit 2.1. http://dx.doi.org/10.1002/0471142301.
ns0201s00.
Smith, C. L. (2011). Basic confocal microscopy. Current Protocols in Neuroscience (Chapter 2),
Unit 2.2. http://dx.doi.org/10.1002/0471142301.ns0202s56.
Spector, D. L., & Goldman, R. D. (2006). Basic methods in microscopy: Protocols and concepts
from cells: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Websites
Davidson, M. W. Molecular Expressions: http://micro.magnet.fsu.edu/.
Nikon Microscopy U: http://www.microscopyu.com.
Olympus Microscopy Resource Center: http://www.olympusmicro.com/.
Zeiss MicroImaging: http://www.zeiss.com/us/micro/home.nsf.
145
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00006-X
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 6
Visualizing Neural Structure
After Reading This Chapter, You Should be Able to:
	l	Explain the process of preparing neural tissue for histological procedures
	l	Describe methods for visualizing gross cellular morphology
	l	Describe methods for visualizing gene and protein expression
	l	Describe methods for visualizing neural circuitry and connections between different
brain regions
Techniques covered:
	l	Tissue preparation: fixation, embedding, sectioning
	l	Visualizing structure: fundamental histological methods (basophilic stains, fiber
stains), Golgi stain, intracellular/juxtacellular labeling
	l	Visualizing gene and protein expression: in situ hybridization, immunohistochemistry,
array tomography, enzymatic histochemistry, reporter genes
	l	Visualizing circuitry: anterograde tracers, retrograde tracers, transsynaptic tracers
A person can learn a lot about a complicated system simply by examining its
structure. For example, imagine a satellite image of a city taken from space. How
might you learn about what it would be like to live in the city just by examining
the city from that vantage point?You could start by separating the image into component
parts—for example, distinguishing buildings from roads. Larger buildings
clustered together could reveal popular hubs of activity, whereas smaller,
scattered buildings might indicate residential communities. You could learn even
more by identifying the different categories of buildings and classifying different
structures as schools, grocery stores, gas stations, shopping centers, and so forth.
Major highways and freeways would indicate routes for long-distance travel; narrower
roads would show local areas of transit. By simply examining the structure
of the city, one could form educated guesses about how the city functions.
Similarly, one can learn a lot about the nervous system simply by examining
its structure. Just like distinguishing between buildings and roads in a city,
a neuroscientist can begin to examine the brain by discriminating between cells
and fiber tracts. Cells, like buildings, can be classified into different structural
and functional groups, with each cell type expressing a unique combination of
genes and proteins that specify its role in the brain. Thick white-matter tracts
show long-distance sites of neural communication, whereas smaller fiber tracts
146  Guide to Research Techniques in Neuroscience
show local communication networks between populations of neurons. Learning
about the structure of distinct brain regions can lead to educated hypotheses
about the function of different neurons and how they influence neural circuits
and behavior. Indeed, in the early twentieth century, Santiago Ramón y Cajal
formed many hypotheses (many of which turned out to be correct) about the
function of neural systems simply by examining their structures.
The goal of this chapter is to survey techniques used to investigate the structure
and connectivity of the nervous system. Using these techniques, it is possible
to classify cells based on location, morphology, gene/protein expression
profiles, and connections with other cells. These methods can be used in combination
with techniques described in Chapter 7 to investigate neural function.
TISSUE PREPARATION
Neural tissue is soft, delicate, and degradable. Therefore, to accurately study
neural structure, an investigator’s first goal is to keep the tissue as close to its
living state as possible. Investigators process tissue by (1) fixing, (2) embedding,
and (3) sectioning before proceeding with visualization methods. Fixing
and embedding methods stabilize tissue to capture its current state, while sectioning
makes the tissue thin enough for light to pass through so that internal
structures can be examined under a microscope.
Fixation
Fixation is the process of using chemical methods to preserve, stabilize, and
strengthen a biological specimen for subsequent histological procedures and
microscopic analysis. This process preserves cells and tissues by strengthening
molecular interactions, disabling endogenous proteolytic enzymes, and killing
microorganisms that might otherwise degrade the specimen. Thus, fixation terminates
any ongoing biochemical reactions by “fixing” proteins into place—a
process that kills cells that are not already dead.
There are two different categories of chemical fixatives: cross-linking fixatives
and dehydrating fixatives. Which fixative an investigator chooses to use depends on
the type of subsequent histology to be performed. Cross-linking fixatives create
covalent chemical bonds between proteins in tissue and include organic compounds
with aldehyde groups, such as formaldehyde, paraformaldehyde, and glutaraldehyde.
These fixatives are good at preserving structure and are often used when
processing cells and tissues for light microscopy. Another cross-linking fixative,
osmium tetroxide, causes molecules to oxidize and is often used as a secondary fixative
for electron microscopy. Dehydrating fixatives disrupt lipids and reduce the
solubility of protein molecules, precipitating them out of the cytoplasmic and extracellular
solutions. Common dehydrating fixatives include methanol and acetone.
A scientist can fix cells in culture simply by adding a chemical fixative
to the culture chamber. Whole brains can be fixed either by immersion or
Visualizing Neural Structure Chapter | 6  147
perfusion. Immersion refers to placing small brains, or even entire animals, in
fixative solutions. The amount of time necessary for the fixative to penetrate
the entire tissue is variable, depending on the size of the brain: a fly brain
may need to be fixed for only a few minutes, while a mouse brain may require
days. Perfusion is the process of delivering a fixative through an animal’s
cardiovascular system. To perfuse an animal, a scientist anesthetizes the animal,
carefully opens the rib cage to expose the heart, and then penetrates the
left ventricle with a needle connected to a perfusion pump. The left ventricle
pumps blood throughout the entire body and can therefore deliver a fixative
to the nervous system. A scientist initially pumps a buffered saline solution to
rinse the blood out of the veins and arteries, and then pumps a cross-linking
reagent to fix all the tissues in the body. Perfusion fixation is thorough, quick
(can be performed in 5–15 min, depending on the animal), and adequately fixes
all cells in the brain if properly performed.
Some histological procedures do not require fixation or even require that
a brain not be fixed. For example, the use of radioactively labeled ligands to
localize neurotransmitter receptor-binding sites depends on those sites remaining
intact, and fixation could disrupt their structure. Each histological method
determines whether fixation is necessary, which fixatives to use, and when the
fixation process should occur.
Embedding
Embedding is the process of surrounding a brain or tissue section with a substance
that infiltrates and forms a hard shell around the tissue. This process
stabilizes the tissue’s structure and makes it easier to cut. Standard embedding
materials include gelatin, paraffin wax, and plastic.
Sectioning
Sectioning is the process of cutting a brain into thin slices for subsequent histological
procedures or microscopic examination. This process is almost always
necessary to examine the structure of neurons within the brains of vertebrate
model organisms. Sectioning the brain allows histological reagents access to
cells within the slice, as well as allows a scientist to examine brain structures
with a microscope. There are three common machines used to section tissue
(Figure 6.1, Table 6.1):
	l	A microtome (micro =  “small,” tome = “cut”) is an instrument used to section
tissue using a retractable blade. A specialized microtome called a freezing
microtome is commonly used to section frozen tissue. Before cutting,
the tissue must be cryoprotected by soaking in a sucrose solution. This minimizes
artifacts caused by freezing. Using a freezing microtome, a scientist
can collect 25- to 100-μm sections off the blade for subsequent histochemical
processing. These sections are usually stored in a buffered saline solution
148  Guide to Research Techniques in Neuroscience
until use. Other types of microtomes are used to section paraffin-embedded
tissue or to create the ultrathin sections required for electron microscopy.
	l	A cryostat is essentially a microtome housed in a freezing chamber that
allows sectioning at temperatures of −20 to −30 °C. Maintaining the tissue and
blade at low temperatures allows a scientist to section nonfixed, frozen tissue.
Cryostats are able to cut thin, 10- to 50-μm sections. A scientist can mount
sections onto slides within the cryostat or store sections in a buffered saline
solution until use.
	l	A vibratome cuts tissue with a vibrating knife, somewhat similar to an electric
vibrating toothbrush. The main function of a vibratome is to section tissues that
are not frozen. The knife vibrates side to side so quickly that a soft brain can be
cut into 100- to 400-μm sections. Thus, vibratomes are useful for avoiding artifacts,
changes in morphology, or interruption of biochemical activities caused
by freezing. A vibratome is also necessary for experiments in which a slice of
brain tissue must be kept alive, as in tissue culture or electrophysiology studies.
FIGURE 6.1  Equipment used to section tissue.  (A)A microtome. (B)A cryostat. (C)A vibratome.
TABLE 6.1  Comparison of Different Brain Sectioning Methods
Essential Features
Standard Section
Thickness
Microtome Brains are frozen
Preserves structures better than cryostat
Medium: 25–100 μm
Cryostat Brains are frozen
Sections are cut in a cold chamber
Thin: 5–50 μm
Vibratome Brains do not need to be frozen
Vibrating knife
Can be used to cut tissue that will be
kept alive
Thick: 100–400 μm
Visualizing Neural Structure Chapter | 6  149
Once a brain is sectioned, a scientist can either mount the sections directly
onto slides or can keep the sections floating in buffered saline for subsequent
histological analysis.
Brains are typically cut in one of three standard orientations: coronal, sagittal,
or horizontal (Figure 6.2). The coronal plane divides the brain in the
anterior-to-posterior direction, revealing a section of the brain that is complete
from top-to-bottom and left-to-right (ear to ear). The sagittal plane shows
the complete top-to-bottom of the brain, cutting it into left and right portions.
A midsagittal cut is a section that perfectly divides the left and right hemispheres
of the brain. The horizontal plane divides the brain into superior (top)
and inferior (bottom) sections, such that an area of the brain is complete from
left-to-right and anterior-to-posterior.
There are some preparations for which brain sectioning is not necessary.
For example, scientists may wish to examine whole-mount preparations of
intact brains or animals. Also, the brains of Drosophila and other invertebrates
are usually small enough such that confocal or two-photon microscopy
(Chapter 5) can provide good images of individual neurons within neural
structures.
A new technique called clear lipid-exchanged acrylamide-hybridized rigid imaging/immunostaining/in
situ hybridization-compatible tissue hydrogel (CLARITY)
fixes tissue using a hydrogel to preserve the physical structure of proteins and nucleic
acids, but allows light-scattering lipids to be removed. This process effectively
makes intact tissue transparent and amenable to the structural visualization methods
FIGURE 6.2  Neuroanatomical planes. Standard orientations for sectioning brain tissue. (A)
Coronal. (B) Sagittal. (C) Horizontal/transverse.
150  Guide to Research Techniques in Neuroscience
described in this chapter. Therefore, CLARITY allows for visualization of nervous
system structure without the need to section the tissue.
Now that we have discussed some common methods of preparing neural tissue
for histological analysis, we describe fundamental methods of visualizing
the gross morphology of the brain.
VISUALIZING MORPHOLOGY
Although lipids, proteins, carbohydrates, and other organic molecules provide
the nervous system with its structure and durability, more than three-quarters
of the mass of the brain is water. This composition imparts significant consequences
for any scientist interested in studying the structure of the brain—
most notably that unprocessed brain sections are almost completely transparent.
Unless treated by histochemical stains, the structure of the nervous system will
remain invisible and appear as a wafer-thin slice of gelatin mounted on a glass
slide. Treating brain sections with various dyes enhances the contrast between
different brain structures and allows an investigator to visualize cells, fibers, or
other features of neural systems (Table 6.2). These methods each provide contrast
to specific structures in the brain so that they stand out from other aqueous
structures in a neural specimen.
Cell Body Stains
A basophilic stain is used to visualize cell bodies. The term basophilic is
derived from baso-, meaning “base” and -philic, meaning “loving” and conveys
the fact that these stains are basic (pH > 8) and good for staining acidic
molecules. As DNA and RNA molecules are acidic, basophilic dyes label
structures enriched in nucleic acids, such as cell nuclei and ribosomes. A scientist
can use these stains to examine the cytoarchitecture of individual neurons
with high magnification or to visualize the macroscopic features of distinct
brain regions, such as layers of cerebral cortex or subdivisions of hypothalamic
nuclei (Figure 6.3A).
Common basophilic stains used in bright field microscopy include hematoxylin
and thionine. A special category of basophilic stains, referred to as Nissl
stains, specifically label RNA within cells, providing contrast to ribosomes and
rough endoplasmic reticulum, which are enriched in neurons. Cresyl violet is
one of the most commonly used Nissl stains. Redistribution of Nissl-stained
structures in injured or regenerating neurons allows Nissl stains to reveal the
physiological state of some neurons.
In fluorescent microscopy, common markers of cell nuclei include DAPI
(4’,6-diamidino-2-phenylindole), Hoechst (bis-benzamide), and propidium
iodide (PI). These dyes intercalate within the helical spirals of DNA in the cell
nucleus. Both DAPI and Hoechst emit blue light when excited by ultraviolet
(UV) light, whereas PI emits red light when excited by green light. PI is useful
Visualizing Neural Structure Chapter | 6  151
TABLE 6.2  Histochemical Stains for Studying Neuroanatomy
Stain Use Appearance Comments
Cresyl violet Cell nuclei,
Nissl stain
Blue to purple Useful for examining
cytoarchitecture; stains
each type of neuron
slightly differently
Hematoxylin Cell nuclei Blue to blue-black Often used in combination
with eosin; known as H&E
Eosin Y Cytoplasm Pink to red Counterstain with
hematoxylin; acidophilic
stain
Thionine Cell nuclei,
Nissl stain
Blue to purple
Methylene
blue
Cell nuclei Blue Can be perfused through
the brain before fixation
Toluidine blue Cell nuclei Nucleus is stained
blue; cytoplasm
light blue
Often used to stain frozen
sections
DAPI Cell nuclei Fluorescent blue Fluorescent DNA
intercalating agent; excited
by UV illumination
Hoechst
(bis-benzamide)
Cell nuclei Fluorescent blue Fluorescent DNA
intercalating agent; excited
by UV illumination
Propidium
iodide (PI)
Cell nuclei Fluorescent red Fluorescent DNA
intercalating agent; excited
by green light illumination
Weigert Myelin Normal myelin
is deep blue;
degenerated
myelin is light
yellow
Combines hematoxylin
with other chemicals to
selectively stain myelin
Weil Myelin Black Combines hematoxylin
with other chemicals to
selectively stain myelin
Luxol fast blue
(LFB)
Myelin Blue
Golgi stain Fills
neuron cell
bodies and
processes
Black Stains individual neurons
at random
152  Guide to Research Techniques in Neuroscience
in assays of cell death because it is membrane impermeant; therefore, it labels
dead but not healthy cells.
Fiber Stains
Fiber stains label white-matter tracts by staining myelin, the fatty substance
that insulates and provides electrical resistance to axons. These stains are
useful for visualizing the major fiber tracts that project throughout the brain
(Figure 6.3B). However, the high density of fibers in most brain regions
makes it impossible to trace individual axons from cell body to synapse.
When used in combination with basophilic stains in adjacent tissues sections,
a scientist can identify more anatomical structures than if either stain
is used alone.
There are various protocols useful for labeling myelin, such as the Weigert
or Weil methods, which stain myelin dark blue or black. A scientist can also
stain myelin using lipophilic fluorescent stains.
Golgi Stain
The Golgi stain is a classic technique used to completely label individual neurons
and their processes (Box 6.1). There are two major advantages to using
a Golgi stain: (1) neurons are labeled in their entirety, including cell bodies,
processes, and even microscopic structures such as dendritic spines; (2) only
5–10% of the total number of cells are labeled. Therefore, individual neurons
stand out in a background of numerous unlabeled cells (Figure 6.4). A scientist
cannot control which neurons in a tissue slice become labeled, but newer techniques
allow a scientist to duplicate the effects of a Golgi stain in a genetically
defined population of neurons (Chapter 12). However, this method is still used
to define and trace the elaborate morphology of neurons, especially in neurodegeneration
studies.
(A) (B)
FIGURE 6.3  Comparison of basophilic and fiber stains.  (A) Cresyl violet Nissl stain of a coronal
mouse brain section. (Reprinted with permission from Rosen, G.D., et al. (2000). The Mouse
Brain Library. International Mouse Genome Conference 14:166.) (B) Coronal section stained for
myelin. (Courtesy of Dr Pushkar Joshi.)
Visualizing Neural Structure Chapter | 6  153
Intracellular and Juxtacellular Labeling
The preceding methods label cell bodies and/or axons after a brain has already
been fixed and sectioned. It is also possible to label individual neurons during
an experiment for later histological analysis so that a neuron’s activity or function
can be coupled to the neuron’s structure and location in the brain. During
electrophysiology experiments, a scientist can use a glass pipette to fill a neuron
of interest with a chemical that can later be visualized using histological
methods. This chemical is usually a variant of a molecule called biotin, such as
biocytin or neurobiotin, which has properties that make it useful for subsequent
histology (Box 6.2). A scientist can intracellularly label cells in vivo or in slices.
During an extracellular recording session, the electrode tip can be apposed to
the neuron’s membrane for juxtacellular labeling. This labeling can completely
fill a neuron’s dendrites or axonal arborizations.
BOX 6.1  Historical Use of the Golgi Stain
The Golgi stain has an important place in the history of neuroscience. In the late
nineteenth and early twentieth centuries, Santiago Ramón y Cajal used this technique
for his seminal studies of the cellular morphology of the nervous system.
Among his many achievements was the discovery that neurons are distinct, independent
cells that communicate via synapses. His work heavily contributed to
the acceptance of the neuron doctrine—the hypothesis that neurons are the basic
structural and functional units of the nervous system. Cajal also used the Golgi
stain to reveal the morphological diversity of neurons, as well as to trace neural
circuits in the retina, hippocampus, cerebellum, and other brain regions. In 1906,
Cajal shared the Nobel Prize with Camillo Golgi, the scientist who developed the
Golgi stain, for elucidating the structure of the nervous system.
FIGURE 6.4  The Golgi stain reveals neuronal structure in fine detail.  Random neurons are
selectively stained in the (A) 100x view of a Golgi-stained mouse cortex and (B) a higher magnification.
Courtesy of Dr Jocelyn Krey.
154  Guide to Research Techniques in Neuroscience
BOX 6.2  Methods of Labeling Molecular Probes
A probe, such as an antibody in immunohistochemistry (IHC) or antisense strand in
in situ hybridization (ISH), is not visible by itself. To be visualized, the probe must
be conjugated to a label that is usually visible on its own or that reacts with other
chemicals to create a visible product. The use of a particular label depends on a
number of factors, including the degree of specificity needed, whether other methods
will be used in combination, and what types of microscopes are available. This
box describes common types of labels (fluorescent, chromogenic, radioactive, and
colloidal gold) and how they can be detected.
Label Type Comments Visualization Method
Fluorescent dyes Green dyes: FITC, Alexa 488, Cy2 Fluorescence microscopy
Red dyes: Rhodamine, Texas red,
Alexa 594, Cy3
Infrared dyes: Cy5
Quantum dots Spectral selectivity based on size Fluorescence microscopy
Alkaline
phosphatase (AP)
NBT/BCIP substrate produces
blue/purple product
Usually light microscopy,
though fluorescent
substrates are available
Horseradish
peroxidase (HRP)
DAB substrate produces brown/
black product
Light or electron
microscopy of colored
DAB product
β-galactosidase
(encoded by
lacZ gene)
X-gal or IPTG substrate produces
blue/purple product
Usually light microscopy,
though fluorescent
substrates are available
Biotin Biotinylated antibodies or proteins
are detected using avidin conjugated
to another label described
(fluorescent, chromogenic, or gold)
Light, fluorescent, or
electron microscopy
depending on type of
label conjugated to
avidin
Digoxygenin Used to label ISH probes,
detected with antidigoxygenin
probe conjugated to another
label, frequently AP
Light or fluorescence
microscopy, depending
on substrate for label
Radioactive isotope
(35S,33P,3H,125I)
ISH probes:35S,33P,3HProteins:
3H,125I
Light microscopy
Gold Electron-dense label used for
detecting immunostaining in EM
Electron microscopy
Fluorescent Labels
Fluorescent molecules are ubiquitous in biological research. Fluorescent proteins,
such as GFP, can be genetically encoded and serve as reporter proteins. Other nonprotein
fluorophores can be attached to a variety of molecular probes. Because
these fluorescent probes have different excitation and emission spectra (Chapter
Visualizing Neural Structure Chapter | 6  155
5), scientists can use multiple fluorescent labels to visualize distinct genes, proteins,
and structures in the same neural specimen.
There are a variety of fluorophores available for labeling probes. Commonly
used fluorescent labels include the organic cyanine and Alexa fluor dyes, which
are commercially available and often used to label antibodies. Quantum dots are
semiconductor nanocrystals that are extremely bright and resistant to photobleaching,
making them particularly useful for live-cell imaging over long time periods.
Fluorescence is a sensitive method of detection, as bound fluorophores will
emit light against a dark background. Attaching multiple fluorophores to a probe
can amplify the signal to enhance detection. However, this amplification also
makes it trickier to quantify exact levels of the probed substance because the
degree of amplification does not necessarily vary linearly with the amount of substance
initially present.
Fluorescently labeled structures can be made visible to light and electron
microscopes through a process called photoconversion. Fluorophores can photoconvert
the chemical diaminobenzidine (DAB), a substrate commonly used for
chromogenic enzymes. Exciting certain fluorophores with the appropriate wavelength
of light in the presence of DAB forms a dark-colored precipitate.
Chromogenic/Colorimetric Labels
Chromogenic or colorimetric labels are enzymes that react with a substrate to produce
a colored product that is visible under a light microscope.The most commonly
used combinations are alkaline phosphatase (AP) with the substrates NBT/BCIP to
produce a blue to purple stain; horseradish peroxidase (HRP) with the substrate DAB
to produce a brown to black stain; and β-galactosidase (genetically encoded by
lacZ) with X-gal or IPTG substrate to produce a blue to purple stain. Commercially
available substrates that produce fluorescent rather than colored products are also
available, though colorimetric substrates are more frequently used.
Although not a chromogenic label itself, biotin is commonly conjugated to
antibodies or incorporated into ISH oligonucleotide probes. The biotinylated probe
can then be detected using the biotin-specific binding partner avidin/streptavidin
conjugated to a chromogenic label such as AP or HRP. Avidin and biotin can
form large complexes and greatly enhance the signal compared to using a probe
alone. Digoxigenin (abbreviated dig, sounds like “dij”) is another common molecule
that is incorporated into ISH probes as an alternative to radioactive labels.
Antidigoxigenin antibodies conjugated to chromogenic enzymes are used to detect
the presence of the dig-labeled probe.
Radioactive Labels
Radioactive isotopes, or radioisotopes, have an unstable nucleus that randomly
disintegrates to produce a different atom. In the course of disintegration, these isotopes
emit either energetic subatomic particles, such as electrons, or radiation,
such as gamma rays. By using chemical synthesis to incorporate one or more radioactive
atoms into a small molecule of interest such as a sugar, amino acid, or neurotransmitter,
the fate of that molecule can be traced during any biological reaction.
BOX 6.2  Methods of Labeling Molecular Probes—cont’d
Continued
156  Guide to Research Techniques in Neuroscience
VISUALIZING GENE AND PROTEIN EXPRESSION
A scientist can classify a cell using its morphology, location in the brain, or its
gene and protein expression profile. The genes expressed by a neuron determine
whether it is excitatory or inhibitory, what kinds of neurotransmitters it releases
and responds to, its physiological characteristics, and many other functional
properties. Therefore, determining the genes and proteins expressed by a cell
provides vital clues to its role in the brain. Conversely, a scientist may identify
a gene or protein of interest, and then want to know where in the brain it is
expressed. The following methods provide tools an investigator can use to visualize
gene and protein expression in the nervous system.
In Situ Hybridization
In situ hybridization (ISH) is used to visualize the expression of nucleic acids,
usually mRNA, allowing a scientist to determine when and where a specific
gene is expressed in the nervous system. This technique is good for identifying
which neurons express a gene, although it does not reveal where the functional
protein product is expressed within the cell.
To perform an ISH experiment, a scientist must first identify the genetic
sequence of an mRNA to be studied. Next, the scientist creates a single-stranded
Radioisotopes are visualized using autoradiography. Specimens are coated with
a photographic emulsion and left in the dark while the radioisotope decays. Each
decay event causes a silver grain to precipitate in the emulsion, so the position of
the radioactive probe is indicated by the position of the developed silver grains.
Because it is a direct chemical reaction, each silver grain represents one decay event.
Therefore, radioactive labels allow quantitative studies of biological probes.
Primarily used to label ISH oligonucleotide probes, 35S and 33P are radioisotopes
incorporated into the probe sequence, allowing sensitive detection, even for
genes expressed at low levels. However, it can be tricky to calibrate the amount of
time needed for exposing the emulsion to get the right signal-to-noise ratio, and the
exposure may require weeks to months to see a signal.
3H and 125I are radioisotopes that are used to label proteins and nucleic acids to
investigate a number of processes, such as receptor localization, proliferation, and protein
trafficking. Radioactively labeled ligands added to a specimen can bind to active
receptors, allowing autoradiographic detection for the presence of those receptors. See
Chapter 7 for a description of autoradiography in proliferation and protein trafficking.
Gold Labels
Colloidal gold labels are conjugated to antibodies for probe detection in electron
microscopes (EM; Chapter 5). Gold provides an electron-dense material that can
be visualized as a dark spot in EM. Avidin/streptavidin can also be conjugated to
gold for detection of biotinylated probes in EM. Gold particles of different sizes can
be used to detect multiple targets in the same sample.
BOX 6.2  Methods of Labeling Molecular Probes—cont’d
Visualizing Neural Structure Chapter | 6  157
nucleic acid probe that has a complementary base-pair sequence. The probe
is tagged with radioactive nucleotides or other molecules that allow detection
(Box 6.2). A tissue sample is exposed to the tagged probes such that the complementary
strands can form a probe-mRNA duplex, marking the location of neurons
that produce the mRNA sequence (Figure 6.5). This tissue sample is almost
always a brain section, although it is possible to perform ISH on cultured cells
or even unsectioned, whole-mount brains and animals.
ISH techniques require specific control experiments. Because an investigator
uses a complementary (“antisense”) strand of mRNA as a probe, a common
negative control is a probe with the exact same (“sense”) sequence of the gene
of interest. This control probe should not produce any signal, as no hybridization
should occur. Another good control to ensure that the antisense strand of mRNA
is specific to the gene of interest is to perform additional experiments with other
antisense strands that hybridize to a different region of the same mRNA.
The Allen Brain Institute in Seattle has provided researchers with a searchable,
high-throughput collection of ISH images for every gene expressed in the
mouse brain. Using this online resource (www.brain-map.org), a scientist can
view a series of brain sections with an ISH reaction for a gene of interest. Alternatively,
a scientist can type in the name of a brain region and receive a list of
the most highly expressed genes in that area of the brain. This resource allows
scientists to quickly obtain information about gene expression throughout the
mouse brain without having to personally perform in situ reactions. The only
limitation to this resource is that it does not examine gene expression for conditions
other than baseline conditions, so gene expression could be different
depending on environmental stimuli.
FIGURE 6.5  In situ hybridization shows where a gene is expressed.  (A) Labeled oligonucleotide
probes are synthesized with a complementary sequence to a gene of interest so they hybridize
to the mRNA when applied to a brain section. (B) Example of an in situ hybridization reaction performed
on a sagittal mouse brain section. The probe was conjugated to digoxigenin and visualized
with a colorimetric reaction.
158  Guide to Research Techniques in Neuroscience
Although it is possible to use fluorescent molecules to label probes for ISH,
fluorescent in situ hybridization generally refers to a specific technique used
to examine the location of DNA within chromosomes rather than tissue. A scientist
can use this method to visualize chromosomal abnormalities, such as
missing, mutated, or dislocated genetic segments.
Immunohistochemistry
Immunohistochemistry (IHC; histo = “tissue”) is used to visualize the expression
of proteins. IHC applied to cells is often referred to as immunocytochemistry
(cyto = “cells”), and IHC using fluorescent reagents is referred to as immunofluorescence.
The root immuno- refers to the fact that these techniques depend on
antibodies to recognize and bind to specific proteins (antigens). By exploiting the
immune system of other animals, such as mice, sheep, rabbits, and goats, scientists
and commercial suppliers have generated a wide number of antibodies for many
proteins. Table 6.3 lists a few of the many antibodies that are commonly used in
neuroscience. See Chapter 15 for details about antibody production.
To perform an IHC experiment, a scientist incubates a sample with an antibody
that recognizes a specific antigen. This primary antibody, the antibody
that binds to the protein, can be conjugated directly with a fluorescent molecule
or chromogenic enzyme. This procedure is known as direct IHC (Figure 6.6A).
Alternatively, in indirect IHC, a scientist adds a secondary antibody that recognizes
the primary antibody (Figure 6.6B). Because multiple secondary antibodies
TABLE 6.3  Commonly Used Antibodies that Label Specific Neural Cell
Types
Cell Type Antibodies
Progenitors/radial glia Nestin; Pax6; RC2; vimentin; NF
(neurofilament)
Young neurons Doublecortin (DCX); NeuroD
Neurons Tuj1 (neuron-specific β-tubulin); NeuN
Dendrites MAP2
Axons Tau-1, L1, Tag-1
Synapses PSD95, synapsin
Neuronal subtypes GAD (GABAergic neurons); vGLUT
(glutamatergic); TH (dopaminergic); 5-HT
(serotonergic); AChE (cholinergic)
Glia GFAP (astrocytes); MBP
(oligodendrocytes, myelin); PLP
Oligodendrocyte progenitor cells (OPC) NG2, A2B5, O4 (late progenitor)
Visualizing Neural Structure Chapter | 6  159
can bind to the primary antibody, the signal is amplified. The secondary antibody
will only react with the primary antibody if it is directed against the correct immunoglobulin
molecule of the animal species in which the primary antibody was
raised. For example, if the primary antibody was produced in a rabbit, then the
secondary antibody must be antirabbit and produced in a different animal. The
secondary antibody is conjugated with a fluorescent molecule or chromogenic
enzyme that allows the scientist to visualize the expression of the protein.
It is critical that an investigator determines whether the result of an IHC
experiment is specific for the protein of interest. Using both a positive control
(a specimen known to contain the antigen of interest) and a negative control (a
specimen known not to contain the antigen of interest) is useful for determining
whether the antibody works properly. Another good control experiment for primary
antibody specificity is to preincubate the primary antibody with a known
solution of its antigen; this process should form antibody/antigen complexes
so there is no free antibody to bind to a sample. In indirect IHC, incubating the
specimen with the secondary antibody but no primary antibody is important to
test for nonspecific binding of the labeled secondary antibody.
While IHC is straightforward in theory, there are a number of steps that must
be optimized for IHC to work in practice. A scientist may need to adjust the
method of fixation, antibody concentration, length of time the antibody is incubated
with tissue, and accessibility to the antigen for good results. Each batch of
antibody is different, even antibodies directed against the same antigen. Therefore,
an investigator should optimize experimental conditions for each new antibody
before performing IHC experiments on multiple samples.
A technique called array tomography uses IHC on ultrathin serial sections to
improve spatial resolution and eliminate out-of-focus fluorescence during imaging.
This method allows investigators to create highly detailed three-dimensional
images of protein expression in the nervous system. An ultramicrotome is used
FIGURE 6.6  Immunohistochemistry (IHC). (A) In direct IHC, primary antibodies are conjugated
directly to a label that can be visualized. (B) In indirect IHC, primary antibodies attract
labeled secondary antibodies, further amplifying the signal.
160  Guide to Research Techniques in Neuroscience
to cut and collect ribbons of ultrathin (50–200 nm) serial sections on glass microscope
slides. A scientist can perform IHC to detect the presence of an antigen, but
then the antibody can be removed and another IHC reaction performed. As many
as 10 IHC reactions can be performed on the same section. Because the sections
are so thin, antibodies have greater access to antigens in the section. The serial
sections can then be reconstructed to provide high-resolution, three-dimensional
information about the spatial relationships of these proteins. In addition, the sections
can be used for electron microscopy, allowing an unprecedented investigation
of protein expression combined with powerful magnification and resolution.
Enzymatic Histochemistry
An enzyme is a biomolecule capable of catalyzing a biological reaction. These
proteins can be detected using IHC; however, another method to detect enzymes
is to use the enzyme’s endogenous activity to create a visible reaction product—
a process known as enymatic histochemistry. The scientist incubates a brain
section in a solution containing a chromogenic chemical capable of serving as
a substrate for the enzyme. A colored byproduct indicates the presence of the
enzyme. For example, endogenous acetylcholinesterase (the enzyme that breaks
down acetylcholine) is often used as a marker for acetylcholine in brain sections.
Another commonly detected enzyme is cytochrome oxidase, which aids
in visualizing metabolically active neurons, such as whisker barrels in rodent
sensory cortex (Figure 6.7).
Reporter Genes
A reporter gene is a nonendogenous gene encoding an enzyme or fluorescent
protein whose expression is controlled by a promoter for a separate gene of
interest. Therefore, it is possible to examine the spatial and temporal expression
of the gene by measuring the expression of the reporter. For example,
imagine that a scientist is interested in studying the expression pattern of
Gene X. The scientist can create a reporter construct (using recombinant DNA
technology methods described in Chapter 10) in which the reporter gene is
placed under the control of the promoter for Gene X. Alternatively, the scientist
can fuse the DNA sequence of the reporter with the DNA sequence for
Gene X and place both under the control of the promoter for Gene X. In either
case, the reporter gene provides the scientist the ability to study the spatial
and temporal expression of Gene X by measuring expression of the reporter
(Figure 6.8).
Common reporters include green fluorescent protein (GFP) and its derivatives,
lacZ (which encodes the enzyme β-galactosidase), and luciferase (the
firefly protein). The expression pattern of reporter genes can reveal information
about the gene on multiple levels, from subcellular localization within a neuron
to patterns of organization in a whole animal.
Visualizing Neural Structure Chapter | 6  161
FIGURE 6.7  Enzymatic histochemistry. (A) In this example, rodent whisker barrels are
enriched in the enzyme cytochrome oxidase, so incubating sections with the substrate (cytochrome
c) and a colorimetric indicator (e.g., DAB) allows the whisker barrels to be visualized. Courtesy of
Dr Pushkar Joshi.
FIGURE 6.8  Using reporter genes to examine gene expression patterns. (A) In this BAC
transgenic mouse line (Chapter 12), the promoter for neurotensin was used to drive expression of the
GFP reporter gene. This mouse allows for visualization of the specific neurons in which neurotensin
is normally expressed. (Reprinted with permission from The Gene Expression Nervous System
Atlas Project.) (B) Reporter genes can also be used to replace a gene when creating a knockout
animal (Chapter 13). Here, LacZ gene expression (shown by β-galactosidase staining) replaces the
gene Satb2, showing where Satb2 is normally expressed. Mutants have altered axonal projections
(arrowheads). (Reprinted from Alcamo E.A. et al. (2008) Satb2 regulates callosal projection neuron
identity in the developing cerebral cortex, Neuron 57(3): 364–377, with permission from Elsevier.)
162  Guide to Research Techniques in Neuroscience
VISUALIZING CIRCUITRY
So far, this chapter has reviewed methods of investigating cells based on their
morphology, location, and gene/protein expression profiles. Of course, a neuron
never functions in isolation; its role in the brain is governed by its unique
combination of inputs and outputs and the neural circuits to which it belongs.
Therefore, scientists often want to know the specific cells in the brain that provide
synaptic input to a neuron of interest, as well as the specific cells to which
a neuron projects. As just described, a Golgi stain can provide initial insight into
the connections formed by neurons, but this method is limited by the random
staining pattern of cells. Other methods allow scientists to more specifically
investigate a neuron’s direct and indirect connections with other neurons in the
nervous system.
Anterograde and Retrograde Tracers
A neuronal tracer is a chemical probe that labels axon paths to illuminate connectivity
in the nervous system. Tracers are described by the direction they travel in
a neuron. Anterograde refers to transport from the cell body through the axon to
the presynaptic terminal; retrograde refers to tracing in the opposite direction—
transport from the synaptic terminal back to the cell body (Figure 6.9). Some
tracers are strictly capable of only anterograde or retrograde transport. Others
work in both directions. Classical tracers include horseradish peroxidase, biotinylated
dextran amine, diamidino yellow, Fluoro-Gold, and fast blue (Table 6.4).
These tracers may be fluorescent or produce a colorimetric product that can
be visualized using light microscopy (Box 6.2). Many scientists use viruses to
deliver a genetically encoded fluorescent protein, such as GFP, to a brain region
of interest for use as an anterograde tracer. The fluorescent protein fills the soma,
but also diffuses down the length of an axon to show anterograde targets.
To use an anterograde or retrograde tracing technique, a scientist almost
always injects the tracer into the brain of a live animal. After a waiting period
(1–7 days), the brain can be removed, processed, and examined for presence
FIGURE 6.9  Anterograde and retrograde tracers.  (A) Anterograde tracers show efferent projections,
revealing regions that receive projections from cells in the labeled area. (B) Retrograde
tracers show afferent projections, indicating regions that project to the labeled area.
Visualizing Neural Structure Chapter | 6  163
TABLE 6.4  Classical Anterograde and Retrograde Tracers
Tracer Direction Comments
Horseradish
peroxidase (HRP)
Retrograde Produces brown precipitate after
reaction with hydrogen peroxide and
DAB (diaminobenzidine)
Fluorescent
microspheres
Retrograde Available in many different colors;
nontoxic
Fluoro-gold Retrograde Widely used, rapid labeling
Diamidino yellow Retrograde Produces yellow fluorescence
Fast blue Retrograde Stable, rapid labeling; produces blue
fluorescence
Cholera toxin,
subunit B (CTB)
Retrograde May also be anterograde
DiI, DiO Anterograde and
retrograde
Lipophilic dye crystals
Biotinylated dextran
amine (BDA)
Anterograde and
retrograde
Widely used; direction of transport
depends on molecular weight and pH;
can be visualized by EM
Phaseolus vulgaris-
leucoagglutinin
(PHA-L)
Anterograde Plant lectin; can be visualized by EM
Tritiated amino
acids (3H-proline,
3H-leucine)
Transsynaptic
(anterograde)
Detected using autoradiography
Wheat germ
agglutinin (WGA)
Transsynaptic Plant lectin; anterograde and
retrograde transport possible; often
conjugated to HRP for detection;
transgene encoding WGA can be used
to label genetically defined neural
circuits
Tetanus toxin,
fragment C (TTC)
Transsynaptic
(retrograde)
Transgene encoding TTC can be used
to label genetically defined neural
circuits; nontoxic fragment
Pseudorabies virus
(PRV)
Transsynaptic Does not infect primates, including
humans; bartha strain most commonly
used for tracing studies; less virulent,
only retrograde transport
Herpes simplex virus
(HSV)
Transsynaptic Broad host range, including humans
164  Guide to Research Techniques in Neuroscience
of the tracer. An anterograde tracer should be present in the cell bodies and
presynaptic terminals of the injected neurons, highlighting areas to which those
neurons project. A retrograde tracer should be present in the cell bodies of neurons
that project to the injection site.
Tracing experiments are often combined with other techniques, such as
electrophysiology or IHC. For example, an investigator might wish to know
which cells in the brain send synaptic input to a neuron immediately following
an in vivo electrophysiology recording. The scientist could inject a retrograde
tracer, such as horseradish peroxidase, wait 3–7 days for transport to take place,
and then perfuse the animal. By performing histology and surveying brain sections
for labeled cells, the scientist could identify which cells projected to the
cell(s) at the recording site.
Transsynaptic Labeling
Traditional anterograde or retrograde tracing experiments label only direct connections
in a neural circuit. However, functional brain circuits are not simply
composed of direct one-to-one connections, but also contain complicated pathways
involving second- or third-order neurons. Scientists wishing to study these
multisynaptic circuits can use a variety of transsynaptic tracers—tracers that
can cross synapses and label multiple neurons in a circuit.
Radioactive Amino Acids
Radioactive amino acids, such as 3H-proline or 3H-leucine (3H is pronounced
“tritiated”), can be used for anterograde transsynaptic labeling. A scientist
injects the amino acids into the local environment of the primary neurons, some
of which are taken up by cells via amino acid transporters. Once inside the cell,
they are incorporated into proteins, some of which are shuttled down axons by
endogenous anterograde transport. Some of these proteins are secreted at the
synaptic terminal and received by a second-order neuron. This process may
repeat itself so that a third-order neuron is labeled. The labeled tracts can be
detected using autoradiography (Box 6.2).
Plant Lectins
Lectins are proteins that exhibit extremely high binding affinities for specific
sugars. Some plant lectins have affinities for specific glycoproteins on neuronal
plasma membranes. After binding, they can be internalized and transported
within the neuron. Some examples of plant lectins that can also be
transported across synapses include wheat germ agglutinin and tetanus toxin
fragment C. These lectins can be detected using antibodies against the lectin
itself or by conjugation with a chromogenic enzyme (Box 6.2). In addition to
the standard method of injecting the tracer into an area of interest, researchers
can create transgenic animals (Chapter 12) that express transneuronal lectin
tracers under the control of neuron-specific promoters to examine cell-type
specific circuits.
Visualizing Neural Structure Chapter | 6  165
Viruses
The use of viruses as transsynaptic tracers exploits the ability of viruses (especially
viruses that naturally infect neural tissue) to invade neurons and then
produce infectious progeny. Some strains of virus release progeny capable of
crossing synaptic junctions and subsequently infecting downstream neurons in
a circuit. The virus acts as a self-amplifying tracer that progressively labels
neurons in a circuit as they become infected. The most common viruses used
to label transneuronal circuits are pseudorabies virus and herpes simplex virus.
The major limitation to using viruses as transsynaptic tracers is that these
viruses can actually kill neurons and lead to generalized infection in the animals.
The longer the animal is infected, the more likely that higher-order neurons
in a circuit will be labeled, but the greater the likelihood that the first- and
second-order neurons will die as the infection proceeds.
CONCLUSION
The organization and structure of the nervous system provide the first clues
about function and how particular structures or classes of cells can give rise to
behaviors. This chapter has surveyed methods of investigating the morphology,
expression patterns, and connectivity of cells. In the next chapter, we will survey
methods that visualize the electrical and biochemical activity within neurons.
SUGGESTED READING AND REFERENCES
Books
Blurry, R. W. (2010). Immunocytochemistry: A practical guide for biomedical research. New York,
NY: Springer.
Chandler, D. E., & Roberson, R. W. (2009). Bioimaging: Current concepts in light and electron
microscopy. Sudbury, MA: Jones and Bartlett Publishers.
Kiernan, J. A. (2008). Histological and histochemical methods: Theory and practice (4th ed.). Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Polak, J. M., & Van Noorden, S. (2003). Introduction to immunocytochemistry (3rd ed.). Oxford:
BIOS Scientific Publishers.
Yuste, R., & Konnerth, A. (2005). Imaging in neuroscience and development: A laboratory manual.
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Yuste, R., Lanni, F., & Konnerth, A. (2000). Imaging neurons. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press.
Review Articles
Enquist, L. W., & Card, J. P. (2003). Recent advances in the use of neurotropic viruses for circuit
analysis. Current Opinion in Neurobiology, 13, 603–606.
Jones, E. G. (2007). Neuroanatomy: cajal and after cajal. Brain Research Reviews, 55, 248–255.
Kobbert, C., Apps, R., Bechmann, I., Lanciego, J. L., Mey, J., & Thanos, S. (2000). Current concepts
in neuroanatomical tracing. Progress in Neurobiology, 62, 327–351.
Massoud, T. F., Singh,A., & Gambhir, S. S. (2008). Noninvasive molecular neuroimaging using reporter
genes: part I, principles revisited. AJNR American Journal of Neuroradiology, 29, 229–234.
166  Guide to Research Techniques in Neuroscience
Pollack, J. D.,Wu, D.Y., & Satterlee, J. S. (2014). Molecular neuroanatomy: a generation of progress.
Trends in Neurosciences, 37, 106–123.
Primary Research Articles—Interesting Examples from the Literature
Alcamo, E. A., Chirivella, L., Dautzenberg, M., Dobreva, G., Farinas, I., Grosschedl, R., et al.
(2008). Satb2 regulates callosal projection neuron identity in the developing cerebral cortex.
Neuron, 57, 364–377.
Braz, J. M., & Basbaum, A. I. (2008). Genetically expressed transneuronal tracer reveals direct
and indirect serotonergic descending control circuits. Journal of Comparative Neurology, 507,
1990–2003.
Chen, B., Schaevitz, L. R., & McConnell, S. K. (2005). Fezl regulates the differentiation and
axon targeting of layer 5 subcortical projection neurons in cerebral cortex. Proceedings of the
National Academy of Sciences of the U.S.A, 102, 17184–17189.
Chung, K., Wallace, J., Kim, S.-Y., Kalyanasundaram, S., Andalman, A. S., Davison, T. J., et al.
(2013). Structural and molecular interrogation of intact biological systems. Nature, 497, 332–337.
Fakhry, A., & Ji, S. (2014). High-resolution prediction of mouse brain connectivity using gene
expression patterns. Methods http://dx.doi.org/10.1016/j.ymeth.2014.07.011.
Faucherre, A., & Lopez-Schier, H. (2014). Dynamic neuroanatomy at subcellular resolution in the
zebrafish. Methods in Molecular Biology, 1082, 187–195.
Micheva, K. D., & Smith, S. J. (2007). Array tomography: a new tool for imaging the molecular
architecture and ultrastructure of neural circuits. Neuron, 55, 25–36.
Takeuchi, A., Hamasaki, T., Litwack, E. D., & O’Leary, D. D. (2007). Novel IgCAM, MDGA1,
expressed in unique cortical area- and layer-specific patterns and transiently by distinct forebrain
populations of Cajal-Retzius neurons. Cerebral Cortex, 17, 1531–1541.
Protocols
Card, J. P., & Enquist, L. W. (2001). Transneuronal circuit analysis with pseudorabies viruses.
Current Protocols in Neuroscience (Chapter 1), Unit 1.5 http://dx.doi.org/10.1002/0471142301.
ns0105s09.
Gerfen, C. R. (2003). Basic neuroanatomical methods. Current Protocols in Neuroscience (Chapter
1), Unit 1.1 http://dx.doi.org/10.1002/0471142301.ns0101s23.
Helmchen, F., Konnerth, A., & Yuste, R. (Eds.). (2011). Imaging in neuroscience: A laboratory
manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Kay, K. R., Smith, C., Wright, A. K., Serrano-Pozo, A., Pooler, A. M., Koffie, R., et al. (2003).
Studying synapses in human brain with array tomography and electron microscopy. Nature
Protocols, 8, 1366–1380.
Tomer, R., Ye, L., Hsueh, B., & Deisseroth, K. (2014). Advanced CLARITY for rapid and highresolution
imaging of intact tissues. Nature Protocols, 9, 1682–1697.
Volpicelli-Daley, L. A., & Levey, A. (2004). Immunohistochemical localization of proteins in
the nervous system. Current Protocols in Neuroscience (Chapter 1), Unit 1.2 http://dx.doi.
org/10.1002/0471142301.ns0102s25.
Wilson, C. J., & Sachdev, R. N. (2004). Intracellular and juxtacellular staining with biocytin.
CurrentProtocolsinNeuroscience(Chapter1),Unit1.12http://dx.doi.org/10.1002/0471142301.
ns0112s26.
Websites
IHC World: http://www.ihcworld.com.
The Antibody Resource: http://www.antibodyresource.com.
Allen Brain Atlas: http://www.brain-map.org.
Mouse Brain Architecture: http://brainarchitecture.org.
167
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00007-1
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 7
Visualizing Neural Function
After Reading This Chapter, You Should be Able to:
	l	Describe techniques for visualizing activity and function in fixed tissue
	l	Describe nonelectrophysiological methods of measuring neural activity
	l	Describe techniques for visualizing protein function
Techniques Covered:
	l	Static measures of activity and function: measuring neural activity using immediate
early genes, measuring cell proliferation with thymidine analogs, measuring protein
trafficking with pulse-chase labeling
	l	Visualizing dynamic neural activity: voltage sensors (voltage-sensitive dyes,
genetically encoded voltage indicators), calcium sensors (calcium-sensitive dyes,
genetically encoded calcium indicators), synaptic transmission sensors (FM dyes,
synaptopHluorin)
	l	Visualizing protein function: reporter genes, fluorescence resonance energy
transfer (FRET), bimolecular fluorescence complementation (BiFC), fluorescence
recovery after photobleaching (FRAP), photoactivation/photoconverstion
Photographs in textbooks and other publications can sometimes create the impression
that an individual cell is a small, static blob—a microscopic collection of
organelles in a cytoplasmic soup that sits silently within a tissue. These individual
snapshots are extremely misleading. The living cell is a dynamic wonder—an
exquisitely complicated machine full of activity that a scientist cannot see with
the naked eye, especially not in a single photograph. A neuron can fire electrical
impulses in a steady tonic rhythm or phasic bursts, depending on the activity
in other cells. Genes are constantly transcribed, with mRNA shuttling out of
the nucleus and ribosomes quick to translate genetic sequences into functional
proteins. Many proteins dash about the cell, changing their function and activity
depending on the other proteins they encounter and the cellular signals transduced
from the plasma membrane.
Methods to visualize the structure and localization of cells and proteins, as
described in the last chapter, offer a good introduction to studying the activity
that takes place in a neuron. However, these methods do not fully reveal
the dynamic electrical and biochemical activity found within the cell. As a
168  Guide to Research Techniques in Neuroscience
metaphor, pretend you know nothing about soccer (or football if you are outside
the United States). To learn more about the sport, you find several photographs
of people playing soccer, mostly photographs taken from a bird’s-eye view of
the field. While these photographs would no doubt teach you some aspects of
the game, the only way to determine the rules, how individual players move, and
how the players work as a team is to watch movies of the game. The same is true
for biological systems; while photographs reveal the structure and localization
of proteins and cells at a snapshot in time, a scientist can learn much more by
observing the dynamic events that take place within neurons.
The purpose of this chapter is to survey methods used to visualize electrical
and biochemical activity in neurons. We begin with a description of how
we can visualize functional processes in fixed tissue. Next, we will survey the
major methods of characterizing electrophysiological properties of neurons
using optical methods. Finally, we survey the growing collection of methods
that can be used to visualize and manipulate biochemical dynamics. Many of
these methods are based on the use of fluorescent dyes and proteins that make
specific components of living cells visible under the microscope.
STATIC MARKERS OF ACTIVITY
Although it is ideal to measure neural activity as it occurs, it is not always
possible or practical. Some cellular functions can be examined through the
use of activity markers in fixed, histological brain sections. There are two
fundamental approaches to examining neural activity after it has already
occurred: (1) measuring activity indirectly by measuring byproducts that
accumulate during specific processes in active neurons and (2) incorporating
a marker into cells that can indicate the presence of activity during
subsequent histological examination. In both methods, a scientist examines
a snapshot of activity, a fixed representation of one moment in a dynamic
process.
Assaying Neural Activity in Fixed Tissue
Neural activity leads to the transient and rapid (within minutes) transcription of a
group of genes known as immediate early genes (IEGs). These genes encode a
diverse range of proteins, including transcription factors (Fos), cytoskeletal-interacting
proteins (Arc), and phosphorylated ribosomal subunits (pS6). By using histological
methods to stain for these markers, a scientist can indirectly determine which
regions were active just prior to the time the brain was fixed (Figure 7.1).A scientist
can use in situ hybridization (ISH) to show expression of the IEGs or immunohistochemistry
(IHC) to show expression of their protein products (Chapter 6).
Because IEG expression patterns and levels vary due to a number of uncontrollable
factors, investigators cannot examine IEGs as stand-alone indicators of
activity. The most reliable evidence compares IEG expression between a group
Visualizing Neural Function Chapter | 7  169
of experimental animals and a control group—for example, demonstrating that
there is significantly higher expression of IEGs in a specific neuron population
in animals that received a stimulus compared to control animals that did not
receive the stimulus. IEG patterns can also be used to screen neurons for the
presence of activities that correlate with specific behaviors. For example, some
brain regions thought to promote sleep states in the brain were discovered by
examining IEGs in animals that were asleep prior to fixation. In these types of
studies, IEGs only provide the first clue as to the function of a brain region. It is
necessary to follow up these studies with other, more reliable measurements of
neural activity to obtain meaningful results.
Assaying Cellular Function in Fixed Tissue
To measure some functional processes in fixed tissue, a scientist can introduce
a marker while the process occurs in live tissue, then detect this marker in subsequent
histological experiments. Two biological functions commonly assayed
using these methods include cell proliferation and protein trafficking.
Assaying Cell Proliferation with Thymidine Analogs
As a cell divides, it progresses through different stages of the cell cycle. Cells in
the DNA synthesis phase of mitosis can be marked by exposing them to tagged
DNA base-pair analogs. BrdU (bromo-deoxyuridine), a synthetic analog of the
DNA base thymidine, or radioactive tritiated thymidine (3H-thymidine), can be
injected into an animal or introduced into cells or tissue in culture media. Cells
that are synthesizing DNA will incorporate BrdU or 3H-thymidine into their
(A) (B)
FIGURE 7.1  Measuring activity in a fixed brain sample.  Overall activity levels can be detected
in fixed tissue by labeling for IEGs using immunohistochemistry or in situ hybridization techniques.
Here, Fos immunoreactivity demonstrates that patterned visual stimulation evokes more activity
in the (A) contralateral visual cortex compared to the (B) ipsilateral visual cortex. Reprinted from
Dotigny, F., et al. (2008). Neuromodulatory role of acetylcholine in visually induced cortical activation:
behavioral and neuroanatomical correlates. Neuroscience, 154(4), 1607–1618, with permission
from Elsevier.
170  Guide to Research Techniques in Neuroscience
newly made DNA in place of endogenous thymidine molecules. In subsequent
histology experiments, BrdU can be detected using IHC, and 3H-thymidine can
be detected using autoradiography.
The presence of BrdU or 3H-thymidine indicates that a cell was dividing
around the time of injection. However, these markers do not indicate whether
the cell continued to proliferate or stopped dividing to become a functional, differentiated
cell. To answer these questions, a scientist performs additional IHC
experiments for proteins that are present only during specific stages of the cell
cycle or in certain types of cells. For example, Ki67 and PCNA are proteins that
are present during the active, proliferating stages of the cell cycle but not during
the resting stage. Therefore, investigators can compare the number of proliferating
cells that differentiated into postmitotic cells to the number of proliferating
cells that continued to divide by combining BrdU/3H-thymidine pulses with
IHC for Ki67 (Figure 7.2).
Assaying Protein Trafficking with Pulse-Chase Labeling
Pulse-chase labeling is used to observe the movement of a substance through
a biochemical or cellular pathway. A labeled probe is injected into animals or
added to cultured cells for a brief period (the pulse) and then washed away and
replaced by unlabeled molecules (the chase). By following changes in the localization
of a marker in different subcellular compartments over time, a scientist
can observe protein trafficking pathways. For example, multiple proteins can
be pulsed at the same time with a radioactively labeled amino acid. By fixing
specimens at different time points, a scientist can use autoradiography to determine
the location of labeled probes at regular intervals to observe changes in
the localization of the incorporated label. Pulse-chase experiments have been
important in elucidating many biochemical pathways, including synthesis and
release of neurotransmitters.
FIGURE 7.2  Measuring cell proliferation
in a fixed brain sample. (A)
In this example, cells that were actively
dividing during the time of BrdU
injection are identified using immunohistochemistry
against BrdU. (B)
Immunohistochemistry against Ki67 in
the same sample identifies cells that were
actively proliferating prior to perfusion.
Courtesy of Dr Sandra Wilson.
BrdU Ki67
Visualizing Neural Function Chapter | 7  171
VISUALIZING NEURAL ACTIVITY
In Chapter 1, we discussed methods of visualizing activity in the whole brain
with a spatial resolution of millimeters (about 100,000 neurons) and a temporal
resolution of several seconds. In Chapter 4, we discussed electrophysiological
methods, which can record neural activity with the spatial resolution of a
single neuron (depending on the type of electrode and location in neural tissue)
and a temporal resolution of milliseconds. The methods of visualizing neural
activity described here combine advantages of both whole-brain imaging and
electrophysiology techniques: it is possible to visualize neural activity with
single-neuron resolution or in thousands of neurons at once while simultaneously
examining the spatial relationships of those neurons. The temporal resolution
is typically on the order of milliseconds to seconds, depending on the specific
technique. These methods can be used to visualize activity in dissociated cells in
culture, tissue slices in bath media, and even intact brains in living organisms.
Visualizing neural activity depends on specialized fluorescent probes that
report changes in membrane potential, calcium concentration, or synaptic vesicle
fusion. These probes can be organic dyes that a scientist adds to a neural
system prior to performing an experiment or a genetically encoded fluorescent
protein stably expressed in a transgenic animal. Dyes tend to exhibit better
temporal properties and signal-to-noise characteristics than proteins. However,
genetically encoded proteins can be targeted to specific cell types in the brain,
allowing a scientist to observe activity in molecularly defined circuits. Table 7.1
compares various methods of visualizing neural activity.
Imaging Voltage
Techniques that visualize changes in membrane potential are the closest analogs to
electrophysiological recordings, as they report voltage changes in neuronal membranes
with a fine temporal resolution. Voltage-sensitive dye imaging (VSDI)
is currently the primary method by which scientists visualize changes in voltage.
However, new genetically encoded voltage indicators (GEVIs) are now making
it possible to image the membrane potential in genetically defined cell types.
Voltage-Sensitive Dyes
Voltage-sensitive dyes shift their absorption or emission fluorescence based on
the membrane potential, allowing a scientist to gauge the global electrical state
of a neuron. Unlike extracellular electrophysiology techniques, it is possible
to detect subthreshold synaptic potentials in addition to spiking activity. These
dyes allow activity measurements in large populations of neurons at once, a task
that would otherwise require impractically large electrode arrays.
There are a variety of voltage-sensitive dyes available, each differing in signal
duration, intensity, signal-to-noise ratio, and toxicity. Most dyes exhibit small
signal changes; the fractional intensity change (ΔI/I or ΔF/F, I = intensity and
172 GuidetoResearchTechniquesinNeuroscience
TABLE 7.1  Commonly Used Neural Activity Sensors
Category Examples Advantages Disadvantages
Voltage-sensitive
dyes
Di-8-ANEPPS,
RH 414
High temporal resolution (microsecond
response time).
Low signal intensity changes (order of 0.1%).
Promiscuous labeling.
Genetically encoded
voltage sensors
ArcLight Targeting specificity. Lower temporal resolution than dyes
(milliseconds).
High background.
Nonratiometric
calcium indicator
dyes
Fluo-3, Fluo-4,
calcium Green-2
Direct fluorescence intensity changes
correlated with Ca2+ concentration.
Prone to artifacts from loading concentration,
photobleaching, and other experiment-specific
conditions.
Cannot be calibrated.
Buffering of intracellular Ca2+ can alter signaling
pathways if present at high concentration.
Ratiometric calcium
indicator dyes
Fura-2, Indo-1 Large signal changes (10%).
Resistant to experiment-specific artifacts, such
as loading concentration and photobleaching.
Enables calibration.
More complicated data acquisition and
measurement than nonratiometric dyes.
Buffering of intracellular Ca2+ can alter signaling
pathways if present at high concentrations.
Genetically encoded
calcium sensors
GCaMP Targeting specificity. Can have long signal decay time, giving low
temporal resolution.
Buffering of intracellular Ca2+ can alter signaling
pathways if present at high concentration.
Dye-based synaptic
vesicle markers
FM dyes (FM1-
43, AM4-64)
Can vary stimulation and exposure to dye to
control number of vesicles labeled.
Labels all membrane surfaces so background can
be high and difficult to wash out of brain slices.
pH-sensitive
fluorescent proteins
SynaptopHluorin Allows study of presynaptic release. Many active release sites can make imaging of
specific synapses difficult.
Vesicle is only visible while fused for exocytosis,
but becomes invisible after endocytosis.
Visualizing Neural Function Chapter | 7  173
F = fluorescence) is in the range of 10−4–10−3 (0.1%), although some dyes may
reach 6% for a large 100-mV change in potential. Thus, there must be very little
noise to detect a reliable signal. Furthermore, activity-dependent changes in
the intrinsic optical absorption and reflection properties of the brain itself can
interfere with the voltage-sensitive dye measurements.
Results fromVSDI tend to look like electrophysiology data, showing changes
in ΔI/I over a certain period of time to report membrane potential changes. This
information can then be encoded to show the magnitude of changes in pseudocolor
maps overlaying the specimen, providing spatial information about the
signal (Figure 7.3).
Genetically Encoded Voltage Indicators
To examine molecularly defined subsets of neurons, a scientist may use a
GEVI. Despite much effort, historically these tools have been inadequate for
precise measurements of membrane potential. However, new tools that have
recently been developed are making GEVIs a reality. For example, ArcLight
is a relatively new GEVI that features improved signal size and signal-to-noise
ratios over previous probes.
Imaging Calcium Dynamics
Intracellular calcium is central to many physiological processes, including
neurotransmitter release, ion channel gating, and second messenger pathways.
In neurons, calcium dynamics link electrical activity and biochemical events.
•
•
∆
FIGURE 7.3  Example of a voltage-sensitive dye imaging experiment.  (A) A craniotomy was
performed over somatosensory cortex (S1) and mortor cortex (M1) in anesthetized mice to deliver
the voltage sensitive dye RH1691. (B) Moving the C2 whiskers evokes transient increases in fluorescence,
first in S1 (black), then in M1 (blue). (C) The spatiotemporal dynamics of voltage changes
across the brain are observed after deflection of the C2 whisker. Reprinted from Ferezou, I., et al.
(2007). Spatiotemporal dynamics of cortical sensorimotor integration in behaving mice. Neuron,
56, 907–923, with permission from Elsevier.
174  Guide to Research Techniques in Neuroscience
Thus, changes in calcium concentration can indirectly indicate changes in electrical
activity. As with voltage-sensitive probes, fluorescent calcium indicators
exist as dyes and genetically encoded calcium indicators.
Like voltage sensitive imaging, data for calcium imaging experiments
typically show changes in fluorescence intensity or the ratio of fluorescence
intensity over time, normalized to initial levels of fluorescence (F0). These
traces are then used to create pseudo-colored maps to show spatial information
about the changes, with hot colors (yellow–red) representing large
changes and cool colors (blue–purple) representing small changes. Compared
to VSDI, calcium imaging produces larger, more robust visible signal changes
(ΔF/F = 1–20%) but with lower temporal resolution (typically milliseconds
rather than microseconds).
Calcium Indicator Dyes
Calcium indicator dyes—organic molecules that change their spectral properties
when bound to Ca2+—can be categorized as ratiometric or nonratiometric
dyes. Ratiometric dyes are excited by or emit at slightly different
wavelengths when they are free of Ca2+ compared to when they are bound to
Ca2+. Thus, they can report changes in Ca2+ through changes in the ratio of
their fluorescence intensity at distinct wavelengths. A common ratiometric
indicator is Fura-2, which emits at 510 nm but can change the wavelength
at which it is excited from 340 to 380 nm in response to calcium binding.
Thus, monitoring the ratio of the fluorescence intensity emitted by Fura-2
at 340 nm to the fluorescence emitted at 380 nm reveals changes in calcium
concentration (Figure 7.4). Ratiometric dyes allow investigators to correct for
background changes in fluorescence unrelated to calcium dynamics, such as
artifacts related to photobleaching, variations in illumination intensity, or differences
in dye concentration. The primary disadvantage of using ratiometric
dyes compared to nonratiometric dyes is that data acquisition and measurements
are more complicated.
Nonratiometric dyes report changes in Ca2+ directly with changes in
either excitation or emission fluorescence intensity. The common nonratiometric
indicators Fluo-3 and Fluo-4 exhibit predictable increases in emission
fluorescence intensity with increases in calcium concentration. While
the direct relationship between fluorescence intensity and calcium concentration
is sensitive for detecting changes due to calcium binding, the measurement
is also prone to detecting changes based on dye concentration and
experiment-specific conditions. However, nonratiometric dyes tend to be
easier to use and quantify.
Because dye-indicators bind to Ca2+, they contribute to buffering of cellular
Ca2+ and can alter Ca2+ homeostasis. Thus, quantitative measurements
of Ca2+-signaling require careful understanding of the indicator properties,
experimental conditions, and potentially interfering binding partners (e.g.,
Mg2+).
Visualizing Neural Function Chapter | 7  175
Genetically Encoded Calcium Indicators
The bioluminescence of the jellyfish Aequorea victoria is due to the presence
of both green fluorescent protein (GFP) and a chemiluminescent protein called
aequorin. Although GFP has received much attention and is ubiquitous in
biological research aequorin has been used for calcium imaging since the late
1960s. This protein emits light when bound to calcium, without requiring illumination
for excitation. Unfortunately, the measurable signal is extremely low
and requires amplification. Thus, it has mostly been superseded by the use of
fluorescent dyes and more robust genetically encoded calcium sensors.
Genetically encoded calcium indicators (GECIs) take advantage of the
conformational changes that occur in certain endogenous calcium-binding
proteins when they bind to calcium. For example, the nonratiometric GECI
GCaMP reports changes in calcium based on a fusion protein between GFP and
the calcium binding protein calmodulin (Figure 7.5). When calmodulin binds to
Ca2+, a conformational change causes an increase in GFP fluorescence. Modern
iterations of the GCaMP protein can report neural activity at the resolution of
single action potentials and reveal subthreshold activity. These probes can also
reveal activity in neural processes, including axons and synaptic terminals.
µ
µ
FIGURE 7.4  Imaging calcium dynamics using the ratiometric dye Fura-2. Fura-2 changes
its emission spectra when bound to calcium.  To examine calcium dynamics, images are collected
after stimulating cells loaded with Fura-2 at (A) 340 nm, dim at resting levels of calcium and (B)
380 nm, brighter at resting levels of calcium, and collecting emission data at 510 nm. The ratio of
340/380 indicates cells with high (bright) or low (dim) concentrations of calcium (C). The ratio can
be converted into known concentrations of free calcium levels (D). Courtesy of Dr Jocelyn Krey.
176  Guide to Research Techniques in Neuroscience
Imaging Synaptic Transmission
A critical component of neural activity is the release of neurotransmitters from
vesicles into the synaptic junction. Electrophysiology first revealed the fusion
and reuptake of synaptic vesicles, but imaging fluorescent dyes and proteins that
report synaptic vesicle activity provides additional details about the dynamic
process of synaptic transmission.
FM Dyes
FM dyes are lipophilic styryl dyes that fluoresce when bound to membranes. The
FM stands for Fei Mao, the chemist who developed these dyes. They are useful for
reporting the exocytosis and endocytosis events that occur during synaptic vesicle
recycling and can stain nerve terminals in an activity-dependent manner. Typically
performed in cell culture preparations, a scientist adds dye to the medium to label
the membrane surface. For activity-dependent labeling, neurons are stimulated
so that all synaptic vesicles undergo exocytosis and endocytosis with the stained
membrane. The stained membranes are internalized during endocytosis. The rest
of the dye is washed away, leaving behind clusters of stained recycled synaptic
vesicles. During an experiment, the investigator observes the destaining process
that occurs as the stained synaptic vesicles release dye during further exocytosis.
FM dyes have been used to characterize neurotransmitter release mechanisms,
measure the kinetics of vesicle recycling, and observe vesicle movements.
pH-Sensitive Fluorescent Proteins
Neurotransmitter release can also be examined using the genetically encoded synaptic
transmission reporter synapto-pHluorin, which uses pH-sensitive mutants
of GFP. The interiors of synaptic vesicles are acidified to a pH of approximately
5.7, an environment that keeps the pHluorins in an off state. When the vesicle
fuses with the plasma membrane during exocytosis, the pH rises to extracellular
levels, switching the pHluorin on and causing it to fluoresce (Figure 7.6). One of
the advantages of using synapto-pHluorins is that the signal regenerates through
FIGURE 7.5  Mechanism behind modern genetically encoded calcium indicators
(GECI).  Modern GECIs, such as GCaMPs, bind green fluorescent protein (GFP) to a calmodulin
(CaM) protein and a peptide sequence called M13. In the presence of Ca2+, a conformational change
causes an increase in GFP fluorescence intensity.
Visualizing Neural Function Chapter | 7  177
multiple rounds of vesicle release and recycling, which permits vesicle recycling
to be imaged in addition to synaptic transmission.
VISUALIZING PROTEIN FUNCTION
Scientists can use fluorescent probes to visualize the activity of specific proteins
within a cell. Using these methods, it is possible to track the subcellular localization
of proteins over time, as well as to detect the interaction between two or
more proteins. These techniques are summarized in Table 7.2.
Time-Lapse Imaging with Reporter Genes
Reporter genes were described in the previous chapter as useful markers to
localize protein expression. Fusing a fluorescent reporter gene, such as GFP or
its variants, to a protein of interest allows a scientist to observe the location and
trafficking of the protein in live cells and tissues (Figure 7.7). In most cases, the
GFP fusion protein behaves in the same way as the original protein. The GFP
fusion protein strategy has become a standard way to determine the distribution
and dynamics of many proteins of interest in living cells. For example, a transcription
factor fused to GFP can reveal changes in localization in response to
growth factors, moving from cytoplasm to nucleus.
Fluorescence Resonance Energy Transfer
Fluorescence (or Förster) resonance energy transfer (FRET) is used to monitor
interactions between proteins (Figure 7.8A). Two molecules of interest are
each labeled with a different fluorophore, chosen so the emission spectrum of one
fluorophore (donor) overlaps with the absorption spectrum of the other (acceptor).
If the two proteins bind, bringing their fluorophores into close proximity
(closer than about 2 nm), the energy of the absorbed light can be transferred
directly from the donor to the acceptor. Thus, when the complex is illuminated
FIGURE 7.6  SynaptopHluorin reports synaptic
transmission dynamics. SynaptopHluorin
does not fluoresce when in acidified synaptic
vesicles (pH ∼ 5.7), but does fluoresce when the
vesicles exocytose and expose the reporter to
physiological pH (pH ∼ 7.4).
pH ~ 5.7
pH ~ 7.4
178  Guide to Research Techniques in Neuroscience
at the excitation wavelength of the first fluorophore, light is emitted at the emission
wavelength of the second. FRET is measured as the ratio of fluorescence
intensity of the donor to fluorescence intensity of the acceptor. This method can
be used with two different spectral variants of GFP as fluorophores to monitor
processes such as the interaction of signaling molecules with their receptors.
FRET has traditionally been exploited to assess distances and orientations
TABLE 7.2  Comparison of Techniques to Optically Investigate Protein
Function
Description
Reporter genes Proteins or organelles can be fluorescently tagged and
observed over time.
Useful for observing behavior, movement and
dynamics of tagged proteins.
Fluorescence/Förster
resonance energy transfer
(FRET)
A donor fluorophore with an emission spectra that
overlaps with the excitation spectra of an acceptor
fluorophore transfers energy to the acceptor when
the donor and acceptor are in close proximity.
Measures the shift in the ratio of donor emission
intensity to acceptor emission intensity.
Useful for observing interactions between two tagged
proteins or two parts of a single protein.
Bimolecular fluorescence
complementation (BiFC)
Two different proteins can be tagged with half a
fluorophore.
The fluorophore will fluoresce only if the two halves
come near enough, reporting a close interaction
between the tagged proteins.
Measures the change in fluorescence intensity to
indicate whether the two proteins are near each other.
Fluorescence recovery after
photobleaching (FRAP)
High-intensity light is used to photobleach a region of
fluorescence.
Measures the kinetics of fluorescence intensity
returning to photobleached region.
Useful for observing dynamics of trafficking, diffusion,
or binding/dissociation of fluorescently tagged proteins.
Photoactivation/
Photoconversion
Photoactivation causes a dramatic increase in
fluorescence intensity after stimulation by light in
specialized fluorophores.
PA-GFP (photoactivatable GFP) becomes fluorescent
after stimulation with UV light.
Photoconversion uses light to change the emission
spectra of a fluorophore.
Useful for tracking localization changes of small
populations or individual cells, organelles, or proteins.
Visualizing Neural Function Chapter | 7  179
′ ′ ′ ′ ′ ′
FIGURE 7.7  Example of time-lapse imaging with reporter proteins.  This migrating neuron
expresses two different reporter proteins with different excitation and emission spectra: a cytoplasmic
fluorescent protein that allows visualization of the cell’s morphology (white) and a GFP fusion
protein that labels early endosomes (green). Observing changes in the position of the endosomes
over time can reveal intracellular trafficking dynamics.
FIGURE 7.8  Fluorescence resonance energy transfer (FRET) and bimolecular fluorescence
complementation (BiFC) are used to determine if and when two proteins interact.  (A) In FRET, one
protein is fused with cyan fluorescent protein (CFP), the other with yellow fluorescent protein (YFP). CFP
is excited by light at ∼436 nm, causing it to emit light at ∼480 nm.YFP can be excited by ∼480 nm light,
so if n emitting CFP-fused protein is nearby,YFP will emit at ∼530nm, indicating that the two proteins are
in close proximity. (B) In BiFC, one fluorescent protein molecule is split in two, and each are fused with a
separate protein. If excited by light when the two proteins are not in proximity, no emission occurs; however,
if excited by light when the two proteins are in proximity, the two subunits can interact to emit light.
180  Guide to Research Techniques in Neuroscience
between separate molecules or different sites within a single macromolecule.
This technique can also be used to report any substrate modification that leads
to a conformational change resulting in the interaction of donor and acceptor
fluorophores.
Bimolecular Fluorescence Complementation
BiFC is similar to FRET in that it relies on the interactions and proximity of two
proteins or parts of a protein to produce a visible change. Rather than two separate
fluorophores, however, two halves of a single fluorophore (e.g., GFP) are
split. Each half of the fluorophore is fused to one protein and does not fluoresce.
When the two proteins are close in proximity, the two halves of the fluorophore
can fold and form the functional fluorophore (Figure 7.8B).
Fluorescence Recovery after Photobleaching
One limitation to using fluorophores is the phenomenon of photobleaching
and decrease in fluorescence intensity over time. However, investigators can
exploit this phenomenon to examine protein turnover rates or trafficking. FRAP
uses strong laser illumination to bleach the fluorescent molecules in a particular
region. The investigator then measures the time course of fluorescence returning
to the bleached region. This can reveal the kinetics of a fluorescently tagged
protein’s diffusion, binding, or dissociation, or active transport into the bleached
region (Figure 7.9).
Photoactivation and Photoconversion
Some fluorescent proteins have been engineered so they are not fluorescent
until hit with a specific wavelength of light (often ultraviolet (UV)) that photoactivates
the fluorophore. Photoactivatable fluorescent proteins can be fused
to a protein and later activated, allowing selected subpopulations of the protein
to be labeled and followed as they move around a cell. This is particularly useful
for observing the behavior and dynamics of a highly abundant protein that
may be difficult to track in a large population of fully fluorescent molecules.
Some sophisticated photoactivatable proteins allow precise control over their
fluorescence. For example, the photoactivatable protein Dronpa can be reversibly
photoactivated; UV light increases fluorescence intensity, while blue light
will quench the fluorescence.
Photoconversion serves a similar purpose as photoactivation, but rather
than activating a previously nonfluorescent molecule, a pulse of light causes a
fluorophore to change its emission spectra from one color to another. This can
be used to examine the timing of protein behaviors. For example, Kaede is a
photoconvertible fluorescent protein that initially emits green light, but emits
red light after UV illumination.
Visualizing Neural Function Chapter | 7  181
CONCLUSION
The development of fluorescent probes to observe neural activity and protein
function allows scientists to view dynamic processes in living cells, organs, and
whole animals. Taken together, these techniques, along with complementary
methods to visualize structure (Chapter 6), record from neurons (Chapter 4), or
investigate intracellular signaling cascades (Chapter 15), constitute a fantastic
toolbox of methods to investigate how proteins determine the properties of neurons,
as well as how neurons determine the properties of circuits and behavior.
SUGGESTED READING AND REFERENCES
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Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Yuste, R., Lanni, F., & Konnerth, A. (2000). Imaging neurons. Cold Spring Harbor, NewYork: Cold
Spring Harbor Laboratory Press.
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Barth, A. L. (2007). Visualizing circuits and systems using transgenic reporters of neural activity.
Current Opinion in Neurobiology, 17, 567–571.
Ellis-Davies, G. C. (2007). Caged compounds: photorelease technology for control of cellular
chemistry and physiology. Nature Methods, 4, 619–628.
Giepmans, B. N., Adams, S. R., Ellisman, M. H., & Tsien, R.Y. (2006). The fluorescent toolbox for
assessing protein location and function. Science, 312, 217–224.
Guzowski, J. F., Timlin, J. A., Roysam, B., McNaughton, B. L., Worley, P. F., & Barnes, C. A.
(2005). Mapping behaviorally relevant neural circuits with immediate-early gene expression.
Current Opinion in Neurobiology, 15, 599–606.
Lippincott-Schwartz, J., Altan-Bonnet, N., & Patterson, G. H. (2003). Photobleaching and photoactivation:
following protein dynamics in living cells. Nature Cell Biology, Suppl. S7–S14.
Miesenbock, G., & Kevrekidis, I. G. (2005). Optical imaging and control of genetically designated
neurons in functioning circuits. Annual Review of Neuroscience, 28, 533–563.
FIGURE 7.9  Fluorescence Recovery after Photobleaching reveals information about
reporter protein kinetics.  Intense illumination is applied to a small region of a cell expressing a
fluorescent reporter, and the recovery of fluorescent signal in the photobleached area is observed
over time. The recovery time can be used to measure the kinetics of protein trafficking or turnover.
182  Guide to Research Techniques in Neuroscience
Tian, L., Akerboom, J., Schreiter, E. R., & Looger, L. L. (2012). Neural activity imaging with
genetically encoded calcium indicators. Progress in Brain Research, 196, 79–94.
Zhang, F., Aravanis, A. M., Adamantidis, A., de Lecea, L., & Deisseroth, K. (2007). Circuitbreakers:
optical technologies for probing neural signals and systems. Nature Reviews
Neuroscience, 8, 577–581.
Zhang, J., Campbell, R. E., Ting, A. Y., & Tsien, R. Y. (2002). Creating new fluorescent probes for
cell biology. Nature Reviews Molecular Cell Biology, 3, 906–918.
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imaging reveals neurophysiological links to behavior in an animal model of depression. Science,
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Angevine, J. B., Jr., & Sidman, R. L. (1961). Autoradiographic study of cell migration during histogenesis
of cerebral cortex in the mouse. Nature, 192, 766–768.
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targeted optical electrophysiology in intact neural circuits. Cell, 154, 904–913.
Chenn, A., & Walsh, C. A. (2002). Regulation of cerebral cortical size by control of cell cycle exit
in neural precursors. Science, 297, 365–369.
Dombeck, D. A., Khabbaz, A. N., Collman, F., Adelman, T. L., & Tank, D. W. (2007). Imaging
large-scale neural activity with cellular resolution in awake, mobile mice. Neuron, 56, 43–57.
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Marek, K. W., & Davis, G. W. (2002). Transgenically encoded protein photoinactivation (FlAsHFALI):
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Marvin, J. S., Borghuis, B. G., Tian, L., Cichon, J., Harnett, M. T., Akerboom, J., et al. (2013). An optimized
fluorescent probe for visualizing glutamate neurotransmission. Nature Methods, 10, 162–170.
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Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C., & Blenis, J. (2002). Molecular interpretation
of ERK signal duration by immediate early gene products. Nature Cell Biology, 4, 556–564.
Ohki, K., Chung, S., Ch’ng, Y. H., Kara, P., & Reid, R. C. (2005). Functional imaging with cellular
resolution reveals precise micro-architecture in visual cortex. Nature, 433, 597–603.
Outeiro, T. F., Putcha, P., Tetzlaff, J. E., Spoelgen, R., Koker, M., Carvalho, F., et al. (2008). Formation
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Arg3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron, 52, 437–444.
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Schroder-Lang, S., Schwarzel, M., Seifert, R., Strunker, T., Kateriya, S., Looser, J., et al. (2007).
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Teather, L. A., Packard, M. G., Smith, D. E., Ellis-Behnke, R. G., & Bazan, N. G. (2005). Differential
induction of c-Jun and Fos-like proteins in rat hippocampus and dorsal striatum after
training in two water maze tasks. Neurobiology of Learning and Memory, 84, 75–84.
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reveals an odor-evoked map of activity in the fly brain. Cell, 112, 271–282.
Protocols
Barreto-Chang, O. L., & Dolmetsch, R. E. (2009). Calcium imaging of cortical neurons using
Fura-2 AM. JoVE, 23. http://dx.doi.org/10.3791/1067. http://www.jove.com/index/details.
stp?id51067.
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dye imaging combined with whole-cell patch recording. Nature Protocols, 3, 249–255.
Fuger, P., Behrends, L. B., Mertel, S., Sigrist, S. J., & Rasse, T. M. (2007). Live imaging of synapse
development and measuring protein dynamics using two-color fluorescence recovery after
photo-bleaching at Drosophila synapses. Nature Protocols, 2, 3285–3298.
Gaffield, M. A., & Betz, W. J. (2007). Imaging synaptic vesicle exocytosis and endocytosis with FM
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185
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00008-3
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 8
Manipulating Neural Activity
After Reading This Chapter, You Should be Able to:
	l	Describe the experimental logic behind gain-of-function and loss-of-function
experiments
	l	Compare different approaches for manipulating neural activity in the nervous
system
	l	Discuss approaches to manipulating neural activity in various organisms, including
humans
Techniques Covered:
	l	Physical manipulation: ablation, thermal cooling
	l	Electrical manipulation: microstimulation, electrolytic lesions
	l	Pharmacological manipulation: agonists, antagonists, drug delivery methods
	l	Genetic manipulation: perturbation of endogenous genes, expression of genes to
ablate neurons
	l	Pharmacogenetics: transgenes from other species, designer receptors exclusively
activated by designer drugs (DREADDs: hM3Dq, hM4Di)
	l	Optogenetics: channelrhodopsin-2 (ChR2), halorhodopsin (NpHR), archaerhodopsin
(Arch), light delivery methods
	l	Neuromodulation in humans: pharmacology, microstimulation, transcranial magnetic
stimulation (TMS), ultrasonic neuromodulation (USNM)
One of the ultimate experimental goals in neuroscience is to perturb a neural system
to test hypotheses about the role of that system for a behavior or phenotype.
For example, if a scientist hypothesizes that a population of neurons in the brain
regulates thirst, a key experiment would be to artificially inhibit or stimulate those
neurons to determine their necessity or sufficiency for water-seeking. The ability
to manipulate neural activity in vivo or in vitro has never been more feasible, and
many techniques have been developed and optimized over just the past 5–10 years
in a variety of model organisms. Importantly, many of these tools have proven to
be relatively cheap and easy to apply in a wide variety of laboratories.
This chapter surveys approaches to artificially inducing or suppressing electrical
activity in neurons (Figure 8.1). Physical methods ablate neural tissue to
determine their role in behavior. Thermal cooling suppresses neural activity by
decreasing the kinetics of cell physiology. Electrical methods apply current to a
tissue preparation to stimulate neural activity or cause a lesion. Pharmacological
186  Guide to Research Techniques in Neuroscience
methods employ chemical compounds to alter the function of ion channels and cell
receptors. Genetically encoded transgenes can be used to ablate neurons or alter
baseline physiological firing properties. A combination of multiple techniques,
such as with pharmacogenetic tools, allows the targeting of nonendogenous receptors
to control neurons with chemical compounds that are normally inert. Finally,
optogenetic methods target light-sensitive ion channels and proteins to neurons
that allow for neuromodulation with unprecedented spatial and temporal precision.
EXPERIMENTAL LOGIC OF MANIPULATING NEURAL ACTIVITY
Before performing a neuromodulation experiment or choosing any specific
technique, it is important to clearly articulate the research goal and comprehend
the meaning of experimental outcomes. In general, neural manipulation
experiments can be classified in two ways: gain-of-function experiments and
loss-of-function experiments. In the context of manipulating neural activity,
a gain-of-function experiment stimulates activity in neurons and tests their
--
- - -
CNO
Physical Electrical Pharmacological
Genetic Pharmacogenetic Optogenetic
FIGURE 8.1  Methods of manipulating neural activity. Neurons in the brain typically reside
within heterogeneous populations composed of many cell types (here denoted by different colors).
Physical lesioning techniques can ablate cells indiscriminately. Electrical techniques can
­microstimulate brain regions or cause electrolytic lesions. Pharmacological techniques inject a psychoactive
drug either centrally or systemically. Genetic methods are capable of affecting very specific
cell types by taking advantage of endogenous promoter regions of genes. Pharmacogenetic techniques
use genetic targeting methods to deliver receptors capable of affecting neural activity to specific cell
types. Optogenetic techniques deliver light-activated neuronal actuators to specific cell types.
Manipulating Neural Activity Chapter | 8  187
sufficiency in causing a phenotype. In contrast, a loss-of-function experiment
inhibits activity in neurons and tests their necessity for a phenotype.
For example, consider a population of neurons that are hypothesized to regulate
hunger. Perhaps these neurons exhibit increased activity following food deprivation
or during conditions of hunger. If artificially stimulating these neurons in
freely moving mice causes an increase in food seeking, the scientist can fairly
conclude that activity in these neurons is sufficient to increase food-seeking behavior.
Alternatively, if artificially inhibiting these neurons decreases food seeking,
especially when an animal is normally hungry, the scientist can fairly conclude
that activity in these neurons is necessary for normal food-seeking behavior. Note
that the concepts of sufficiency and necessity are completely separable, and it
is possible for a neural system to be sufficient but not necessary (or vice versa).
For example, perhaps stimulating neurons could increase food-seeking behavior
while inhibiting neurons would have no significant effect. This result could suggest
that there are other neurons in the brain that also regulate food seeking, and
no single neural population is necessary for food seeking to occur.
Scientists must be careful not to make inappropriate conclusions from gainof-function
and loss-of-function experiments. For example, if inhibiting a population
of neurons decreases food intake, a scientist would be correct that these
neurons are necessary for food-seeking behavior, but it would be unwise to conclude
that these neurons function as “hunger neurons” or “food intake neurons.”
Perhaps these neurons are also necessary for wakefulness or motor activity, and
animals eat less simply because they move less. Thus, concluding that a neural
system is necessary for a behavior might not, by itself, be informative. Such a
result should impel a scientist to perform other experiments to rule out other
potential roles for that neural system in other biological processes.
PHYSICAL MANIPULATION
A physical manipulation is any method that physically disrupts the activity of
neurons due to direct physical contact. Two of the most common methods of
physically manipulating neural activity are through ablation experiments and
thermal cooling.
Ablation
Ablation, or creating a brain lesion, is the oldest technique used to study the
necessity of a particular brain region for a biological function. A scientist can
physically lesion the brain by excising or aspirating a piece of brain tissue during
a stereotaxic surgery (Chapter 3).
Despite their place in the history of neuroscience research, producing physical
lesion is a relatively crude method for performing loss-of-function experiments
in the brain. It is often difficult to control the precision of the lesion or the
impact of removing tissue on the remaining brain structures. Because ablating
188  Guide to Research Techniques in Neuroscience
an area damages not only cell bodies but also fibers of passage (the axons traveling
through that region of the brain), it is difficult to know if the effects of
the lesion are due to the neurons in the ablated area or neurons in a distant area
whose fibers have been damaged. Sometimes ablating tissue can also affect the
local circulatory system and cause systemic changes in the entire organism.
Finally, an obvious limitation to physical ablation is that it is completely irreversible
(see Table 8.1 for a comparison of neural lesioning techniques).
Physical ablation techniques can also be used in invertebrate model organisms.
For example, individual cells in Caenorhabditis elegans can be eliminated
by lasers under a dissection microscope. These lesions are cleaner and more precise
than physical ablations in vertebrates; however, they are also irreversible.
Thermal Cooling
Neurons in the brain function at an optimal temperature, and precise homeostatic
mechanisms ensure that the brain remains at an optimal 37 °C. Cooling
neurons a few degrees can temporarily slow down the kinetics of ion channels
and cell physiology, effectively decreasing neural activity. Neurons can be easily
cooled in in vitro preparations by slightly cooling the temperature of the
bath solution. Neurons can be cooled in vivo by placing a piezoelectric implant
in the brain. In contrast to physically removing brain tissue, cooling a brain
region is reversible—assuming that the altered temperatures do not cause longlasting
damage. Therefore, each animal can serve as its own internal control,
and behavior can be compared during cooled and noncooled conditions.
TABLE 8.1  Methods of Lesioning the Nervous System
Category Tool Precision
Physical Surgical blade Not very precise. Cannot
discriminate between cell
types or fibers of passage
Electrical Microelectrode More spatially precise than
surgical (physical) lesions, but
cannot discriminate between
cell types or fibers of passage
Pharmacological Toxins (e.g., ibotenic
acid)
Spatial precision determined
by quality and specificity of
injection. Fibers of passage
are unaffected
Genetic Genetically encoded
proteins (e.g., ataxin,
diphtheria toxin receptor)
Cell-type specificity
Manipulating Neural Activity Chapter | 8  189
Although reversible, cooling the brain is not very spatially precise because
it is not possible to discriminate among neurons in a given area. Cooling is also
not temporally precise, as it takes a variable amount of time for the neurons to
cool down and then warm up when the experiment is complete. Finally, thermal
manipulation techniques are much more feasible for loss-of-function experiments
than gain-of-function experiments because heating the brain more often
causes damage rather than an increase in neural activity.
ELECTRICAL MANIPULATION
Microelectrodes are used during electrophysiology experiments to record electrical
activity from neurons, but they can also be used to deliver electrical current
into the brain or to neurons in culture in a process called microstimulation.
A microelectrode is inserted into the brain or next to a neuron of interest and
current is applied at a fixed frequency and time. The electrode elicits action
potentials by changing the extracellular environment such that voltage-gated
ion channels open, depolarizing the neuron. If too much current is applied to
the electrode, the scientist can kill nearby cells. Often, this is done deliberately
to create an electrolytic lesion that permanently ablates neurons. Therefore,
microelectrodes can be used to either reversibly stimulate neural activity or irreversibly
lesion neural tissue. The spatial resolution of electrical manipulation
is limited, as injection of current affects all neurons in a local area, regardless
of cell type, and can affect fibers passing through the area of stimulation as
well. Microstimulation exhibits very precise temporal resolution because the
frequency and timing of current delivery is under the control of the investigator.
PHARMACOLOGICAL MANIPULATION
A scientist can influence neural activity pharmacologically using one of hundreds
of compounds that selectively bind to membrane-bound receptors
(Figure 8.2). An agonist is a compound that can bind to and cause activation of
a receptor, thus mimicking an endogenous ligand or neurotransmitter. A partial
agonist is a compound that can produce an agonist-like effect, but not to the
maximal extent of the full agonist or endogenous ligand.An antagonist binds to
the receptor but does not cause activation. Therefore, it prevents the endogenous
ligand from binding, inhibiting its biological activity.
Where do pharmacological compounds come from? Some drugs are engineered
in pharmaceutical laboratories to target specific receptors or other proteins.
However, many plants and animals naturally produce pharmacological
compounds and neurotoxins for self-defense. Scientists have purified several of
these compounds to manipulate neural activity in vitro and in vivo. For example,
many fish of the order Tetraodontiformes (the most famous being the pufferfish)
produce a compound called tetrodotoxin. This compound is an antagonist
of voltage-gated sodium channels, and therefore can be used to inhibit
190  Guide to Research Techniques in Neuroscience
action potentials. Another commonly used agent to inhibit action potentials is
tetraethylammonium, a potassium-selective ion channel antagonist.
Many commonly used pharmacological agents target inhibitory GABA
receptors. GABA-receptor antagonists include bicuculline and metrazol; they
have a net effect of increasing neural excitability, as they prevent the normal
inhibitory effects of GABA signaling. GABA-receptor agonists decrease neuronal
excitability. For example, muscimol is a common GABA-receptor agonist
that is often used during behavioral and/or in vivo electrophysiology experiments
to inhibit neural activity.
Some pharmacological agents can be used to irreversibly ablate neurons.
For example, ibotenic acid causes excitotoxic effects when directly injected
into the brain. Pharmacological ablation of neurons is preferable over physical
or electrical ablation methods because it spares fiber tracts crossing through the
target area. Therefore, if an injection of ibotenic acid is precise, a scientist can
make better conclusions about any resulting effects on behavior or physiology.
For in vitro experiments, drugs can be delivered in cell culture media or perfused
in the bath solution incubating a brain slice. For in vivo experiments, some
drugs can be delivered to the brain via the bloodstream, allowing a scientist to
perform an intraperitoneal (i.p.) injection. However, many drugs do not cross
the blood–brain barrier. In this case, a scientist can perform an intracerebroventricular
(i.c.v.) injection into the lateral ventricles of the brain. Substances
in the cerebrospinal fluid in the ventricles can then diffuse throughout the extracellular
milieu of the brain. Alternatively, a scientist can perform a small, local
injection into a discrete brain region.
To inject a drug into the brain just before a behavioral experiment, a scientist
can first implant a cannula above the brain region of interest during a surgery
(Chapter 3), then subsequently inject the drug through the cannula. Injections into
Ligand Agonist Partial Agonist Antagonist
Binding of ligand to
its receptor causes
receptor activation
Binding of agonist to
a receptor causes
receptor activation
Binding of partial
agonist to a
receptor causes
some activation, but
not full effect
Binding of antagonist
to a receptor does
not cause activation
and prevents ligand
from binding
FIGURE 8.2  Compounds used in pharmacology: ligands, agonists, partial agonists, and
antagonists.
Manipulating Neural Activity Chapter | 8  191
the brain can be performed either by pressure injection or microiontophoresis,
the process of using a small electrical current to drive substances out of a glass
pipette. The advantage to microiontophoresis is that it allows for careful delivery
of small volumes to discrete, local areas of the brain. For sustained, chronic delivery
of a drug over multiple days or weeks, a scientist can use specially implanted
osmotic minipumps that infuse the drug through an implanted cannula.
Manipulating neural activity using pharmacological agents is usually reversible
(except in the case of neurotoxins such as ibotenic acid). These agents have
varying degrees of spatial precision depending on the specific receptors they
target and the receptor expression patterns in the area of injection. They are
typically not very temporally precise in vivo because, once injected, they require
several minutes or even hours to clear from the system.
GENETIC MANIPULATION
Unlike physical, electrical, and pharmacological techniques, genetic methods
of modulating neural activity can target specific cell types in the brain by taking
advantage of endogenous promoters and other genetic regulatory elements
that limit expression of genes to specific populations of neurons (Chapter 12).
Therefore, if a cell type of interest exists in a heterogeneous population of neurons
with one or more other cell types, it is possible to selectively target these
neurons and not surrounding cell types using modern transgenic and gene targeting
technologies (Chapters 12 and 13).
To permanently ablate a specific cell type, a scientist can express a transgene
that causes the death of the cell. For example, the ataxin transgene efficiently
causes apoptosis when expressed in neurons. One potential limitation to using
the ataxin transgene is that the neurons die immediately upon expression, which
typically occurs during development or even in utero. Therefore, the brain may
compensate and another brain region may assume the functions of the lost neurons.
To gain more temporal control over expression of the ataxin gene, it is
possible to use transgenic technologies that allow for controlled expression at
specific time points (Chapter 12). Alternatively, it is also possible to express the
diphtheria toxin receptor to ablate neurons at a time point of the investigator’s
choosing. This receptor can cause the death of neurons, but only in the presence
of the diphtheria toxin. Therefore, a scientist can target the diphtheria toxin
receptor to a specific cell type and ablate those neurons at any time point during
an animal’s lifespan by injecting the toxin.
Another method of modulating neural activity using genetics is to overexpress
or knockout a naturally occurring ion channel or membrane-bound receptor
that regulates cellular excitability. For example, knocking out an endogenous
GABA receptor subunit might increase neuronal excitability. However, neurons
have a remarkable ability to compensate for alterations in cell physiology, and
other proteins affecting cell physiology may be upregulated or downregulated to
return the intrinsic excitability to normal. Furthermore, even if no compensatory
192  Guide to Research Techniques in Neuroscience
mechanisms occur, genetically manipulating neurons does not allow for a finetune
control over the neurons’ firing properties.
In the last few years, scientists have developed ingenious methods of targeting
genetically encoded actuators to neurons that allow a scientist to reversibly
stimulate or inhibit neural activity with exceptional temporal precision. Two
major categories of genetically encoded neuromodulation tools include pharmacogenetics
and optogenetics.
PHARMACOGENETIC MANIPULATION
Pharmacogenetics refers to a set of membrane-bound receptors that are activated
by chemical compounds (“pharmaco-”) and are genetically encoded
(“-genetics”). The overall strategy is to genetically target neurons with nonendogenous
receptors that are capable of being activated by a normally inert
ligand. Because other cell types in the brain do not naturally respond to the
ligand, only the cells that express the receptor are affected.
Some pharmacogenetic strategies use receptor-ligand systems from other
species. For example, the mammalian capsaicin receptor (TrpV1) is a ligandgated
channel that depolarizes a neuron upon exposure to capsaicin. Flies and
other invertebrates do not normally express this channel, so capsaicin exposure
will depolarize only those neurons that express the receptor. A similar strategy
can be used to inhibit activity and hyperpolarize neurons in the presence of a
ligand. For example, the insect allatostatin receptor (Alstr) is not expressed in
mammals. In the presence of the insect protein allatostatin, this receptor inhibits
neural activity. Thus, if Alstr is expressed in the mammalian brain, addition of
allatostatin will reversibly inactivate genetically targeted populations of neurons.
These strategies have proven useful in many studies, but there are substantial
disadvantages to using these systems in rodents. The capsaicin receptor
is endogenous in rodents and causes a painful sensation when activated, so its
use has been limited to TrpV1 knockout mice. The allatostatin receptor is not
endogenous in rodents, however, allatostatin does not cross the blood–brain barrier,
so it must be injected centrally through a surgically implanted cannula.
In recent years, a new set of pharmacogenetic tools have been specifically
engineered for use in a wide range of model organisms. Referred to as designer
receptors exclusively activated by designer drugs (DREADDs), these tools
consist of artificially engineered G-protein coupled receptors that activate
intracellular signaling pathways to affect neuronal activity. The hM3Dq receptor
(human M3 muscarinic DREADD coupled to Gq) takes advantage of the
endogenous Gq signaling pathway to increase neuronal excitability and induce
firing, whereas the hM4Di receptor (human M4 muscarinic DREADD coupled
to Gi) takes advantage of the endogenous Gi signaling pathway to decrease neuronal
excitability and silence neural activity. These receptors are sensitive to the
normally inert ligand clozapine-N-oxide (CNO). Importantly, CNO is capable
of crossing the blood–brain barrier, so animals can be injected intraperitoneally
Manipulating Neural Activity Chapter | 8  193
during in vivo experiments. The effects of CNO typically last 6–10 h, the time
it takes CNO to be metabolized and neuronal signaling cascades to return to
baseline. For in vitro culture experiments using cells or tissue expressing DREADDS,
CNO can be directly added to the bath solution.
Pharmacogenetic techniques are spatially precise because neuromodulation
is restricted to the genetically targeted population of cells. However, these techniques
do not offer temporal precision greater than traditional pharmacological
approaches.
OPTOGENETIC MANIPULATION
Optogenetics refers to a set of neuromodulation tools that are activated by light
(“opto-”) and are genetically encoded (“-genetics”). Like other transgenes, optogenetic
tools can be delivered with high spatial precision to specific cell types in
the nervous system. However, because scientists can deliver light to neurons at any
duration or frequency, optogenetics allows for unprecedented spatial and temporal
precision. Since their development and application in the past few years, hundreds
of laboratories now routinely use optogenetics to make major advances in
our understanding of neural systems and circuits in a variety of model organisms.
Optogenetic Transgenes
The most commonly-used optogenetic probes are engineered versions of microbial
opsins, proteins that translocate ions across membranes in response to stimulation
by specific frequencies of light (Figure 8.3).
FIGURE 8.3  Genetically encoded actuators used in optogenetics. (A) Channelrhodopsin-2
(ChR2) is a nonspecific cation channel that opens in response to blue light. (B) Halorhodopsin
(NpHR) is a chloride pump that actively translocates chloride ions from outside to inside a cell in
response to yellow light. (C) Archaerhodopsin (Arch) is a hydrogen (proton) pump that actively
translocates hydrogen ions from inside to outside a cell in response to yellow light.
194  Guide to Research Techniques in Neuroscience
Optogenetic Stimulation
Channelrhodopsin-2 (ChR2) is a nonspecific cation channel derived from the
green algae Chlamydomonas reinhardtii. ChR2 absorbs blue light, causing a
conformational change that allows H+, Na+, K+, and Ca+ ions to passively diffuse
down their concentration gradients. When expressed in neurons, the opening
of these channels causes a rapid depolarization of the plasma membrane that
can cause action potentials. Importantly, the channel also closes very rapidly
when the blue light is switched off. Therefore, single action potentials can be
generated with a brief pulse of blue light, with no long-lasting residual effects
of stimulation. ChR2 is capable of driving neuronal activity at frequencies from
1 to 40 Hz.
Scientists have made slight genetic modifications to ChR2 to create other
channels with desirable neuromodulatory properties. For example, a mutation
in the coding sequence of ChR2 causing a single amino acid substitution allows
for stimulation of frequencies up to 200 Hz (scientists have named this tool
“ChETA”). To cause activation of neurons over longer time periods without the
need to continuously deliver light, scientists have created amino acid substitutions
in ChR2 that cause the channel to remain open for several minutes or even hours
in response to a single pulse of blue light. Called “step function opsins,” these
ChR2-derived tools can be closed by a single pulse of yellow light. Scientists
have also engineered mutations that alter the wavelength of light used to activate
ChR2. The ability to express different versions of ChR2 that react to different
colors of light allow for combinatorial experiments in which neuronal activity in
multiple cell types can be distinctly manipulated in the same individual.
Optogenetic Inhibition
There are two major transgenes used for optogenetic inhibition. Halorhodopsin
(NpHR) is a chloride pump derived from the halobacterium Natronomonas
pharaonis. NpHR actively pumps Cl− ions into cells in response to yellow
light. Archaerhodopsin-3 (Arch) is derived from Halorubrum sodomense and
actively pumps hydrogen ions outside of the cell in response to yellow light. The
translocation of Cl− ions into the cell or H+ neurons out of the cell effectively
hyperpolarizes neurons.
One potential limitation of using optogenetic techniques to inhibit neurons
is the need for continuous light illumination. Unlike ChR2, for which blue
light is typically applied at regular pulses to mimic action potential trains, yellow
light must be continuously delivered to neurons for sustained hyperpolarization.
For experiments in which a scientist wants to inhibit neural activity
for minutes or even hours, it is not practical to continuously deliver yellow
light due to potential side effects due to heat that may cause neuronal damage.
Therefore, a scientist must carefully consider whether NpHR or Arch are the
best tools for a given experiment. For experiments in which neural inhibition
is necessary over multiple hours, perhaps a pharmacogenetic strategy may be
the best approach.
Manipulating Neural Activity Chapter | 8  195
Methods of Delivering Light to the Nervous System
Optogenetic methods require the delivery of light to targeted neurons of interest.
For in vitro applications, such as recording neurons in acute brain slice preparations
(Chapter 14), scientists can position a light source directly next to the bath or culture
plate. Alternatively, light can also be directly coupled to the light path of a microscope,
and specialized shutters can be installed to deliver light at specific frequencies.
For experiments in invertebrate model organisms, light is typically delivered to
the entire housing chamber and passes into the brain directly. In vivo light delivery
to rodents is more challenging because of the need to implant a light delivery device
capable of penetrating the skull. Light must be delivered very close to the target
neurons because brain tissue scatters light exponentially, with only about 10% light
intensity remaining at a distance 500 μm from the light source. Furthermore, light
delivery systems must not weigh more than a freely moving animal can carry.
The most common method for delivering light to freely moving rodents
is implanting a guide cannula for placement of a lightweight fiber optic cable
(Figure 8.4A). This strategy allows for the targeting of relatively deep brain structures.
During an experiment, the exposed cannula implanted on the skull is coupled
to a fiber-optic cable (usually about 2 m in length) connected on the other end to
a light delivery source, often a laser. For optical modulation of superficial cortical
neurons, small light emitting diodes (LEDs) can be mounted directly above the
brain over a cranial glass window (Figure 8.4B). These LEDs are powerful enough
to deliver light to all cortical layers. Some scientists have even devised methods of
coupling a small LED to a fiber-optic implant for delivery to deep brain structures
so that the animal does not require tethering to a fiber-optic cable.
Fiber optic cable Light emitting diode (LED)
Cannula
Optical fiber
Light from
laser diode
Optical window
LED
Connector
(A) (B)
FIGURE 8.4  Methods of targeting light to neurons in the brain.  (A) The most common form
of light delivery in optogenetic experiments is to implant a cannula above a cell population of interest,
then deliver light via a fiber-optic cable inserted through the cannula. The animal is connected to fiberoptic
cable that itself is connected to a laser or other light source. (B) Light-emitting diodes (LEDs) are
useful for illuminating greater areas of neural tissue at once. Some LED implants do not need to be tethered
to a power source at all, allowing an animal to freely move about its cage with no tether.
196  Guide to Research Techniques in Neuroscience
Strategies for Studying Neural Circuits Using Optogenetics
In a typical optogenetic experiment (typical does not mean easy), a scientist
transduces a population of neurons with an optogenetic transgene and then
investigates the effects of directly stimulating or inhibiting those neurons
with light. In addition to stimulating the cell bodies of transduced neurons,
it is also possible to stimulate fiber projections to distinct downstream target
regions (Figure 8.5). The ability to selectively stimulate individual downstream
projections from a neuronal population provides scientists with an
unprecedented neural dissection tool. For example, if stimulating a population
of neurons is sufficient to cause a phenotype, a scientist can test the hypothesis
that this effect is mediated by a specific projection to a downstream brain
region. The only caveat to these experiments is that stimulating fiber projections
may cause antidromic effects that propagate action potentials up axon
tracts back to neuronal cell bodies; therefore, scientists should perform careful
controls to ensure that their stimulation effects are truly specific to the targeted
projections of interest.
FIGURE 8.5  Stimulation of projections
using optogenetics. (A) Optogenetic transgenes
can be delivered to a cell population of
interest, and scientists can directly test the effects
of stimulation or inhibition of the cell soma.
Alternatively, scientists can test hypotheses
about specific downstream pathways by implanting
a fiber-optic cable above discrete projection
fields (B, C).
Manipulating Neural Activity Chapter | 8  197
NEUROMODULATION TECHNIQUES USED IN HUMANS
The rapid development of cutting-edge neuromodulation tools begs the question
about their applications in humans. The tools described in this chapter have
obvious therapeutic potential in neurodegenerative diseases and neuropsychiatric
disorders. Interestingly, they also have potential for “neuroenhancement”—
improving human attention, memory, and cognitive abilities in those with
already normal function. These applications in humans are highly controversial
and hotly debated among neuroethicists.
Which neuromodulation techniques have immediate potential for use in
humans? Physical methods are still in use, primarily for trying to limit epileptic
electrical activity from spreading by surgically removing the seizure-generating
regions or disrupting the connections through which the activity spreads. Electrical
stimulation is used to treat severe symptoms in intractable human diseases,
including rigidity in Parkinson’s disease and serious cases of depression.
Pharmacological techniques have been used over several decades to treat pain,
depression, anxiety, attention-deficit disorders, and other neurological causes of
human suffering. Most psychoactive agents were developed many years ago, and
new pharmacological treatments for neuropsychiatric conditions are relatively rare.
Chapter 1 described two methods of manipulating neural activity in humans
without penetrating the skull. Transcranial magnetic stimulation (TMS) is
capable of altering neural activity by applying a magnetic field outside the skull,
inducing weak electric currents at a specific focal point on the surface of the
brain. Depending on the brain region and the strength of the magnetic field
impulse, the electrical activity may either stimulate neural activity or cause a
hyperpolarized state in which neural activity is temporarily inactivated. This
technique has a spatial resolution of millimeters but can only be applied to superficial
neurons near the surface of the skull, such as cortical neurons. Another
technique, ultrasonic neuromodulation (USNM), uses focused sound waves
to heat the brain in a defined area. Like TMS, this technique is currently only
efficient in superficial neurons and also has a spatial resolution of millimeters.
Both of these techniques have been applied in clinical and experimental settings.
Neuromodulation tools that require genetically encoded transgenes, such as
optogenetics, have not been applied to humans, mostly due to the need for delivering
DNA to human cells in vivo. The only gene delivery strategy that can efficiently
target transgenes to human neurons is viral gene delivery (Chapter 11). Although
commonly used adeno-associated virus and lentiviral vectors are relatively safe
in animal models, scientists have been reasonably reluctant to inject these vectors
into human subjects. Perhaps in the future, other methods of gene delivery will
be possible so that cell-type specific modulation of neural activity in humans can
become a possibility.
CONCLUSION
The past decade has seen a dramatic expansion in the toolkit used to manipulate
neural activity in model organisms. There are multiple methods of stimulating
(Table 8.2) and inhibiting (Table 8.3) neurons in a wide variety of model
198 GuidetoResearchTechniquesinNeuroscience
TABLE 8.2  Methods of Stimulating Neural Activity
Category Tool Invasiveness Spatial precision Temporal precision
Electrical Microelectrode Surgical implantation Cannot distinguish
between cell types or
fibers of passage
Milliseconds
Pharmacological Various agonists
and antagonists of
membrane-bound
receptors
Intraperitoneal injection
or surgical implantation
of cannula
Depends on specificity of
receptor expression
Effects can last minutes to hours
Genetic Modulation of
endogenous
membrane-bound
receptors
Not invasive unless viral
gene delivery
Depends on specificity of
receptor expression
Permanent effects
Pharmacogenetic Ligand-gated receptors
(e.g., hM3Dq, TrpV1)
Not invasive unless
viral gene delivery;
need to inject drugs
intravenously
Cell-type specificity Effects can last minutes in vitro or
hours in vivo
Optogenetic Channelrhodopsin-2
(ChR2)
Surgical implantation;
attachment to fiber-optic
cable
Cell-type specificity Milliseconds
ManipulatingNeuralActivity Chapter|8 199
TABLE 8.3  Methods of Inhibiting Neural Activity
Category Tool Invasiveness Spatial precision Temporal precision
Electrical Microelectrode (causes
electrolytic lesion)
Surgery necessary Cannot distinguish
between cell types or
fibers of passage
Permanent lesion
Pharmacological Various agonists and antagonists
of membrane-bound receptors
Intraperitoneal injection
or surgical implantation
of cannula
Depends on specificity of
receptor expression
Effects can last minutes
to hours
Genetic Modulation of endogenous
membrane-bound receptors
Not invasive unless viral
gene delivery
Depends on specificity of
receptor expression
Permanent effects
Pharmacogenetic Ligand-gated receptors (e.g.,
hM4Di, AlstR)
Not invasive unless viral
gene delivery; need to
inject drugs intravenously
Cell-type specificity Effects can last minutes
in vitro or hours in vivo
Optogenetic Halorhodopsin (NpHR),
Archaerhodopsin (Arch)
Surgical implantation;
attachment to fiber-optic
cable
Cell-type specificity Milliseconds
200  Guide to Research Techniques in Neuroscience
organisms, each with their own advantages and disadvantages. The recent
development of genetically encoded tools that can reversibly affect neural activity
with unprecedented spatial and temporal resolution has allowed scientists to
probe many previously intractable questions about the role of specific neural
systems and circuits in behavior.
SUGGESTED READING AND REFERENCES
Books
Knopfel, T., & Boyden, E. S. (2012). Optogenetics: Tools for controlling and monitoring neuronal
activity. Progress in brain research (Vol. 196). Elsevier.
Meyer, J. S., & Quenzer, L. F. (2013). Psychpharmacology: Drugs, the brain, and behavior (2nd
ed.). Sinauer Associates, Inc.
Nestler, E., Hyman, S., & Malenka, R. (2011). Molecular neuropharmacology: A foundation for
clinical neuroscience (2nd ed.). McGraw Hill.
Review Articles
Cheney, P. D., Griffin, D. M., & Van Acker, G. M. III. (2013). Neural hijacking: action of highfrequency
electrical stimulation on cortical circuits. Neuroscientist, 19, 434–441.
Luo, L., Callaway, E. M., & Svoboda, K. (2008). Genetic dissection of neural circuits. Neuron, 57,
634–660.
Sternson, S. M., & Roth, B. L. (2014). Chemogenetic tools to interrogate brain functions. Annual
Review of Neuroscience, 37, 387–407.
Warden, M. R., Cardin, J. A., & Deisseroth, K. (2014). Optical neural interfaces. Annual Review of
Biomedical Engineering, 16, 103–129.
Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., & Deisseroth, K. (2011). Optogenetics in
neural systems. Neuron, 71, 9–34.
Primary Research Articles—Interesting Examples from the Literature
Adamantidis,A. R., Zhang, F.,Aravanis,A. M., Deisseroth, K., & de Lecea, L. (2007). Neural substrates
of awakening probed with optogenetic control of hypocretin neurons. Nature, 450, 420–424.
Alon, T., & Friedman, J. M. (2006). Late-onset leanness in mice with targeted ablation of melanin
concentrating hormone neurons. Journal of Neuroscience, 26, 389–397.
Carter, M. E., Soden, M. E., Zweifel, L. S., & Palmiter, R. D. (2013). Genetic identification of a
neural circuit that suppresses appetite. Nature, 503, 111–114.
Garner, A. R., Rowland, D. C., Hwang, S. Y., Baumgaertel, K., Roth, B. L., Kentros, C., et al.
(2012). Generation of a synthetic memory trace. Science, 335, 1513–1516.
Gradinaru, V., Mogri, M., Thompson, K. R., Henderson, J. M., & Deisseroth, K. (2009). Optical
deconstruction of parkinsonian neural circuitry. Science, 324, 354–359.
Lin, D., Boyle, M. P., Dollar, P., Lee, H., Lein, E. S., Perona, P., et al. (2011). Functional identification
of an aggression locus in the mouse hypothalamus. Nature, 470, 221–226.
Long, M. A., & Fee, M. S. (2008). Using temperature to analyse temporal dynamics in the songbird
motor pathway. Nature, 456, 189–194.
Luquet, S., Perez, F. A., Hnasko, T. S., & Palmiter, R. D. (2005). NPY/AgRP neurons are essential
for feeding in adult mice but can be ablated in neonates. Science, 310, 683–685.
Marella, S., Fischler, W., Kong, P., Asgarian, S., Rueckert, E., & Scott, K. (2006). Imaging taste
responses in the fly brain reveals a functional map of taste category and behavior. Neuron, 49,
285–295.
Manipulating Neural Activity Chapter | 8  201
Noudoost, B., & Moore, T. (2011). Control of visual cortical signals by prefrontal dopamine.
Nature, 474, 372–375.
O’Doherty, J. E., Lebedev, M. A., Ifft, P. J., Zhuang, K. Z., Shokur, S., Bleuler, H., et al. (2011).
Active tactile exploration using a brain-machine-brain interface. Nature, 479, 228–231.
Salzman, C. D., Britten, K. H., & Newsome, W. T. (1990). Cortical microstimulation influences
perceptual judgements of motion direction. Nature, 346, 174–177.
Winkowski, D. E., & Knudsen, E. I. (2006). Top-down gain control of the auditory space map by
gaze control circuitry in the barn owl. Nature, 439, 336–339.
Protocols
Dong, S., Rogan, S. C., & Roth, B. L. (2010). Directed molecular evolution of DREADDs: a generic
approach to creating next-generation RASSLs. Nature Protocols, 5, 561–573.
Zhang, F., Gradinaru, V., Adamantidis, A. R., Durand, R., Airan, R. D., de Lecea, L., et al. (2010).
Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures.
Nature Protocols, 5, 439–456.
Websites
DREADD wiki: http://pdspit3.mml.unc.edu/projects/dreadd/wiki/wikistart.
Optogenetics resource center: http://web.stanford.edu/group/dlab/optogenetics/index.html.
Optogenetics wiki: OpenOptogenetics.org.
203
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00009-5
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 9
Identifying Genes and Proteins
of Interest
After Reading This Chapter, You Should be Able to:
	l	Understand basic concepts in genetics, such as transcription and translation
	l	Compare and contrast the strengths and limitations of commonly used genetic
model organisms
	l	Describe methods for identifying genes and proteins important to a biological
phenotype, including genetic, in silico, and molecular screening techniques
Techniques covered:
	l	Genetic screens: forward and reverse genetic screens
	l	In silico screens: BLAST, Ensembl
	l	Molecular screens: cDNA microarray, RNAi screen
Virtually every cell in your body contains a copy of your genome, the complete
set of genetic information embedded in DNA that is necessary for your
development, metabolism, homeostasis, and plasticity. The functional unit of the
genome is the gene, a segment of DNA that codes for a functional protein product.
Proteins are the molecular machines responsible for virtually all of the cell’s
structural and functional properties. To understand how the brain works, a neuroscientist
must understand the anatomy and physiology of neurons—and to understand
the anatomy and physiology of neurons, a neuroscientist must understand
the genes and proteins that give rise to their properties.
One of the overarching questions in neuroscience is how an animal’s genotype,
the genetic constitution of an animal, determines that animal’s phenotype, an
observable trait or set of traits. In other words, what genes influence a trait, such
as proper development of an axon toward its target or an appropriate behavioral
response following a stimulus? Researchers regularly publish a variety of studies
either identifying a novel gene and demonstrating its important role in an
animal’s phenotype or showing that a previously discovered gene also contributes
to a separate phenotype. How do scientists identify genes that function as
the molecular substrates of behavior?
The purpose of this chapter is to describe methods useful for identifying which
genes or proteins are important for a specific biological phenotype. These methods
204  Guide to Research Techniques in Neuroscience
usually involve screening thousands of genes to identify just a few that contribute
to a neurobiological phenomenon. In previous chapters, we examined techniques
that generally study whole brains, behaviors, or the activity of cells. Starting with
this chapter, we begin to study genetic and molecular neuroscience—the contribution
of genes and molecules to development, physiology, and behavior. Therefore,
we start the chapter with a brief introduction to studying genes and proteins, as
well as a survey of genetically tractable model organisms. The second half of the
chapter describes various methods for identifying genes or proteins of interest:
using genetic screens, in silico screens, and molecular screens. Once these genes
are identified, scientists can perform other experiments to further understand their
contribution to a phenotype, as described in later chapters.
A BRIEF REVIEW OF GENES AND PROTEINS
The number of genetic and molecular techniques used in the literature can often
be intimidating to neuroscientists without formal training in genetics or molecular
biology. Fortunately, the background information necessary to understand
these techniques and why they are used is not complex; it can be summarized in
terms of the flow of information within each cell. Highly detailed descriptions
of this information flow can be found in other textbooks. Here, we provide the
essential information that a neuroscientist needs to know to understand genetic
and molecular neuroscience, the subject of the remaining chapters in this book.
The Central Dogma of Molecular Biology
The central dogma of molecular biology is a model of the flow of information
within a cell. Described by Francis Crick in 1958, this model posits that information
in a nucleic acid, encoded by the sequence of specific DNA or RNA bases,
can be transferred to another nucleic acid or to a protein, but information in a
protein, encoded by the sequence of specific amino acids, is not transferred back
to another protein or to a nucleic acid. Many of us learned the simplest version
of the central dogma suggesting a unidirectional flow of information—-specific
sequences of DNA code for molecules of RNA—and these RNA molecules, in
turn, code for proteins (Figure 9.1). This model lays the foundation for all of
molecular biology research. While scientists have elaborated on this model over
the past 40 years and found more complex situations that contradict the simple
version of the central dogma, such as the discovery of microRNAs and other
RNA-based methods of DNA regulation, it continues to serve as the framework
from which all molecular neuroscientists design and execute experiments.
Deoxyribonucleic Acid
DNA is the ultimate source of all genetic information within a cell. Residing
within the cell nucleus, a DNA molecule is a long polymer made up of repeating
units called nucleotides (Figure 9.2). These nucleotides are composed of a
DNA is
transcribed
into mRNA
mRNA
leaves the
nucleus
mRNA is
translated
into protein
DNA
Nucleus
mRNA mRNA
translated by
ribosome into
amino acid
polypeptide
Protein
FIGURE 9.1  The flow of information within a cell.  DNA resides in the nucleus and codes for
relatively short sequences of mRNA. After transcription, an mRNA molecule leaves the nucleus
for the cytoplasm, where it is translated by ribosomes into proteins. Thus, the molecular flow of
information starts with nucleic acids and ends with amino acids.
O
OH
OH
O
O
O
O
O
O
O
O
O
O
OO
O
O
O
O
O
O
O
O
OO
O
O
O
OO
O
O
OO
O
O
O
O O
O
O
O
OO
O
O
O
OO
O
P
P
P
P
P
P
P
P
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
NN
N
N
NH2
NH2
NH2
HN
HN
H2N
H2N
H2N
NH
NH
_
_
_
_
_
_
_
_
_
_
Adenine
Thymine
Guanine
Cytosine
FIGURE 9.2  The molecular structure of DNA.  DNA is composed of a sugar-phosphate backbone
on the outside and nitrogenous bases on the inside. The four nitrogenous bases form hydrogen
bonds in the interior of the DNA molecule and hold the two complementary strands together. The
two strands are oriented in opposite directions, with one strand running in the 5′ to 3′ direction and
the other running in the 3′ to 5′ direction.
206  Guide to Research Techniques in Neuroscience
phosphate group, a sugar molecule (2-deoxyribose—what gives DNA its name),
and a nitrogenous base. The phosphate and sugar molecules form the backbone
of a DNA polymer. There are four types of nitrogenous bases found in DNA that
define the properties of the nucleotide: adenine (abbreviated A), thymine (T),
guanine (G), and cytosine (C).
In all eukaryotic organisms, DNA exists as a tightly associated pair of two
long strands that intertwine, like two handrails of a spiral staircase, forming the
shape of a double helix. The two strands of DNA are stabilized by hydrogen
bonds between the nitrogenous bases attached to the two strands. A base on one
strand forms a bond with only one type of base on the opposite strand: A only
bonds with T, and C only bonds with G. The arrangement of two nucleotides
binding together across the double helix is referred to as a base pair. Strands of
DNA that form matches among base pairs are called complementary strands.
Hydrogen bonds, unlike covalent bonds, are relatively weak and can be broken
and rejoined relatively easily. This allows other enzymes to unwind DNA during
DNA replication or gene transcription.
In addition to being complementary, the two strands of DNA are also antiparallel,
oriented in opposite directions. The directionality of a DNA strand
is defined by the carbon atom in the 2-deoxyribose molecule that attaches to
another phosphate molecule in a nucleotide monomer. One strand is said to
be oriented in the 5′ (pronounced “five prime”) to 3′ (“three prime”) direction,
while the other strand is oriented in the 3′ to 5′ direction (Figure 9.2). This orientation
has important consequences in DNA synthesis, as DNA can only be
synthesized in the 5′ to 3′ direction. This orientation also has consequences for
RNA synthesis, as RNA can also only be synthesized in the 5′ to 3′ direction.
Transcription
Transcription is the synthesis of a messenger RNA (mRNA) molecule from
a DNA template. There are three major differences between an RNA molecule
and a DNA molecule: (1) RNA molecules use the sugar ribose instead of
2-deoxyribose; (2) RNA molecules are single-stranded; and (3) RNA molecules
use the nitrogenous base uracil (U) instead of thymine.
In transcription, only one of the two strands of DNA, called the template
strand, is transcribed. The other strand, the coding strand, has a sequence that
is the same as the newly created RNA transcript (with U substituted for T). An
enzyme called RNA polymerase reads the template strand in the 3′ to 5′ direction
and synthesizes the new mRNA in the 5′ to 3′ direction (Figure 9.3).
Transcription begins when various proteins called transcription factors
interact with regions of the genome called promoters. A promoter is a specific
sequence of DNA that facilitates the transcription of a specific gene. These
promoters are often located adjacent and upstream of the genes they regulate,
toward the 5′ region of the coding strand. When a transcription factor binds to
a promoter, multiple proteins are recruited to begin the transcription process.
Identifying Genes and Proteins of Interest Chapter | 9  207
Coding strand
Template strand
Coding strand
Template strand
Coding strand
Template strand
Coding strand
Template strand
TF
TF
TF
TF
RNA
Polymerase
RNA
Polymerase
RNA
Polymerase
FIGURE 9.3  The transcription of DNA into RNA. (A) The process of transcription begins
when specific transcription factors interact with promoter sequences on the genome. These factors
recruit other proteins that are necessary to attract RNA polymerase. (B) RNA polymerase binds to
the complex between transcription factors and DNA. Once bound, the enzyme begins prying the two
strands of DNA apart. (C) RNA polymerase proceeds down the length of the DNA, simultaneously
unwinding DNA and adding nucleotides to the 3′ end of the growing RNA molecule. (D) When
transcription is complete, RNA polymerase dissociates from the DNA and the two DNA strands
reform a double helix conformation. A new mRNA is produced that is complementary to the DNA
template strand.
208  Guide to Research Techniques in Neuroscience
After transcription terminates, a newly formed RNA molecule receives further
processing in the form of RNA splicing before leaving the nucleus. At
this point, the RNA is composed of exons and introns flanked by untranslated
regions (UTRs) at either end (Figure 9.4). An exon is a sequence of an RNA
strand that remains after splicing occurs, while an intron is a sequence that is
lost. The 5′ and 3′ UTRs are sequences that cap the RNA molecule and are not
translated into amino acids. RNA splicing is catalyzed by a macromolecular
complex known as a spliceosome, which cuts exon and intron sequences apart
and then joins the exons back together to form a single strand. After RNA splicing,
a mature mRNA molecule leaves the nucleus and migrates to the cytoplasm,
where it can be translated into a protein.
Translation
Translation is the process of using an mRNA molecule as a template to produce
a protein. This process is performed by specialized macromolecules in the
cytoplasm called ribosomes. A ribosome reads an mRNA molecule in the 5′ to
3′ direction and uses this template to guide the synthesis of a chain of amino
acids to form a protein. Amino acids are the building blocks of all proteins and
are assembled as a long strand, much like nucleotides are assembled as a long
strand in a nucleic acid molecule. Each sequence of three nucleotides of an
mRNA molecule code for one amino acid. The three nucleotides collectively
form a codon, a translation of genetic information into an amino acid monomer.
There is a precise genetic code for the conversion of codons into amino acids
(Figure 9.5), with each codon precisely coding for one specific amino acid. One
codon, AUG, codes for the amino acid methionine, which is the start of any protein
sequence. Other codons (UAA, UAG, and UGA) serve as “stop” codons and
terminate translation. After translation ends, the newly formed polypeptide chain
folds upon itself to assume its native protein conformation. These proteins can
FIGURE 9.4  RNA splicing. Before mRNA leaves the nucleus, macromolecular complexes
called spliceosomes remove noncoding intron sequences and rejoin exon sequences. Noncoding
sequences remain at the 5′ and 3′ ends.
Identifying Genes and Proteins of Interest Chapter | 9  209
be further processed by other organelles, such as the endoplasmic reticulum, or
receive posttranslational modifications from other proteins.
This incredibly brief survey of DNA, transcription, and translation is no
match for much more detailed descriptions found in genetics or molecular
biology textbooks. However, this information is the foundation on which most
molecular neuroscience techniques are based.
GENETIC SCREENS
A genetic screen is a process of identifying genes that contribute to a phenotype.
Genetic screens fall into two categories: forward or reverse screens.
A forward genetic screen is a screen used to identify genes important for a
biological phenotype. In these screens, an investigator selects a scientifically
interesting phenotype and seeks to determine which genes are necessary for
the generation of this phenotype. A reverse genetic screen is the opposite: an
investigator selects an interesting gene and seeks to determine which phenotypes
result from its absence. Forward genetic screens often test thousands of
genes at once, while a reverse genetic screen tests only a single gene. Forward
genetic screens have been compared to fishing because a scientist is never quite
sure which genes he or she will catch; reverse genetic screens have been compared
to gambling because a scientist places all of his or her hopes on that gene
producing an interesting phenotype. Forward genetic screens can be considered
Stop
Stop
Stop
U C A G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
U
C
A
G
Firstletter
Second letter
Thirdletter
UUU
UUC
UUA
UUG
CUU
CUC
CUA
CUG
AUU
AUC
AUA
AUG
GUU
GUC
GUA
GUG
UCU
UCC
UCA
UCG
CCU
CCC
CCA
CCG
ACU
ACC
ACA
ACG
GCU
GCC
GCA
GCG
UAU
UAC
UAA
UAG
CAU
CAC
CAA
CAG
AAU
AAC
AAA
AAG
GAU
GAC
GAA
GAG
UGU
UGC
UGA
UGG
CGU
CGC
CGA
CGG
AGU
AGC
AGA
AGG
GGU
GGC
GGA
GGG
Phe
Leu
Ile
Leu
Met
Val
Ser
Pro
Thr
Ala
Tyr
His
Gln
Asn
Lys
Asp
Glu
Cys
Trp
Arg
Ser
Arg
Gly
FIGURE 9.5  The translation of mRNA into proteins. (A) A ribosome translates the genetic
sequence of an mRNA molecule into an amino acid sequence that forms a polypeptide. mRNA
sequences are processed in the 5′ to 3′ direction. Each series of three nucleotides, called codons,
translates into a single amino acid. (B) The genetic code for the conversion of codons into amino
acids. Each codon translates into a specific amino acid (abbreviated here in three letters, such as
Phe, which is the abbreviation for phenylalanine). Because there are more possible codons than
amino acids, some codons translate into the same amino acids, such as UUU and UUC. The AUG
codon translates into methionine and signals the start of translation. The UAA, UAG, and UGA
codons do not translate into amino acids, instead serving as a stop signal and terminating translation.
210  Guide to Research Techniques in Neuroscience
“hypothesis generating,” because they typically produce multiple candidate
genes that may be important for a phenotype; reverse genetic screens can be
considered “hypothesis testing,” because a scientist can test specific hypotheses
about the role of a gene in a phenotype. Here, we briefly describe the process of
performing a forward or reverse genetic screen.
Forward Genetic Screen
In a forward genetic screen, a scientist mutagenizes animals to produce thousands
of lines that may have a mutation in a single gene, and then screens each
individual to identify animals with an abnormal phenotype. These screens are
typically performed in flies and worms, although they have also rarely been performed
in mice (Box 9.1). There are six main steps in performing a forward
genetic screen:
	1.	Design an assay to measure a phenotype. The first step in determining which
genes may be important for a phenotype is to design a specific, quantitative
assay to discriminate between wild-type individuals and animals with an
aberrant phenotype. This requires fully characterizing the wild-type phenotype
and choosing measurable parameters to identify individuals with an
abnormal phenotype. For example, a scientist performing a forward genetic
screen to determine genes important for axon guidance might choose a specific
axon and characterize its normal growth and development over time.
When screening individuals, the scientist could look for axons that mistarget
to abnormal locations in the brain. Note that this screen requires the scientist
to identify the same axon in thousands of individuals. Alternatively, a
scientist could choose a behavioral phenotype, characterizing the normal
behavioral response to a stimulus and then examining individuals with an
abnormal behavior. The abnormal behavior must be statistically different
from the normal variation in behavior between individuals.
	2.	Mutagenize eggs/larvae. There are three methods of developing mutant lines
of organisms. In chemical mutagenesis, a scientist applies a mutagenizing
chemical, such as ethyl methane sulfonate or N-ethyl-N-nitrosourea, to
thousands of eggs/larvae, which statistically creates lines of animals with
mutations in a single gene in the genome. In irradiation mutagenesis, a
scientist achieves the same goal with high-intensity ultraviolet light, which
damages DNA at a rate such that animals statistically have a mutation in a
single gene in the genome. Finally, in insertional mutagenesis, a scientist
uses various methods to insert mobile genetic elements called transposons
into the genomes of offspring (Chapter 12). These transposons insert at random
locations in the genome, occasionally disrupting an endogenous gene.
Therefore, they can be used to test thousands of animals, each with a potential
loss-of-function of a single gene.
	3.	Screen for abnormal phenotypes. After hundreds or thousands of mutant
lines are produced, a scientist screens each individual in a phenotypic assay,
Identifying Genes and Proteins of Interest Chapter | 9  211
Box 9.1  Genetic Model Organisms
Hundreds of studies are regularly published in peer-reviewed journals that use
genetic tools to investigate the nervous system. Interestingly, nearly all of these
studies use the same five species as model organisms: the worm (Caenorhabditis
elegans), the fly (Drosophila melanogaster), the zebrafish (Danio rerio), the mouse
(Mus musculus), and the human (Homo sapiens). There are occasional exceptions,
such as the frog (Xenopus laevis) or the sea slug (Aplysia californica), but
these species collectively represent less than 1% of published studies. It is obvious
why neuroscientists would want to learn more about the genes of humans,
as the ultimate goal of neuroscience is to better understand our own species. But
why did the other four species become such tractable organisms for genetic studies?
Why use roundworms and fruit flies? Why not flatworms, bees, goldfish, or
gerbils? Many of these other organisms have the same short breeding time, high
number of offspring, and other factors that might make them attractive model
species.
The answer can essentially be summarized in a single word: history. There
really is not a good reason why scientists started using mice instead of gerbils,
but when multiple laboratories started performing genetic experiments on these
animals, it made sense for other laboratories to follow. It is useful for multiple
laboratories to study the same species: when dozens or hundreds of laboratories
each study the same organism, a community develops that allows researchers to
better share results, reagents, and genetic tools.
There is no better example of a community forming around a genetic model
organism than the community of scientists that study Drosophila. Ever since
Thomas Hunt Morgan began using the fly as a model organism to study genetics
in 1910, a growing number of scientists learned more and more about ideal
conditions and practices for using flies in experiments. As the knowledge about
fly maintenance increased, so did the number of genetic tools amenable to fly
genetic studies. In the late 1960s, Seymour Benzer used Drosophila to study the
link between genes and behavior. In the 1980s and 1990s, scientists developed
dozens of molecular genetic tools for carefully dissecting genetic circuits in flies,
and in 2000, the fly became the second eukaryotic organism to have its genome
sequenced.
A similar history developed for the worm: the prominent scientist Sydney
Brenner began using Caenorhabditis elegans as a model organism in the 1960s.
After years of study, scientists have given all 302 neurons specific names and
mapped out the connections of all 7000 synapses. There is now an energetic
C. elegans community just as there is a vibrant Drosophila community, with
many established methods for maintaining and manipulating genetic strains of
worms.
Table 9.1 summarizes some of the traits and benefits of these commonly used
genetic organisms. We mention these species not only because they are highly
represented in the literature, but also because virtually all novel genes and proteins
are discovered in these species. The methods described in this chapter to identify
functionally relevant genes depend on the decades-long development of these
organisms for biological research.
212  Guide to Research Techniques in Neuroscience
one mutant at a time. This is easily the most time-consuming aspect of a
forward genetic screen. Any mutant lines that show an abnormal phenotype
are maintained and bred for future experiments. The vast majority of mutant
lines that do not show aberrant phenotypes are either used for other genetic
screens or discarded.
TABLE 9.1  Relative Advantages and Disadvantages of Different Organisms
Used for Genetic Studies
Species Advantages Disadvantages
Worm
(Caenorhabditis
elegans)
Simple, multicellular organism
Genome sequenced
Short lifespan
Relatively easy to manipulate
genes and screen for genes of
interest through mutagenesis
Can be frozen
All neurons and their connections
are known
Invertebrate
Primitive nervous system
Fruit fly
(Drosophila
melanogaster)
Complex, multicellular organism
Genome sequenced
Short lifespan, rapid reproduction
rate
Relatively easy to manipulate
genes
Many mutant lines available
Invertebrate
Primitive nervous system
Zebrafish
(Danio rerio)
Genome sequenced
Can use powerful invertebrate
techniques like Gal4/UAS system
Good for imaging studies during
development because eggs are
clear
Large number of offspring
Not a mammal
Mouse
(Mus musculus)
Complex, higher-order organisms
Genome sequenced
Possible to manipulate genes in
whole organism or specific tissues
Nervous system is homologous to
humans
Relatively long lifespan
and reproductive time
Genetic manipulations
are lengthy and costly
Greater genetic
redundancy than
invertebrates
Human
(Homo sapiens)
Complex, higher-order organism
Genome sequenced
Can observe naturally occurring
genetic mutations and
polymorphisms
Long lifespan
Not tractable for
experimental genetic
manipulation
Identifying Genes and Proteins of Interest Chapter | 9  213
	4.	Perform complementation analysis. Once a genetic mutant is isolated, investigators
often perform a complementation test to determine if the mutation
is unique or has been previously described. This approach is used when a
mutation results in the same phenotype as a previously described mutation.
The two strains could have mutations in the same gene or in two different
genes. To differentiate between these two possibilities, the two strains are
mated and the phenotypes of the offspring are identified. If the offspring
exhibits the wild-type phenotype, a scientist concludes that each mutation is
in a separate gene. However, if the offspring does not express the wild-type
phenotype but rather a mutant phenotype, the scientist concludes that both
mutations occur in the same gene.
	5.	Map the gene. Just because a scientist has discovered a mutation in a gene
that alters a phenotype does not mean that the scientist knows the molecular
identity of that gene. The only way to determine the molecular sequence of
the gene and its location in the genome is to map the gene. If mutagenesis was
caused by chemical or irradiation methods, investigators typically perform a
linkage analysis to map the gene. This technique is used to identify the relative
position of a novel gene in relation to the location of known genes. To
map the gene, a scientist mates the mutant animals with animals of a separate
mutant phenotype. The two traits are then assessed in the offspring to determine
if they are inherited together at relatively high rates. When two traits
are closely linked in future offspring, they appear together more frequently
and there is a greater likelihood that they are located close to each other on a
chromosome. Because many genomes have been sequenced, a scientist can
identify potential regions on a chromosome where the mutant gene may be
found. If mutagenesis was caused by insertional mutagenesis methods, it is
much easier to identify the molecular identity of the mutated gene. A scientist
simply uses the transposon’s genetic sequence to design primers for DNA
sequencing, and then sequences DNA in either direction of the transposon to
identify the region of the genome where it landed (Chapter 10).
	6.	Clone the gene. The final step in the process of identifying novel genes for
a phenotype is to clone the DNA encoding the gene. This allows a scientist
to perform future genetic experiments that manipulate the gene in vitro or
in vivo. We will discuss methods of cloning genes in Chapter 10.
Reverse Genetic Screen
In a reverse genetic screen, a scientist identifies a gene of interest and then
perturbs the gene to determine its role in various phenotypes. For example, if
a scientist hypothesizes that a certain gene is necessary for proper formation
of neuromuscular junctions in the mouse, the scientist could make a knockout
mouse, removing the gene from the genome and examining whether proper synapses
form between peripheral nerves and motor units. We describe the process
of performing these kinds of experiments in Chapters 12 and 13.
214  Guide to Research Techniques in Neuroscience
IN SILICO SCREENS
Using modern technology, it is possible to use bioinformatics and genomics
databases to identify genes and proteins that may be relevant for a phenotype of
interest. An in silico screen is a computer-based method that identifies and compares
similar DNA and/or protein sequences from multiple species. Suppose that
a scientist identifies a gene encoding a novel transmembrane receptor. The scientist
may be able to identify and discover other receptors by looking for similar
sequences in the genome. Alternatively, if a gene is discovered in one species, a
scientist can use bioinformatics to identify orthologous sequences in other species.
These genes may have the same function or very different functions. Two
examples of publicly available genetic databases are BLAST and Ensembl.
BLAST
The Basic Local Alignment Search Tool (BLAST) is a tool used to compare the
nucleotide sequences of DNA or the amino acid sequences of proteins. The power
of BLAST is the ability to compare a DNA or protein sequence with the entire
library of known sequences from all species. The user can set a threshold for the
degree to which two sequences form a match. Therefore, if a scientist wants to identify
genes with high sequence similarity to a gene of interest, the BLAST program
can identify similar genes with high sequence similarity, and potentially similar
functions. Many genes in the mouse genome have been identified due to their
similarity with other structures, especially neuropeptides and ion channels. The
best way to learn about BLAST is to try it: http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Ensembl
Ensembl is a bioinformatics browser that functions much like BLAST but
with a greater emphasis on genomic analysis. This tool provides the actual genomic
sequences for a huge diversity of animal models, allowing comparative
genomic searches to be performed. Additionally, this program can be used for
analyzing sequence alignment, to investigate gene homology, and to obtain specific
gene sequences to be used for subsequent experiments. Ensembl has the complete
genomes and rough drafts of genomes for dozens of animals, including standard
model organisms, as well as relatively esoteric organisms, such as the bush
baby, the bat, and the platypus. Again, the best way to learn about this genetic
tool is to try it: http://www.ensembl.org/index.html.
MOLECULAR SCREENS
A molecular screen uses molecular biology techniques to identify genes important
for a specific biological phenotype. Unlike standard genetic screens, molecular
screens use high-throughput strategies and thousands of nucleic acid probes
to identify the molecular components of a phenotype.
Identifying Genes and Proteins of Interest Chapter | 9  215
cDNA Microarray Screen
A microarray is a device used to measure and compare gene expression profiles
from biological samples. It can be used to analyze gene expression from
a single sample or to compare gene expression between two different samples.
Because gene expression is tightly regulated, genes are only expressed at times
when they serve a useful function. Therefore, a scientist can potentially identify
a gene of interest based on how its expression changes over time or if its expression
changes in response to a stimulus or environmental condition. The power
of a microarray is that it is possible to assay all genes in the genome at the same
time in a relatively small device that can fit in the palm of your hand.
A microarray consists of an array of thousands of tiny microscopic spots
of DNA sequences, often sequences that are parts of genes (Figure 9.6).
A scientist takes a biological sample, extracts the RNA, and then uses a special
enzyme called reverse transcriptase to make complementary DNA (cDNA)
FIGURE 9.6  A cDNA microarray.  A microarray is
used to analyze gene expression in a biological sample
and compare expression patterns with other samples.
mRNA is extracted from a sample and reverse transcribed
to produce cDNA. The cDNA is labeled and
then hybridized to a microarray chip containing thousands
of potentially complementary strands. A computer
detects the amount of signal from each specific
sequence. Thus, a microarray can be used to detect
which specific mRNA sequences were expressed in the
original sample.
Obtain sample
Extract RNA
Use reverse
transcriptase
to make cDNA
Hybridize cDNA
to microarray
Analyze
hybridization
data
216  Guide to Research Techniques in Neuroscience
from the RNA strands. The cDNA is labeled and incubated with the surface
of the DNA array. The cDNA is allowed time to hybridize with the
sequences on the array. A computer subsequently scans the entire array and
detects hybridization between cDNA in the sample and spots on the array.
Because the computer contains a database of every spot on the array, a scientist
can learn which genes were present in a sample and which genes were
undetected.
A microarray can be used to identify novel genes for a neurobiological phenotype.
For example, suppose a scientist wishes to identify genes that respond
to light/dark cycles. The scientist can collect samples from a part of the brain
known to play a role in regulating light/dark cycles, such as the suprachiasmatic
nucleus. Some samples can be collected from animals during the day,
while other samples are collected at night. A comparison in the gene expression
profile between day/night genes will produce a list of genes that show differential
expression during those two periods. Most microarray experiments usually
result in the identification of dozens, if not hundreds of genes. Sometimes these
lists can be overwhelming to an investigator, and much follow-up is required to
reduce the list to a more manageable set of candidates for future experiments.
For example, a scientist can repeat the experiment several times to reduce the
rate of false-positive identification of genes, or more precisely control the environmental
conditions during which the original RNA was extracted.
RNAi Screen
RNA interference (RNAi) is a technique used to knock down the expression
of mRNA molecules (Chapter 13). RNAi screens use thousands of RNAi molecules
to disrupt individual gene function in a variety of organisms, most notably
worms, or cultured cells. For example, in a multiwell-based RNAi screen,
hundreds of unique RNAi molecules are each added to the bottom of 384-well
plates, with a separate RNAi molecule in each well. Cells are added to the wells
and the scientist observes changes in various parameters, such as morphology,
proliferation, or death rate. Thus, an investigator can determine which RNAi
molecules, and therefore which genes, are necessary for a cellular process to
occur.
CONCLUSION
This chapter describes the first steps in identifying new genes and their protein
products. These techniques are often the beginning steps of discovering the role
of a gene in a biological process or phenotype. Other methods are necessary
to validate this discovery, such as methods that perturb the expression of the
gene to identify its necessity and/or sufficiency for a phenotype. The techniques
described in the remaining chapters use molecular and genetic methods to gain
insight into the roles of newly discovered genes in vitro and in vivo.
Identifying Genes and Proteins of Interest Chapter | 9  217
SUGGESTED READING AND REFERENCES
Books
Greenspan, R. J. (2004). Fly pushing: The theory and practice of drosophila genetics (2nd ed.).
Cold Spring Harbor Laboratory Press.
Griffiths, A. J. F., Wessler, S. R., & Carroll, S. B. (2010). Introduction to genetic analysis (10th ed.).
New York: Freeman.
Krebs, J. E., Goldstein, E. S., & Kilpatrick, S. T. (2012). Lewin’s genes IX (9th ed.). Sudbury, MA:
Jones and Bartlett.
Review Articles
Goldowitz, D., Frankel, W. N., Takahashi, J. S., et al. (2004). Large-scale mutagenesis of the mouse
to understand the genetic bases of nervous system structure and function. Brain Research.
Molecular Brain Research, 132, 105–115.
Medland, S. E., Jahanshad, N., Neale, B. M., & Thompson, P. M. (2014). Whole-genome analyses
of whole-brain data: working within an expanded search space. Nature Neuroscience, 17,
791–880.
Paddison, P. J., Silva, J. M., Conklin, D. S., et al. (2004). A resource for large-scale RNA-interferencebased
screens in mammals. Nature, 428, 427–431.
Ryder, E., & Russell, S. (2003). Transposable elements as tools for genomics and genetics in Drosophila.
Briefings in Functional Genomics & Proteomics, 2, 57–71.
Wu, S.,Ying, G., Wu, Q., & Capecchi, M. R. (2007). Toward simpler and faster genome-wide mutagenesis
in mice. Nature Genetics, 39, 922–930.
Primary Research Articles—Interesting Examples from the Literature
Crick, F. H. (1970). Central dogma of molecular biology. Nature, 227, 561–563.
Devineni, A. V., Eddison, M., & Heberlein, U. (2013). The novel gene tank, a tumor suppressor
homolog, regulates ethanol sensitivity in Drosophila. Journal of Neuroscience, 33, 8134–8143.
Husken, U., Stickney, H. L., Gestri, G., Bianco, I. H., et al. (2014). Tcf7l2 is required for left-right
asymmetric differentiation of habenular neurons. Current Biology, 24, 2217–2227.
Schuldiner, O., Berdnik, D., Levy, J. M., et al. (2008). piggyBac-based mosaic screen identifies
a postmitotic function for cohesin in regulating developmental axon pruning. Developmental
Cell, 14, 227–238.
Tracey, W. D., Jr, Wilson, R. I., Laurent, G., & Benzer, S. (2003). Painless, a Drosophila gene essential
for nociception. Cell, 113, 261–273.
Ule, J., Ule, A., Spencer, J., et al. (2005). Nova regulates brain-specific splicing to shape the synapse.
Nature Genetics, 37, 844–852.
Walsh, T., McClellan, J. M., McCarthy, S. E., et al. (2008). Rare structural variants disrupt multiple
genes in neurodevelopmental pathways in schizophrenia. Science, 320, 539–543.
Zarbalis, K., May, S. R., Shen,Y., Ekker, M., Rubenstein, J. L., & Peterson, A. S. (2004). A focused
and efficient genetic screening strategy in the mouse: identification of mutations that disrupt
cortical development. PLoS Biology, 2, E219.
Protocols
Bökel, C. (2008). EMS screens: from mutagenesis to screening and mapping. Methods in Molecular
Biology, 420, 119–138.
Lehner, B., Tischler, J., & Fraser, A. G. (2006). RNAi screens in Caenorhabditis elegans in a
96-well liquid format and their application to the systematic identification of genetic interactions.
Nature Protocols, 1, 1617–1620.
218  Guide to Research Techniques in Neuroscience
Nolan, P. M., Kapfhamer, D., & Bucan, M. (1997). Random mutagenesis screen for dominant
behavioral mutations in mice. Methods, 13, 379–395.
Ramadan, N., Flockhart, I., Booker, M., Perrimon, N., & Mathey-Prevot, B. (2007). Design and
implementation of high-throughput RNAi screens in cultured Drosophila cells. Nature Protocols,
2, 2245–2264.
Websites
BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi.
Ensembl: http://www.ensembl.org/index.html.
219
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00010-1
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 10
Molecular Cloning and
Recombinant DNA Technology
After Reading This Chapter, You Should be Able to:
	l	Describe tools used to manipulate and recombine DNA
	l	Explain how to create novel DNA constructs
Techniques covered:
	l	Isolation of DNA fragments: restriction enzymes, polymerase chain reaction
(PCR), reverse transcription PCR (RT-PCR), quantitative real time PCR (qRT-PCR),
gel electrophoresis
	l	Cloning DNA fragments: vectors, ligation, transformation, DNA purification
	l	Identifying DNA fragments: DNA sequencing, Southern blot, northern blot
Modern molecular biologists manipulate DNA much like classic filmmakers edit
movies. If a filmmaker wishes to take a scene from one film reel and add it to another
film reel, he or she can locate the scene, physically cut it out, splice it together into
the new film reel, and run the film to ensure that the sequence has integrated correctly.
Likewise, a molecular biologist may want to cut out one DNA sequence from
a specialized DNA storage system, called a vector, and splice it into a new vector.
A scientist can do this in the same way as the filmmaker, with specific molecular
tools that take the place of scissors, tape, and a sequence identification system.
Identifying and cutting a piece of DNA out of the genome for future use is
referred to as molecular cloning. Cloning allows the isolation and amplification
of a specific DNA sequence. This sequence can then be manipulated using
the tools of recombinant DNA technology to form novel sequences, mutate
endogenous genes, tag genes with reporter constructs, and much more. In other
words, modern molecular techniques allow a scientist to manipulate DNA in
just about any way that he or she would like.
In particular, aspects of two molecular cloning have revolutionized a scientist’s
ability to manipulate DNA: (1) the ability to cut, alter, and join DNA
molecules in vitro and (2) the ability to copy and mass produce recombinant
DNA molecules in host/vector systems.
220  Guide to Research Techniques in Neuroscience
The purpose of this chapter is to describe these tools and how they are used
to clone and manipulate DNA constructs. First, we will discuss methods of isolating
DNA fragments from the genome and other DNA vectors. Then we will
describe molecular cloning: how these isolated fragments are inserted into storage
vectors and mass produced using bacteria. Next, we describe how DNA
fragments or recombinant DNA constructs are purified from other DNA molecules.
Finally, we describe methods of reading the molecular sequence of a
DNA construct. After creating and purifying a novel DNA construct, a scientist
can deliver this construct into various cell types (Chapter 11) or use the construct
to produce a transgenic or knockout animal (Chapters 12 and 13).
ISOLATING DNA FRAGMENTS
In Chapter 9, we discussed methods of identifying genes that could be important for
a neural phenotype. Once a gene has been identified, isolating the DNA sequence
that encodes the gene is critical for subsequent experiments and techniques used to
manipulate and recombine the DNA in useful ways. There are two primary methods
used to isolate DNA sequences from their location in the genome or a storage
vector: (1) restriction digests that cut DNA into fragments and (2) polymerase chain
reaction (PCR), which amplifies DNA sequences of interest.
Restriction Enzymes
The discovery of restriction enzymes (also called restriction endonucleases)
that recognize and cut specific double-stranded DNA sequences is one of the
most important advances in biology that makes recombinant DNA technology
possible. Restriction enzymes are like targeted molecular scissors, cutting double-stranded
DNA at unique base pair sequences called recognition (or restriction)
sites. These enzymes are naturally produced by various strains of bacteria,
which normally produce these enzymes to cleave foreign DNA molecules in
their environment. Molecular biologists have identified and purified hundreds
of unique restriction enzymes from many species of bacteria, and now these
enzymes can be easily ordered from a variety of commercial manufacturers.
Each enzyme recognizes a unique sequence of about 4–8 nucleotides and
cuts at specific locations within these recognition sites in a process called a
restriction digest. Some restriction enzymes digest DNA in a way that produces
blunt ends on the newly cut strands (Figure 10.1A). Others produce
staggered cuts that leave sticky or cohesive ends with short single-stranded
tails hanging over the ends of each fragment (Figure 10.1B). Each tail can form
complementary base pairs with the tail at another end produced by the same
enzyme. This allows any two DNA fragments to be easily joined together, as
long as the fragments were generated with the same restriction enzyme (or
another enzyme that leaves the same sticky ends). These different ends can be
thought of as puzzle pieces whose shape is determined by the particular enzyme
Molecular Cloning and Recombinant DNA Technology Chapter | 10  221
used. Therefore, if the same enzyme is used to cut two different pieces of DNA,
then the resulting pieces will fit with each other because they have the same
corresponding sticky ends that complement each other.
Polymerase Chain Reaction
PCR is a biochemical reaction that uses controlled heating and cooling in the
presence of DNA synthesizing enzymes to exponentially amplify a small DNA
fragment. It requires only tiny amounts of DNA to be used as a template from
which a selected region will be amplified a billion-fold, effectively purifying
this DNA fragment away from the rest of the template DNA.
Short single-stranded oligonucleotides (about 20–30 nucleotides in length)
called primers are essential ingredients in PCR. Primers specify what fragment
will be amplified by binding to the template in the region flanking the
sequence of interest (Figure 10.2). Where do these primers come from? They
are synthesized in vitro using what is called the “solid-phase” method. In this
method, short DNA strands are synthesized by adding activated monomers to
a growing DNA chain linked to an insoluble support. DNA chains of as many
as 100 nucleotides can be synthesized using this automated method. This type
of chemical DNA synthesis can also be used to create labeled oligonucleotides
for nucleic acid hybridization techniques. DNA sequences can also be modified
using PCR either by adding restriction sites to the ends of a DNA fragment or
by introducing mutations in the sequence to characterize the function of the
DNA fragment. Laboratories do not often chemically synthesize DNA fragments
themselves but rather order them from a company or core facility at their
institution that specializes in oligonucleotide synthesis.
Standard PCR
Standard PCR starts with a DNA template, often from a genomic or complementary
DNA (cDNA) library (Box 10.1), and uses primers to selectively
amplify a specific DNA fragment of interest. The ingredients of a PCR reaction
include a DNA template containing the target sequence to be amplified; a pair
of oligonucleotide primers that hybridize with the sequences flanking the target
sequence; all four deoxyribonucleoside triphosphates (dNTPs—A, T, G, C);
FIGURE 10.1  Restriction enzymes. Acting
like molecular scissors, restriction enzymes
recognize specific sequences and cut at specific
locations within those sequences. They
can either (A) leave blunt ends, such as the
enzymes SmaI and HpaI or (B) make uneven
cuts that result in sticky ends, such as the
enzymes EcoRI and HindIII.
222  Guide to Research Techniques in Neuroscience
and a heat-stable DNA polymerase, an enzyme that adds nucleotides onto the
end of a forming DNA strand.
A PCR cycle consists of three steps (Figure 10.2):
	1.	Strand separation. The two strands of the template DNA molecule are separated
by heating the solution to 95 °C for about 15 s so that each strand can
serve as a template.
	2.	Hybridization of primers. The solution is abruptly cooled to about 50–55 °C
to allow each primer to hybridize to a DNA strand. One primer hybridizes
FIGURE 10.2  Polymerase chain reaction (PCR).  Double-stranded DNA is heated to separate
the strands. The temperature is lowered to approximately 50–55 °C to allow primers to hybridize
to their complementary sequences on the separated DNA strands. The temperature is then raised to
approximately 70–75 °C so that Taq DNA polymerase can add nucleotides to the growing 3′ end of
the primer. This cycle is repeated 20–30 times until the target DNA sequence between the primers
is greatly amplified.
Molecular Cloning and Recombinant DNA Technology Chapter | 10  223
BOX 10.1  DNA Libraries
A DNA library is a comprehensive collection of cloned DNA fragments from a
cell, tissue, or organism. DNA libraries can be used to isolate a specific gene of
interest, as they generally include at least one fragment that contains the gene.
Libraries can be constructed using any of a variety of different vectors (plasmid,
phage, cosmid, or artificial chromosomes) and are generally housed in a population
of bacterial cells. Nowadays, it is rare for a single scientist or laboratory to
produce a DNA library. Instead, if a scientist wants to isolate a particular genomic
sequence, he or she can order a vector from a DNA library repository.
Plasmid DNA libraries are created using the same process used to clone genes
into expression vectors (Figure 10.5). Plasmid and cellular DNA are both cut with
restriction enzymes and annealed via their sticky ends to form recombinant DNA
constructs. These recombinant molecules containing foreign DNA inserts are then
covalently sealed with DNA ligase, and bacteria are transformed using these recombinant
DNA plasmids. Each bacterial cell that was initially transformed contains, in theory,
a different foreign DNA insert, replicated in all the clones of the bacterial colony.
For many years, plasmids were used to clone DNA fragments of 1–30 kb. Larger
DNA fragments are more difficult to handle and thus were harder to clone. Then
researchers began to use artificial chromosomes—larger DNA vectors, such as
yeast artificial chromosomes (YACs), which could handle very large pieces of
DNA (100-1000 kb). Today, new plasmid vectors based on the naturally occurring
F plasmid of the bacteria Escherichia coli are used to clone DNA fragments of
150–350 kb. Unlike smaller bacterial plasmids, the F plasmid—and its derivative,
the bacterial artificial chromosome (BAC)—is present in only one or two copies
per E. coli cell. Because of their stability, ability to accept large DNA inserts, and
ease of handling, BACs have been the preferred vector for building DNA libraries of
complex organisms—including those representing the human and mouse genomes.
Genomic DNA Libraries
Cleaving the entire genome of a cell with a specific restriction enzyme and cloning
each fragment is sometimes called the “shotgun” approach to gene cloning. This
technique can produce a very large number of DNA fragments that will generate
millions of different colonies of transformed bacterial cells. Each of these colonies
is composed of bacteria derived from a single ancestor cell and therefore contains
a particular stretch of the fragmented genome. Such a plasmid is said to contain
a genomic DNA clone, and the entire collection of plasmids is called a genomic
DNA library. Because the genomic DNA is cut into fragments at random, only
some fragments contain genes that encode functional protein products. Many of
the genomic DNA clones obtained from mammalian DNA samples contain only
noncoding DNA, which makes up most of the DNA in such genomes. By making
genomic libraries based on mRNA rather than DNA, investigators can produce
cDNA libraries that contain only the DNA sequences used to produce proteins.
cDNA Libraries
By starting with mRNA rather than genomic DNA, only DNA sequences that correspond
to protein-encoding genes are cloned. These libraries are created by extracting
mRNA from cells and then using RT-PCR to make a cDNA copy of each mRNA
Continued
224  Guide to Research Techniques in Neuroscience
to the 3′-end of the target on one strand, and the other primer hybridizes to
the 3′-end on the complementary target strand so that DNA extension from
the primers will be directed toward each other. This amplifies the region
between the primers. Parent DNA duplexes do not form because the primers
are present in great excess.
	3.	 DNA synthesis. The solution is heated to about 70–75 °C, which is the optimal
temperature for Taq DNA polymerase. This heat-stable polymerase is
derived from Themus aquaticus, a thermophilic bacterium found naturally
in hot springs. The polymerase elongates both primers in the direction of
the target sequence because DNA synthesis is in the 5′ to 3′ direction; new
dNTPs are added to the growing 3′ end of each primer. DNA synthesis takes
place on both strands and extends so that the target DNA sequence is fully
extended to the length between each of the two primers.
These three steps—strand separation, hybridization of primers, and DNA
synthesis—constitute one cycle of PCR amplification. A standard PCR protocol
consists of 25–30 cycles, and each cycle effectively doubles the amount of the
target DNA from the previous cycle. A single cycle requires only about 2–3 min
(depending on the length of the PCR template to be amplified), and the entire
procedure can be easily automated using modern PCR machines, also known as
thermocyclers. The end result of a PCR reaction is a solution in which the predominant
DNA species are several million copies of the target DNA sequence
dictated by the position of the two primers.
molecule present, and inserting this cDNA into a vector. Each clone obtained in
this way is called a cDNA clone, and the entire collection of clones derived from
one mRNA preparation constitutes a cDNA library. There are important differences
between genomic DNA clones and cDNA clones. Genomic clones represent
a random sample of all the DNA sequences in an organism and, with very
rare exceptions, are the same regardless of the cell type used to prepare them. In
contrast, cDNA clones contain only those regions of the genome that have been
transcribed into mRNA. Because cells of different tissues produce distinct sets of
mRNA molecules, a distinct cDNA library is obtained for each cell type used to
prepare the library. For example, a cDNA library derived from mouse neurons will
be different than a cDNA library derived from mouse fibroblasts.
A major advantage to using a cDNA library over a standard genomic DNA
library is that a cDNA library contains only the uninterrupted coding sequence
of a gene—only the exons that code for proteins, free from noncoding intron
sequences. Thus, when the aim of cloning is either to deduce the amino acid
sequence of a protein from the DNA sequence or to produce a protein in high
quantities by expressing the cloned gene in a bacterial or yeast cell, it is preferable
to start with cDNA. However, genomic DNA libraries are useful for examination of
noncoding regions in a genome.
BOX 10.1  DNA Libraries—cont’d
Molecular Cloning and Recombinant DNA Technology Chapter | 10  225
Reverse Transcription PCR
Reverse transcription PCR (RT-PCR) uses mRNA rather than DNA as the
starting template. First, the enzyme reverse transcriptase uses the mRNA template
to produce a complementary single-stranded DNA strand called cDNA
in a process known as reverse transcription. Next, DNA polymerase is used
to convert the single-stranded cDNA into double-stranded DNA. These DNA
molecules can now be used as templates for a PCR reaction as described above.
The value of RT-PCR is that it can be used to determine if an mRNA species is
present in a sample or to clone a cDNA sequence for a subsequent experiment.
Quantitative Real-Time PCR
To compare the relative amount of mRNA or DNA present in different samples,
a scientist can employ a modified version of PCR called quantitative realtime
PCR (qRT-PCR, not to be confused with RT-PCR described above). This
technique is based on detecting a fluorescent signal produced in proportion to
the PCR product during each cycle of DNA amplification. There are multiple
methods of producing a fluorescent signal during a qRT-PCR reaction. In the
TaqMan® assay (Figure 10.3), a fluorogenic probe within the region to be amplified
contains a fluorescent reporter tag on one end and a fluorescence quencher
on the other end. During each cycle of amplification, DNA polymerase cleaves
the fluorescent probe molecule, removing the quencher and allowing the reporter
to emit fluorescence. The increase in fluorescence is proportional to the amplification
during the exponential phase of amplification. An alternate method uses
a fluorescent dye called SYBR Green® that binds to double-stranded DNA; the
resulting DNA-dye complex is capable of absorbing blue light and emitting
green light, indicating the concentration of amplified double-stranded DNA.
A sensitive probe within the PCR chamber can measure and record the fluorescent
signal after each PCR cycle and produce a real-time measure of the concentration
of double-stranded amplified DNA fragments. Thus, a scientist can
compare the relative concentration between different samples.
Nucleic acid hybridization techniques such as Southern (DNA) or northern
(RNA) blots (discussed later in the chapter) are alternative methods to
DNA synthesis
displaces the probe
Probe is cleaved
by DNA polymerase
Quenched fluorogenic
probe binds template
FIGURE 10.3  Quantitative real-time PCR (qRT-PCR) using a TaqMan® assay.  A probe containing
a fluorescent reporter, R, is initially fused to a quencher, Q. During a PCR reaction, DNA
synthesis of the growing strand cleaves the probe, causing removal of the quencher and increasing
the fluorescent signal. Each round of amplification produces fluorescence that can be quantified to
determine the amount of nucleic acid template present in the sample.
226  Guide to Research Techniques in Neuroscience
quantitatively compare amounts of DNA or RNA present in samples. However,
these methods are not as quantitative as qRT-PCR and typically require large
concentrations of starting material.
Modifying DNA Sequences Using PCR
Although the primary purpose of PCR is to make exact copies of a DNA
sequence, this technique can also be used to modify a DNA sequence for
subsequent experiments. For example, a scientist may want to introduce a
point mutation into a sequence of DNA or add base pairs to a sequence
to introduce restriction sites. To modify DNA sequences using PCR, scientists
design primers that not only specify the DNA fragment to be amplified,
but also add or alter base pairs within the primer region itself. Although
the primers may not hybridize with the template DNA strands as tightly for
the first few PCR cycles, with each successive cycle, the added or mutated
sequences in the primers will be copied and incorporated into the amplified
PCR product.
Isolation and Characterization of DNA Fragments Using Gel
Electrophoresis
Once DNA fragments have been generated using restriction enzymes or PCR
amplification, they must be purified and separated from other DNA fragments
that are not of interest. The separation and identification of DNA fragments
based on their size is possible using a ubiquitous tool called gel electrophoresis.
Gel electrophoresis is used to isolate, identify, and characterize properties of
DNA fragments (Figure 10.4).
First, a scientist uses molds to make an agarose gel with wells at one end
for placing DNA samples. The gel is placed in an electrophoresis chamber and
DNA samples are added to the wells. The electrophoresis apparatus produces
a small electrical field, driving negatively charged DNA strands away from the
cathode (the negative end) and toward the anode (the positive end). The mobility
of the DNA fragments through the gel is inversely proportional to the logarithm
of the number of base pairs in the fragments. Therefore, smaller DNA
fragments will migrate much more quickly through the gel than large fragments.
One of the lanes of the gel typically contains a DNA ladder—DNA that has
been previously digested and characterized, producing fragments with known
base pair sizes. Therefore, it is possible to determine the sizes of the fragments
in the DNA samples by comparing their location to the location of the fragments
in the ladder.
The DNA bands on agarose gels are invisible unless the DNA is labeled
or stained. A common method of staining agarose gels is briefly incubating
them in a solution containing ethidium bromide, a DNA intercalating agent that
fluoresces under ultraviolet light when bound to DNA. After identifying DNA
Molecular Cloning and Recombinant DNA Technology Chapter | 10  227
fragments in a gel, it is possible to physically cut the DNA fragments out of the
gel and purify the DNA from the agarose using simple, commercially available
kits and protocols. After purifying the DNA, a scientist can clone the fragment
into a storage vector.
CLONING DNA
Once a DNA fragment is isolated using restriction enzymes or PCR techniques,
it is necessary to “store” the fragment into a vector for future use and
amplification. The process of isolating a piece of DNA, ligating it into a storage
vector, and producing enough copies of the vector for subsequent experiments
is called molecular cloning (Box 10.2). The process of transferring a
DNA sequence that already exists within a vector to a new vector is called
subcloning.
Vectors
A DNA vector is a nucleic acid storage system that can hold an isolated DNA
sequence of interest. The essential features of a vector are that it can replicate
autonomously in a host species, usually bacteria, and that it can be combined
with other pieces of DNA. Often, the inserted DNA fragment comes from a
different organism than the vector DNA, and the fusion is called a recombinant
DNA construct. Two commonly used vectors are plasmids, naturally occurring
circles of DNA that act as independently replicating accessory chromosomes in
bacteria, and bacteriophage or phage, a virus that can deliver its genetic cargo
to bacteria. It is much more common for scientists performing recombinant
DNA techniques in the laboratory to use plasmids over phage vectors. Many
BOX 10.2 The Definition of Clone
The word clone is used often in molecular biology. Unfortunately, this word has
distinct definitions that can often cause confusion. In general, the precise definition
of a clone is an exact copy. In reference to a piece of DNA, this term implies
that a scientist will make exact copies of a DNA sequence. In reference to a bacterial
cell, this term implies that a bacterium will multiply to eventually produce
millions of genetically identical bacteria, each harboring the same DNA as the
initial cell. Nowadays, it is possible to produce clones of larger organisms, including
mammals—meaning that the animals share identical copies of genomic DNA.
The term “cloning a gene” usually refers to the process of isolating a particular
DNA sequence, placing the sequence in a vector, and producing many copies of
the vector for use in subsequent experiments. For example, if a scientist identifies
a novel gene after performing a screen (Chapter 9), he or she could clone the gene
using PCR techniques, placing the gene in a vector, and using recombinant DNA
techniques to modify the gene in subsequent experiments.
228  Guide to Research Techniques in Neuroscience
plasmids have been ingeniously modified to enhance the delivery of recombinant
DNA molecules into bacteria and to facilitate the selection of bacteria
harboring these vectors.
Because there is a size limit to the number of base pairs that can be stably
inserted into a plasmid or phage vector (typically 1–25 kb), scientists sometimes
use other vectors that can hold larger amounts of DNA. A cosmid is a plasmid
that contains phage sequences that allow the vector to be packaged and transmitted
to bacteria like a phage vector. Cosmids are more stable than regular
plasmids, and therefore they can hold larger inserts (30–50 kb). Artificial chromosomes
containing telomeric and centromeric regions can hold even larger
inserts. Because cosmids and artificial chromosomes can stably hold large DNA
inserts, they are often used to create DNA libraries (Box 10.1)
Common Vector Features
Useful vectors contain three main features:
	l	An origin of replication. An origin of replication is a specific DNA sequence
at which DNA replication is initiated. The origin is therefore absolutely essential
for the amplification of the vector inside a bacterial host.
	l	 Useful restriction enzyme sites. Restriction sites are needed to place a DNA
sequence of interest into a vector. Because vectors are designed to be easy storage
systems for cloned DNA, a standard feature in many vectors is the presence
of a multiple cloning site (MCS), a region with multiple useful restriction
enzyme sites to make a compatible digest of the vector and DNA fragment
more feasible. Both the vector and the DNA fragment are cut using the same
restriction enzymes so that sticky ends from the DNA fragment match the
sticky ends on the vector (Figure 10.5). The two molecules can then be stitched
together through DNA ligation. The recombinant DNA molecule comprised of
the vector and the DNA insert is also known as a DNA construct.
	l	 A selectable marker.A selectable marker is a method of allowing bacteria containing
the vector to be readily identified and purified. Plasmids often confer
antibiotic resistance to the bacteria, so only bacteria containing the vector will
survive treatment with antibiotics. Common antibiotics used in the laboratory
include ampicillin and kanamycin, and many plasmids contain genes that confer
ampicillin and kanamycin resistance.
Types of Vectors
Cloning vectors provide a backbone for the DNA insert to be reproduced and
propagated in bacteria; however, these vectors are only useful for storing a
genetic sequence. By themselves, they are incapable of allowing for transcription
and translation of the gene into a functional protein product.
For a gene to give rise to a protein product, an expression vector must be
used that contains the necessary elements for a host cell to transcribe and translate
the gene. In the case of a mammalian cell, a standard mammalian expression
Molecular Cloning and Recombinant DNA Technology Chapter | 10  229
vector will contain an origin of replication, MCS, and selectable marker, as
described above. However, the expression vector will also need a promoter
found in mammalian cells that can drive the expression of the gene. The
coding DNA needs other features to be transcribed and translated, such as the
polyadenylation tail that normally appears at the end of transcribed pre-mRNA
and a sequence that attracts the ribosome for translation.
Ligation
To place a DNA fragment into a vector, both the fragment and vector are usually
cut using the same restriction enzymes so that they have the same sticky
end fragments. The fragment can then be “pasted” into the vector using DNA
ligase, an enzyme normally used by cells for DNA repair (Figure 10.5). It is
also possible to ligate DNA fragments and vectors together from restriction
enzyme reactions that produce blunt end fragments. However, because the
blunt ends can join in multiple orientations and configurations, there is a high
chance of ligation in the wrong orientation, or even the potential for multiple
DNA fragments to insert into the same vector backbone. Some vectors, such as
FIGURE 10.4  Gel electrophoresis. (A) DNA samples are loaded into individual wells of an
agarose gel. (B) When an electric current is passed through the gel, negatively charged DNA travels
away from the cathode (−) and toward the anode (+) in a size-dependent manner. (C) A DNA ladder
(left side) of DNA fragments of known sizes is usually run adjacent to the samples to identify the
sizes of the sample fragments.
230  Guide to Research Techniques in Neuroscience
bacterial artificial chromosomes (BACs, Box 10.1), are so large that it is nearly
impossible to use standard restriction enzymes and DNA ligase protocols to
produce recombinant DNA. An alternative to this molecular cutting and pasting
is recombineering (Box 10.3).
The vector and DNA
fragment to be inserted
are cut with the same
restriction enzymes
The “sticky” ends of
the linearized vector
and the DNA insert
are complementary
DNA ligase joins
the complementary
ends of the vector
and insert together
Competent bacteria
are transformed with
the recombinant DNA
vector
Successfully transformed
bacteria have resistance to
an antibiotic grown on the
culture plate. These bacteria
contain recombinant DNA
molecules
FIGURE 10.5  Steps in making a recombinant DNA plasmid.
Molecular Cloning and Recombinant DNA Technology Chapter | 10  231
Transformation
After a DNA fragment and vector are ligated together, the construct is introduced
into competent cells, which are host cells (usually bacteria) that have
been made transiently permeable to DNA (Figure 10.5). The process of introducing
a DNA vector into competent cells is called transformation. Bacteria
can be used to grow large quantities of a single recombinant DNA molecule
because they divide and grow exponentially, doubling in number every 30 min.
When the host cell divides, the recombinant plasmids also replicate such that a
high concentration of the desired DNA will be produced.
After transformation, the bacteria are usually grown on agar plates with an
antibiotic such that only bacteria successfully carrying the vector (containing an
antibiotic resistance gene) survive.
Purifying DNA from Host Cells
After transforming bacterial host cells to grow many copies of DNA, the recombinant
DNA plasmid must be purified from the host cell DNA and proteins.A scientist
typically takes a sample of a bacterial colony from an agar plate and grows
the bacteria in fresh culture media so that the bacteria continue to divide and
produce many copies of the desired DNA. Depending on the volume of bacterial
culture, different amounts of DNA can be purified, ranging from micrograms to
grams. A scientist performs a routine protocol to lyse the bacteria, extract the
DNA, and separate plasmid DNA from host cell DNA. In fact, many kits are now
available from commercial manufacturers that greatly simplify the process of
purifying DNA from transformed bacteria. This procedure is known as the miniprep
(for small amounts derived from 1 to 3 ml starting bacterial culture), maxiprep
(for 200–300 ml starting culture), or gigaprep (for 1–2 l starting culture).
IDENTIFYING DNA
After a complete cloning or subcloning experiment (Box 10.4), it is a good idea
to verify that the recombinant DNA product turned out as planned, typically by
BOX 10.3  Recombineering
Recombineering (“recombination-mediated genetic engineering” or “recombinogenic
engineering”) is a method used to create constructs without the use of restriction
enzymes and DNA ligase. This process uses a bacterium’s natural process of
homologous recombination (Chapter 13) to recombine DNA without the “cutting
and pasting” of traditional molecular biology techniques. These techniques can be
useful when it is difficult to find unique restriction enzyme sites in cloning vectors
or genomic DNA. Furthermore, recombineering allows for recombinant DNA
products using large cosmids or BACs, for which finding unique restriction enzyme
sites would be nearly impossible.
232  Guide to Research Techniques in Neuroscience
BOX 10.4  Walkthrough of a Subcloning Experiment
Imagine that you have identified an interesting gene called devX from a microarray
experiment (Chapter 9) to screen for genes that decrease over the course of neural
development. You identified the sequence of this gene and know that it is highly
expressed in neurons.You would like to examine the subcellular trafficking of devX
in neurons using time-lapse imaging, so you want to produce a reporter construct
(Chapter 7) by tagging devX with green fluorescent protein (GFP) to produce devXGFP.
Once created, this DNA construct can be introduced into cells or animals
using the transfection methods described in Chapter 11.
To create your devX-GFP sequence, you must first choose an appropriate vector
to build your construct and determine the best restriction enzymes to use. Because
you want to create a GFP-fusion construct, you could use a commercially available
GFP-containing plasmid vector called pEGFP-N1. This vector contains a promoter
that constitutively drives gene expression (called CMV); the eGFP (enhanced green
fluorescent protein) coding sequence; a gene that confers antibiotic resistance to
kanamycin; and a MCS, all contained within a circular plasmid. The MCS is located
in front of the eGFP coding sequence, meaning the devX coding sequence will be
attached to the N-terminus of eGFP (this is what the N1 in the vector’s name refers to).
An MCS contains many unique restriction sites to choose from, so compatible
restriction enzymes can be used on both the vector and the insert. The primary
factor to consider when choosing the appropriate restriction enzyme to use is
whether the site is unique. You obviously do not want to cut your DNA of interest
in unexpected or undesired areas. Because you will be using the same restriction
enzyme to open up the vector that you will be using to generate the ends of your
insert, you must consider the sequences of both items. After using software that
tells you where various restriction sites are located in your DNA plasmid, you end
up choosing the common enzymes EcoRI and BamHI, because devX does not
contain those recognition sites within its coding sequence.
The next major step is to generate a devX fragment with the appropriate restriction
sites to prepare the insert to go into the vector. There are multiple ways to
generate the small fragment of DNA that represents devX. If you already had a vector
containing the segment of devX, you might be able to directly cut it out of this
vector to paste into a new vector. Alternatively, you could use PCR to amplify the
appropriate fragment from a cDNA template, adding the recognition sequences for
EcoRI and BamHI to the ends of the devX fragment.
After choosing a vector and preparing the insert, you will need to make them
compatible by cutting them with the same restriction enzymes. You need to insert
the devX fragment in the proper direction and the same reading frame as the eGFP
so that they form a single protein together. You have done this by choosing two different
restriction enzymes (EcoRI and BamHI) that leave “sticky” ends after digestion
and by making sure that the frame of the devX PCR product after digestion is
the same as the eGFP coding sequence within the vector.
To isolate your digested products, you run the digested vector and insert on an
agarose gel. You should be able to identify the appropriate bands based on their
size. Then, you can physically cut out these bands and purify the DNA from the gel
using simple gel purification protocols or kits.
Molecular Cloning and Recombinant DNA Technology Chapter | 10  233
sequencing the DNA product. DNA sequencing identifies the individual base
pairs that make up the DNA strand. Another common method of DNA identification
is to use nucleic acid hybridization techniques to probe whether a specific
sequence is present in a sample.
DNA Sequencing
DNA sequencing is used to determine the exact sequence of nucleotides
(A, G, C, T) in a strand of DNA. The Sanger dideoxy chain-termination
method can determine the sequence of nucleotides with high fidelity for a
stretch of approximately 200–500 base pairs in any purified DNA sample.
Based on in vitro DNA synthesis, the Sanger method synthesizes short pieces
of DNA in the presence of nucleotide bases to which other bases cannot be
added: chain-terminating dideoxyribonuceoside triphosphates. These chainterminating
nucleotides are labeled and mixed with regular nucleotide bases
so that fragments of DNA will be created at many different lengths, each
You mix the purified digested products with DNA ligase, which will ligate the
fragments together. The EcoRI digest on the 5′ end of the devX insert matches the
EcoRI digest on the 3′ end of the plasmid, while the BamHI digest on the 3′ end of
the devX insert matches the BamHI digest on the 5′ end of the plasmid. Thus, the
devX sequence should be inserted in the proper direction into the plasmid.
Next, you can transform competent bacteria to grow many copies of your
recombinant DNA plasmid. You mix the ligation product with competent cells
and then spread the cells on plates containing nutrients—to help cells grow—and
kanamycin, the antibiotic that cells containing your construct should be resistant
against. The next morning, you should see clonal colonies of bacteria growing on
the plate, and you can select individual colonies whose member cells contain
identical copies of the construct.
If you place a bit of the colony in a new tube with bacterial culture medium,
you can allow the bacteria to multiply over a period of 8–12 h. Then, you can
purify the plasmid DNA from each of the clones using a “miniprep” kit. You lyse
the bacteria, extract the DNA from the lysed bacteria, and purify the plasmid DNA
from the bacterial DNA using purification columns.
The purified plasmid DNA can be examined using restriction enzyme analysis.
Using restriction digests, you can ensure the presence of the insert, whether the
insert was ligated in the correct direction, and whether there is more than one
insert present.These questions can be answered by choosing appropriate restriction
enzymes, which will not necessarily be the ones used to join the insert and vector.
Finally, you verify that the sequence in your construct matches the DNA sequence
of devX-GFP by performing sequencing reactions (Figure 10.6).
Now that you have produced and verified your devX-GFP construct, you can
introduce your vector into cells using techniques that will be described in the next
chapter.
BOX 10.4  Walkthrough of a Subcloning Experiment—cont’d
234  Guide to Research Techniques in Neuroscience
randomly stopped by the addition of a chain-terminating nucleotide. The four
chain-terminating nucleotides are each labeled with a different colored fluorescent
dye. Thousands of fragments of different lengths are run on a gel,
and an automated fluorescence detector can quickly scan the gel to read the
identity of the last, terminating nucleotide (Figure 10.6). Modern molecular
biology laboratories tend not to run sequencing reactions themselves, as it is
typically faster and less expensive to send DNA samples off to a dedicated
facility for sequencing.
The Sanger method is the gold standard for DNA sequencing, and it is still
commonly used for the day-to-day verification of molecular biology experiments.
However, DNA sequencing methods have evolved rapidly over the past
decade in what are referred to as next-generation sequencing (NGS) techniques,
which make sequencing large stretches of DNA cheaper and faster. NGS
techniques have enabled large-scale sequencing of entire genomes and allowed
for the rapid development of the field of genomics.
Nucleic Acid Hybridization Techniques
The term hybridization refers to the phenomenon whereby two complementary
nucleic acid strands bind to each other. Any fragment of DNA with a known
base-pair sequence can be detected by labeling a probe strand with the complementary
sequence with a radioactive label or biochemical tag that can produce
a colorimetric or fluorescent signal.
A
C
G
T
G G C T G C A A G A C G C C T T C T C T G C C A
Signal intensity
at band position
FIGURE 10.6  DNA sequencing.  Each chain-terminating nucleotide is labeled with a different
fluorescent dye, and many fragments of DNA are synthesized. When all of the synthesized DNA
fragments are separated using gel electrophoresis, they will be separated by size and a detector can
automatically detect which label is at each position by measuring the intensity of each fluorescent
signal.
Molecular Cloning and Recombinant DNA Technology Chapter | 10  235
Southern Blot
Developed by and named after Edward Southern, a Southern blot is used to
identify if a known DNA sequence is present in a sample by using a specific
probe to identify the sequence in a large mixture of other DNA fragments.
A DNA sample (typically genomic DNA) is digested with restriction enzymes,
and the mixture of fragments is separated by gel electrophoresis. The DNA
is denatured to form single-stranded DNA, and the DNA is transferred to a
nitrocellulose membrane (Figure 10.7). The positions of the DNA fragments
FIGURE 10.7  The Southern and northern blot methods detect specific sequences of nucleic
acids.  (A) In a Southern blot, restriction enzymes are used to digest DNA into fragments. (B)A northern
blot uses RNA molecules as its starting material. The nucleic acids are separated by size using gel electrophoresis,
then transferred to a membrane. The membrane is incubated with labeled probes capable of
hybridizing to specific nucleic acid sequences. The bands can then be visualized using autoradiography.
236  Guide to Research Techniques in Neuroscience
in the gel are preserved on the nitrocellulose membrane so a scientist can identify
fragments based on their size. The membrane is exposed to a radioactive
32P-labeled single-stranded DNA probe corresponding to the sequence of interest.
This probe hybridizes with the restriction fragment containing the complementary
sequence, and autoradiography reveals the position of the restriction
fragment-probe duplex. Therefore, a specific fragment in a mixture of millions
of other fragments can be identified on the membrane.
Southern blots are often used to demonstrate a successful gene knockout by
using a probe against the region of the gene that was deleted from the genome
(Chapter 13). PCR techniques are more sensitive and a faster alternative to identifying
a DNA fragment, but these techniques are often used in conjunction with
Southern blots to confirm a result.
Northern Blot
A technique similar to a Southern blot used to identify the presence of RNA has
been humorously named the northern blot. Northern blots directly compare
the relative amounts of mRNA levels between different samples. Intact mRNA
molecules are separated on the basis of their size using gel electrophoresis, and
a procedure identical to that used in a Southern blot is performed to visualize the
size and relative expression of the mRNA species.
Alternative methods for examining mRNA expression levels include qRT-PCR
and microarrays (Chapter 9). Performing experiments with RNA typically requires
more care in protecting the samples because RNases (enzymes that degrade RNA)
are ubiquitous in the environment and RNA molecules are much less stable than
DNA.An analogous technique to Southern and northern blots used to detect protein
expression levels using an antibody is called the western blot (Chapter 15).
CONCLUSION
Molecular cloning and recombinant DNA technology have provided scientists
with the ability to manipulate genes and the proteins they encode. These tools
allow us to alter DNA sequences, cutting and pasting them in novel ways for
many subsequent experiments. The power of these methods is complemented by
other techniques that allow for the delivery of DNA to cells and creation of transgenic
and mutant organisms, as will be discussed in the next several chapters.
SUGGESTED READING AND REFERENCES
Books
Dale, J. W., von Schantz, M., & Plant, N. (2011). From genes to genomes: Concepts and applications
of DNA technology (3rd ed.). West Sussex, UK: Wiley.
Drlica, K. (2004). Understanding DNA and gene cloning: A guide for the curious (4th ed.).
Hoboken, NJ: Wiley.
Griffiths, A. J. F. (2010). Introduction to genetic analysis (10th ed.). New York: Freeman.
Molecular Cloning and Recombinant DNA Technology Chapter | 10  237
Nicholl, D. S. T. (2008). An introduction to genetic engineering (3rd ed.). Cambridge: Cambridge
University Press.
Review Articles
Bras, J., Guerreiro, R., & Hardy, J. (2012). Use of next-generation sequencing and other wholegenome
strategies to dissect neurological disease. Nature Reviews Neurology, 13, 453–464.
Campbell, T. N., & Choy, F. Y. (2002). Approaches to library screening. Journal of Molecular
Microbiology and Biotechnology, 4, 551–554.
Copeland, N. G., Jenkins, N. A., & Court, D. L. (2001). Recombineering: a powerful new tool for
mouse functional genomics. Nature Reviews Genetics, 2, 769–779.
Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K., & MArdis, E. R. (2013). The nextgeneration
sequencing revolution and its impact on genomics. Cell, 155, 27–38.
Marziali, A., & Akeson, M. (2001). New DNA sequencing methods. Annual Review of Biomedical
Engineering, 3, 195–223.
Primary Research Articles—Interesting Examples from the Literature
Agate, R. J., Choe, M., & Arnold, A. P. (2004). Sex differences in structure and expression of the
sex chromosome genes CHD1Z and CHD1W in zebra finches. Molecular Biology and Evolution,
21, 384–396.
Bacon, A., Kerr, N. C., Holmes, F. E., Gaston, K., & Wynick, D. (2007). Characterization of an
enhancer region of the galanin gene that directs expression to the dorsal root ganglion and
confers responsiveness to axotomy. Journal of Neuroscience, 27, 6573–6580.
Brenner, S., Williams, S. R., Vermaas, E. H., Storck, T., et al. (2000). In vitro cloning of complex
mixtures of DNA on microbeads: physical separation of differentially expressed cDNAs. Proceedings
of the National Academy of Sciences of the United States of America, 97, 1665–1670.
Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., et al. (2003).
A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.
Nature, 425, 917–925.
Hsu, S. Y., Kaipia, A., McGee, E., Lomeli, M., & Hsueh, A. J. (1997). Bok is a pro-apoptotic Bcl-2
protein with restricted expression in reproductive tissues and heterodimerizes with selective
anti-apoptotic Bcl-2 family members. Proceedings of the National Academy of Sciences of the
United States of America, 94, 12401–12406.
Paisan-Ruiz, C., Jain, S., Evans, E. W., Gilks, W. P., et al. (2004). Cloning of the gene containing
mutations that cause PARK8-linked Parkinson’s disease. Neuron, 44, 595–600.
Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, et al. (2003). High-throughput engineering
of the mouse genome coupled with high-resolution expression analysis. Nature Biotechnology,
21, 652–659.
Protocols
Choi, S., & Kim, U. J. (2001). Construction of a bacterial artificial chromosome library. Methods in
Molecular Biology, 175, 57–68.
Lee, S. C., Wang, W., & Liu, P. (2009). Construction of gene-targeting vectors by recombineering.
Methods in Molecular Biology, 530, 15–27.
Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Websites
New England BioLabs catalog: http://www.neb.com/nebecomm/tech_reference.
Primer-BLAST: primer designing tool: http://www.ncbi.nlm.nih.gov/tools/primer-blast/.
Addgene plasmid repository: http://www.addgene.org/.
239
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00011-3
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 11
Gene Delivery Strategies
After Reading This Chapter, You Should be Able to:
	l	Describe common methods of delivering recombinant DNA into cells
	l	Compare and contrast methods of DNA delivery in various in vitro and in vivo
preparations
Techniques Covered:
	l	Physical gene delivery: microinjection, electroporation, biolistics
	l	Chemical gene delivery: calcium phosphate transfection, lipid-based transfection
	l	Viral gene delivery: adenovirus, adeno-associated virus (AAV), lentivirus, herpes
simplex virus (HSV), canine adenovirus (CAV)
The previous chapter discussed methods for making and manipulating DNA constructs
using recombinant DNA technology. Of course, the ultimate goal of creating
a DNA construct is to deliver the manipulated sequence into living cells so
that the endogenous cellular machinery can transcribe and translate the sequence
into functional proteins. A neuroscientist might want to deliver a DNA sequence
into a population of cells in culture, in a brain slice, or in the brain of a living
animal. The purpose of this chapter is to survey the common methods of delivering
recombinant DNA into cells in each of these environments.
There are three general categories of DNA delivery: physical, chemical,
and viral. Transfection refers to nonviral methods of delivering DNA to cells,
including physical and chemical methods. Infection refers to viral DNA delivery,
in which viruses attach themselves to cells and inject their DNA cargo.
Each method has its own relative advantages and disadvantages that make it
particularly well-suited for delivering DNA in different experimental contexts
(Table 11.1). Major factors that dictate the choice of DNA delivery method
include cellular environment, cell type, and experimental goals. Additionally,
these DNA delivery methods vary in terms of the number of cells they affect, levels
of gene expression they induce, and the length of gene expression over time.
PHYSICAL GENE DELIVERY
Physical gene delivery methods deliver DNA into a cell by physically penetrating
the cell membrane with force. These methods are often highly efficient
240  Guide to Research Techniques in Neuroscience
(transfected cells express high levels of the delivered gene) and can be used
with any cell type. However, they can sometimes disrupt the integrity of the cell
membrane, traumatizing the cell and leading to its death. Therefore, care must
be taken in moderating the amount of force applied to deliver DNA to cells.
Three common physical methods include microinjection, electroporation, and
biolistics.
Microinjection
A scientist can microinject a solution containing DNA into a cell by piercing
the cell membrane with a small glass needle and then applying pressure (Figure
11.1). If the scientist uses an extremely thin needle and withdraws the needle
carefully, the cell membrane remains intact, and the cell has an opportunity to
incorporate the injected DNA into its genome. This process requires a microscope
to view the living cell, as well as fine micromanipulators to precisely
place the needle adjacent to the cell membrane. Each cell must be injected one
at a time (in contrast to other methods that deliver DNA to millions of cells
at once), making microinjection laborious and low throughput. This method
requires technical skill and is therefore usually performed by trained technicians
or laboratory personnel who use these techniques regularly. Microinjection
is rarely used to transfect neurons because their small shape and sensitivity
makes them more difficult to inject than other cell types.
TABLE 11.1  Categories of Gene Delivery Strategies
Method Advantages Disadvantages
Physical High-efficiency gene transfer
No limitations on construct size
No cell type dependency
Low throughput
Requires specialized equipment
Can physically harm cells
Chemical High efficiency in vitro
No limitations on construct size
Relatively easy to perform
Rapid
High throughput
Low immunogenicity
Limited in vivo applications
Efficiency depends on cell type
Can be toxic to cells
Transient expression
Viral High-efficiency gene transfer
Cell-specific targeting is possible
Long-term expression
Can be used in vitro and in vivo
Complex cloning required
More expensive than
transfection methods
Safety concerns regarding
production of infectious viruses
in humans
May provoke immune response
Laborious preparation
Limited construct size
Gene Delivery Strategies Chapter | 11  241
DNA microinjection is the method used to make transgenic animals. To
make a transgenic mouse, a scientist microinjects the transgenic DNA construct
into newly fertilized mouse eggs. In some of the new eggs, the transgene
randomly incorporates into the mouse genome. These cells are injected into a
female mouse to carry the egg to gestation. The full details of creating transgenic
mice, as well as other transgenic animals, are discussed in Chapter 12.
Electroporation
Electroporation is the process of using an electric pulse to transfect cells with
DNA (Figure 11.2). Applying an electric field to cells is thought to induce temporary
pores in the cell membrane, allowing the cell to take up DNA sequences.
The electric field also drives negatively charged DNA strands away from the
cathode (negative end) toward the anode (positive end) of an electric field.
Therefore, an electric pulse causes some of the DNA to enter the cell. After the
electric field is switched off, the cell membrane reseals, trapping some of the
electroporated DNA within the cell.
Electroporation can be used to efficiently deliver DNA to cells for in vitro
conditions using special containers with electrical contacts. In a process known
as ex vivo electroporation, a scientist removes a brain from an animal, applies
electroporation techniques, and then sections the brain for slice cultures.
In neuroscience research, electroporation has been particularly useful for
in utero preparations (Figure 11.3). In utero electroporation surgeries begin by
exposing the embryonic pups of a pregnant animal, usually a rodent. DNA is
injected into the pups’ ventricles, and electrode paddles direct the uptake of
the injected DNA construct(s) toward the anode and away from the cathode.
The pups are placed back inside the mother, who is sutured and allowed to
recover. Days later, pups are removed or the mother gives birth, and the neurons
of the offspring express the introduced DNA construct. Similar methods can
be used in the chicken model system in a process called in ovo electroporation.
These methods are useful for delivering genes to brain regions adjacent to the
FIGURE 11.1  DNA microinjection.  An extremely thin glass needle punctures the plasma membrane,
allowing DNA to be injected into the cell.
242  Guide to Research Techniques in Neuroscience
nervous system’s fluid-filled ventricles, as the DNA-containing solution must
be injected into the ventricle. Thus, they have primarily been used in studies of
chick spinal cord and rodent cerebral cortex. A scientist can control both spatial
and temporal specificity by controlling the position of the electrode paddles and
the timing of electroporation. For example, a scientist can target gene transfection
to distinct cortical layers in the mammalian cerebral cortex by electroporating
at specific embryonic time periods.
Electroporation has numerous advantages, such as the ability to transfect
cells in many different in vivo and in vitro environments. Furthermore, investigators
can vary expression levels of a transfected gene by varying the strength
and pattern of the electric field pulses.
FIGURE 11.2  Electroporation.  (A) Cells
are placed in a special chamber with a solution
containing the DNA to be transfected.
(B)Applying an electric field drives the negatively
charged DNA strands away from
the cathode and toward the anode. (C)After
the electric field is switched off, the cell
membrane reseals, and some DNA remains
in the cell.
Gene Delivery Strategies Chapter | 11  243
Biolistics
Biolistics, short for “biological ballistics” and also known as particle-mediated
gene transfer, is the method of directly shooting DNA fragments into cells
using a device called a gene gun.
To use a gene gun, a scientist first mixes a DNA construct with particles
of a heavy metal, usually tungsten or gold. These fine particles stick to the
negatively charged DNA. The DNA/metal particles are loaded onto one side
of a plastic bullet (Figure 11.4). A pressurized gas, usually helium, provides
the force for the gun. Gas pressure builds up until a rupture disk breaks, driving
the plastic bullet down a shaft. The plastic bullet is abruptly stopped at
the end of the shaft, but the DNA/metal particles emerge from the gun with
great speed and force. If the gun is aimed at biological tissue, some of the
metal particles will penetrate the cell membranes and deliver DNA constructs
to cells.
A neuroscientist can use biolistic technology to cause efficient gene expression
in neurons. This technology can produce a dispersed transfection pattern,
similar to a Golgi stain, in which only individual cells receive the foreign DNA
in a background of untransfected cells. Another advantage to using biolistic
technology is that a gene gun can deliver DNA through relatively thick tissue,
such as a tissue slice in culture. Therefore, it is possible to transfect cells
within a slice that would be difficult to target using other gene delivery methods.
This technique has not yet been successful in transfecting mammalian neurons
in vivo, although it has been used in vivo to transfect liver and skin cells. The
main disadvantage of using this technology is that it may cause physical damage
to cells. Optimization is required to limit the amount of tissue damage caused by
the force of impact of the projectiles.
+
_
Pregnant rodent
Developing
embryos
Injection of DNA
construct
Application of
electric field
FIGURE 11.3  In utero electroporation. A scientist performs a surgical procedure on a pregnant
female, exposing the embryonic pups. DNA is injected into the pups’ ventricular system, and
paddles generate electric field pulses to introduce DNA into the cells lining the ventricles.
244  Guide to Research Techniques in Neuroscience
CHEMICAL GENE DELIVERY
Chemical gene delivery is the process by which a scientist uses a chemical
reaction to deliver DNA into cells. Negatively charged DNA can form macromolecular
complexes with positively charged chemicals. These complexes
can then interact with a cell’s membrane, promoting uptake through endocytosis
or fusion. Chemical gene delivery strategies are useful in that they
are incredibly high-throughput and often simple to perform. However, they
are generally inefficient for in vivo delivery and therefore mainly used for
cell culture experiments (Chapter 14). Expression of the target gene is usually
transient, lasting for days to weeks depending on the cell type. Common
chemical gene delivery strategies include calcium phosphate transfection and
lipid transfection.
Calcium Phosphate Transfection
The calcium phosphate method of DNA transfection is one of the simplest and
least expensive methods of chemical gene delivery (Figure 11.5). This method
requires two chemical solutions: calcium chloride, which serves as a source of
Pressurized gas
Rupture disk
Bullet
DNA-coated
metal particles
Filter screen
Gas breaks
rupture disk,
driving bullet
down the shaft
Bullet blocked by
filter screen,
DNA-coated metal
particles emerge
Gun aimed at
biological
specimen
FIGURE 11.4  A gene gun.  DNA-coated metal particles are placed on the front end of a bullet.
High-pressured gas drives the bullet down a shaft. At the end of the shaft, the bullet is blocked, but
the DNA-coated particles emerge with great speed and force.
Gene Delivery Strategies Chapter | 11  245
calcium ions, and HEPES buffered saline (HBS), which serves as a source of
phosphate ions. First, the calcium chloride solution is mixed with the DNA to be
transfected, and then the HBS is added. When the two solutions are combined,
the positively charged calcium ions and negatively charged phosphate ions form
a fine precipitate. The calcium ions also cause the DNA to precipitate out of
solution. After a few minutes, the solution with precipitate is directly added to
cells in culture. By a process that is not entirely understood, the cells take up
some of the precipitate, and with it, the DNA. It is thought that the DNA precipitate
enters cells by endocytosis, but the exact mechanism remains a mystery.
This method works best for a cell monolayer so that the DNA precipitate covers
the cells evenly.
The advantages to using the calcium phosphate method are that it is relatively
easy, reliable, and cheap. It is useful for transient expression or creating
stable cell lines from immortalized cells (Chapter 14). The main disadvantage
is that transfection efficiencies are low in neurons (1–3%), and this method does
not work at all for transfecting neurons in intact tissue. Some immortalized cell
Solution with DNA and
calcium chloride
HEPES buffered
saline solution
DNA precipitates
out of solution
Solution with precipitate
added to cells
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+Ca2+
Ca2+Ca2+
Ca2+
Ca2+
Ca2+
FIGURE 11.5  Calcium phosphate transfection.  A scientist mixes DNA, calcium chloride, and
HEPES-buffered saline. The chemical reaction forms a DNA-calcium phosphate precipitate that is
able to pass through the cell membrane and deliver DNA to the nucleus.
246  Guide to Research Techniques in Neuroscience
lines, such as HEK 293T and HeLa cells (Chapter 14), exhibit high transfection
efficiencies (90–100%), which is one reason for their ubiquitous use in the life
sciences.
Lipid Transfection
Lipofection, also known as “lipid transfection” or “liposome-based transfection,”
uses a lipid complex to deliver DNA to cells. Lipids are a broad class of
fat-soluble biomolecules, such as fats, oils, and waxes. The cell membranes of
animal cells are composed of a bilayer of phospholipids with hydrophilic surfaces
facing the cytoplasm and extracellular environment (Figure 11.6A). Lipofection
technology uses tiny vesicular structures called liposomes that have the
same composition as the cell membrane (Figure 11.6B). A scientist performs a
simple reaction that forms a liposome around the DNA sequence to be transfected
(Figure 11.6C). Depending on the liposome and cell type, the liposome
can be endocytosed (Figure 11.6D) or directly fuse with the cell membrane to
release the DNA construct into cells (Figure 11.6E).
The advantage to lipofection is that it works in many cell types, including
cultured neurons. Commercially available kits allow transfection reactions to
be performed within 30 min and gene expression to be assayed within hours.
FIGURE 11.6  Lipofection. (A) The cell membrane is composed of a lipid bilayer, with a
hydrophobic interior and hydrophilic exterior. (B) Liposomes are also composed of a lipid bilayer
arranged as a spherical shell. (C) A scientist performs a brief reaction that allows liposomes to
form around DNA. (D) Cells in culture can endocytose the liposome, digesting it within vesicles
to release DNA. (E) Alternatively, liposomes can directly fuse with the plasma membrane, directly
releasing DNA into cells.
Gene Delivery Strategies Chapter | 11  247
However, like the calcium phosphate method, lipofection is almost exclusively
used in cell culture experiments.
VIRAL GENE DELIVERY
Viral gene delivery uses one of many available viral vectors to deliver DNA to
cells in vitro or in vivo. A virus can be thought of as a tiny molecular machine
whose entire purpose is to attach to cells and inject genetic material. In nature,
this genetic material encodes the proteins necessary to make more virus, so
the infected cell essentially becomes a virus-making factory. In the laboratory,
many viruses have been manipulated so that they can no longer replicate
inside a host cell on their own. Additionally, the coding region of viral DNA
is exchanged with a gene of interest to make the virus deliver a transgene to a
cell without forcing that cell to produce more virus particles. This process of
using a nonreplicating viral vector to deliver foreign DNA into a cell is called
transduction.
Scientists can either produce virus in their own laboratories or outsource
viral production commercially (Figure 11.7). To begin, a scientist uses recombinant
DNA technology (Chapter 10) to place a DNA sequence of interest
into a plasmid containing the necessary sequences to incorporate into a virus
particle. Note that this plasmid is not the same as a virus itself but is simply
a piece of DNA with the necessary sequences to incorporate into a virus particle,
provided the other necessary proteins are present. To produce a batch of
virus, cultured cells are used to produce all the necessary viral components.
A scientist transfects these cells with the recombinant plasmid, as well as
additional DNA sequences coding for the necessary viral proteins. Because
viral DNA has been engineered so that it cannot replicate more virus on its
Transfect cells with
plasmids encoding
viral particles
After 2 days,
collect viral-containing
supernatant
Centrifuge to
collect viral pellet
Resuspend pellet
in buffer
FIGURE 11.7  Virus production. A scientist transfects cultured cells with DNA encoding the
necessary proteins to make virus. Over 2–3 days, the cells produce virus and release it into the
culture medium. The medium is collected and centrifuged to collect a viral pellet. The pellet is
resuspended in buffer and frozen until use.
248  Guide to Research Techniques in Neuroscience
own, these viral proteins are necessary to produce functional, infectious viral
particles. Immortalized cell lines, such as HEK 293T cells, are the best cells to
serve as virus packaging cells because they grow quickly, are transfected relatively
easily, and can produce large amounts of virus. One to two days after
transfection, the packaging cells produce many virus particles and release
them into the extracellular medium. The final step of virus production is to
collect the medium, filter out cell debris, and centrifuge the medium to collect
the viral pellet. The pellet is dissolved in a buffer and, depending on the needs
of the investigator, either used immediately to infect the target cells of interest
or frozen until needed.
Viral infections are robust, highly efficient, and can lead to long-term expression.
Viruses have been used to deliver genes to cultured cells, brain slices, tissue
explants, and brain regions in vivo. They are the tools of choice to deliver
genes into the brains of rodents, as most other methods of gene delivery are
incapable of working efficiently in vivo. However, there are also disadvantages
and limitations to using viral gene delivery. The infected cells, especially cells
in vivo, may not begin expressing the transduced gene until 7–14 days after
exposure to the virus. Also, the size of the viral vector restricts the size of the
DNA construct that a scientist can transduce. Finally, scientists must take great
care and safety when using viruses because it is possible for scientists to accidentally
infect themselves.
There are a variety of viral vectors that are useful to neuroscientists
(Table 11.2). They vary in terms of infection efficiency, expression levels,
duration of expression, time to start of expression, host cell toxicity, and
host cell preference. Adeno-associated virus and lentivirus are the most
commonly used viral vectors due to long-term expression of transgenes and
low toxicity in neurons.
Adenovirus
Adenovirus can infect both dividing and postmitotic cells in a broad range of
species and cell types. It can carry DNA constructs that are 7.5–30 kb (kilobases)
long. The DNA does not integrate into the genome, so expression is transient,
lasting weeks or months. It is useful for high-level transient expression in
neurons, but it can often cause an inflammatory response and cell death.
Canine Adenovirus
Canine adenovirus (CAV) is a form of adenovirus that is highly infectious
in dogs. This virus has a high carrying capacity of 7.5–30 kb and is used in
neuroscience research because of its retrograde properties. Upon injection into
the brain, CAV can be taken up by the presynaptic membrane and retrogradely
transported to cell soma. Therefore, this viral vector is useful for targeting
neurons based on their connectivity.
Gene Delivery Strategies Chapter | 11  249
Herpes Simplex Virus
Herpes simplex virus (HSV) is a neurotropic virus that naturally targets
neurons as host cells. It has a very large genome compared to other viruses
(100–200 kb) that allows delivery of long DNA sequences. While the DNA does
TABLE 11.2  Viral Vectors Used in Neuroscience
Vector
Insert
size Features
Adenovirus 7.5–30 kb Infects dividing and nondividing cells
Transient expression—does not integrate in
the genome
High transduction efficiency
Onset of expression in days
Triggers immune response
Canine adenovirus
(CAV)
7.5–30 kb Exhibits retrograde transport properties
Infects dividing and nondividing cells
Transient expression—does not integrate in
the genome
High transduction efficiency
Onset of expression in days
Triggers immune response
Herpes simplex virus
(HSV)
>30 kb Neuron specific
Stable, long-term expression—does not
integrate in the genome
Low transduction efficiency
Safety concerns
Complex production
Onset of expression in hours
Adeno-associated virus
(AAV)
5 kb Naturally replication-deficient—safe
Infects dividing and nondividing cells
Stable expression—integrates in the
genome
High transduction efficiency
Onset of expression in weeks
Minimal toxicity
Labor-intensive production
Lentivirus 8 kb Form of retrovirus that can infect
nondividing cells in addition to dividing
cells
Stable expression—integrates in the
genome
Safety concerns
250  Guide to Research Techniques in Neuroscience
not integrate into the host genome, it exists stably in the nucleus to produce
long-term (months to years) expression. The disadvantages are that it can be
difficult to engineer HSV because of its large size, and it can be toxic to cells.
There are also safety concerns because it is highly infectious.
Adeno-Associated Virus
Adeno-associated virus (AAV) is naturally replication deficient, requiring
a helper virus to replicate, so it is safer to use than other viral strains. AAV
can infect both dividing and postmitotic cells, but, unlike adenovirus, does
integrate into the host genome, permitting long-term gene expression. AAV
also seems to be less toxic to neurons than adenovirus. However, AAV production
is labor-intensive and the carrying capacity is much less than adenovirus,
about 5 kb.
Lentivirus
Lentivirus belongs to a class of virus called retrovirus that has an RNA genome
rather than DNA. To produce functional gene products, the virus also contains
the enzyme reverse transcriptase, which produces cDNA from the RNA template
(Chapter 10). When a cell endocytoses a lentivirus particle, the RNA is
released and reverse transcriptase produces cDNA. The DNA migrates to the
nucleus, where it integrates into the host genome.
Most retroviruses only infect dividing cells, making them useful for
studying neuronal development and cell fate. However, lentivirus is capable
of infecting both dividing and postmitotic cells (e.g. neurons) and is therefore
widely used in neuroscience experiments. Lentivirus is based on the
human immunodeficiency virus and has an 8-kb carrying capacity. Because
the DNA integrates into the genome, lentivirus delivery leads to long-term
expression.
CONCLUSION
This chapter has surveyed common methods used to deliver DNA sequences
into cells. A scientist chooses one method over another based on a number
of factors, such as the cell type and the cell’s environment, as well as
the specific goals of the experiment (Table 11.3). For cell culture experiments,
chemical, electroporation, or viral gene delivery strategies all work
well. For in vivo DNA delivery to adult animals, viral gene delivery is the
most efficient option. Now that we have surveyed these methods, the next
two chapters will focus on methods of manipulating the genomes of living
organisms.
Gene Delivery Strategies Chapter | 11  251
SUGGESTED READING AND REFERENCES
Books
Heiser, W. C. (Ed.). (2010a). Gene delivery to mammalian cells. Volume 1: Nonviral gene transfer
techniques. New York, NY: Humana Press.
Heiser, W. C. (Ed.). (2010a). Gene delivery to mammalian cells. Volume 2: Viral gene transfer techniques.
New York, NY: Humana Press.
Twyman, R. M. (2005). Gene transfer to animal cells.Abingdon, Oxon, UK: Garland Science/BIOS
Scientific Publishers.
Review Articles
Bonetta, L. (2005). The inside scoop—evaluating gene delivery methods. Nature Methods, 2,
875–883.
Callaway, E. M. (2008). Transneuronal circuit tracing with neurotropic viruses. Current Opinion in
Neurobiology, 18, 617–623.
Luo, L., Callaway, E. M., & Svoboda, K. (2008). Genetic dissection of neural circuits. Neuron, 57,
634–660.
Washbourne, P., & McAllister, A. K. (2002). Techniques for gene transfer into neurons. Current
Opinion in Neurobiology, 12, 566–573.
TABLE 11.3  Choosing a Gene Delivery Strategy
Environment Considerations
Commonly used
gene delivery
methods
Cells in the intact nervous
system of living animals
Cells within the brain
are notoriously resistant
to most transfection
methods.
Electroporation, virus
Cells in brain slices Gene delivery must be
fast and efficient and may
need to go through thick
tissue.
Electroporation, biolistics
Cells in dissociated
cultures
Gene delivery must be
highly efficient and high
throughput to transfect/
transduce thousands or
millions of cells.
Electroporation, biolistics,
chemical transfection,
virus
Individual cells Cells are often valuable,
such as extracted
embryos or newly
fertilized eggs, so care is
taken to efficiently deliver
DNA to each cell.
Microinjection,
electroporation
252  Guide to Research Techniques in Neuroscience
Primary Research Articles—Interesting Examples from the Literature
Carter, M. E., Soden, M. E., Zweifel, L. S., & Palmiter, R. D. (2013). Genetic identification of a
neural circuit that suppresses appetite. Nature, 503, 111–114.
Chen, J. L., Carta, S., Soldado-Magraner, J., Schneider, B. L., & Helmchen, F. (2013). Behaviourdependent
recruitment of long-range projection neurons in somatosensory cortex. Nature, 499,
336–340.
Krashes, M. J., Shah, B. P., Madara, J. C., Olson, D. P., Strochlic, D. E., et al. (2014). An excitatory
paraventricular nucleus to AgRP neuron circuit that drives hunger. Nature, 507, 238–242.
Pekarik, V., Bourikas, D., Miglino, N., Joset, P., Preiswerk, S., & Stoeckli, E. T. (2003). Screening
for gene function in chicken embryo using RNAi and electroporation. Nature Biotechnology,
21, 93–96.
Tsai, H. C., Zhang, F., Adamantidis, A., Stuber, G. D., Bonci, A., de Lecea, L., et al. (2009).
Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning. Science, 324,
1080–1084.
Wilson, S. P.,Yeomans, D. C., Bender, M. A., Lu,Y., Goins, W. F., & Glorioso, J. C. (1999). Antihyperalgesic
effects of infection with a preproenkephalin-encoding herpes virus. Proceedings of
the National Academy of Sciences of the United States of America, 96, 3211–3216.
Protocols
Lappe-Siefke, C., Maas, C., & Kneussel, M. (2008). Microinjection into cultured hippocampal neurons:
a straightforward approach for controlled cellular delivery of nucleic acids, peptides and
antibodies. Journal of Neuroscience Methods, 175, 88–95.
O’Brien, J., & Lummis, S. C. (2004). Biolistic and diolistic transfection: using the gene gun to
deliver DNA and lipophilic dyes into mammalian cells. Methods, 33, 121–125.
Walantus, W., Castaneda, D., Elias, L., & Kriegstein, A. (2007). In utero intraventricular injection
and electroporation of E15 mouse embryos. Journal of Visualized Experiments, 6. http://dx.doi.
org/10.3791/239. http://www.jove.com/index/details.stp?id=239.
Websites
University of North Carolina Vector Core: http://www.med.unc.edu/genetherapy/vectorcore.
University of Pennsylvania Vector Core: http://www.med.upenn.edu/gtp/vectorcore/.
253
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00012-5
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 12
Making and Using Transgenic
Organisms
After Reading This Chapter, You Should be Able to:
	l	Describe and categorize commonly used transgenes
	l	Describe the necessary components of a transgenic DNA construct
	l	Understand the benefits and utility of binary transgenic expression systems
	l	Explain the process of creating viable transgenic organisms
Techniques covered:
	l	Common transgenes in neuroscience: reporter genes, genes used to measure neural
activity, genes used to manipulate neural activity, genes used to ablate neurons,
genes used to disrupt endogenous genes
	l	Binary transgenic systems: Gal4/UAS, Cre/lox, Flp/Frt, Tet-off/Tet-on
	l	Making transgenic organisms: mice, flies, worms
One of the modern marvels of genetic engineering technology is the ability to
take a gene from one species and place it in the genome of another species. The
rich diversity of life on Earth provides an extraordinary variety of genes that not
only help organisms survive unique niches on the planet but also help neuroscientists
probe the structure and function of neural systems. For example, the
jellyfish Aequorea victoria evolved a gene capable of emitting photons of light
for bioluminescence. This gene, known as green fluorescent protein (GFP),
has been used in thousands of studies to examine the structure and function of
the nervous system. A variety of species have evolved other genes useful for
visualizing neurons and manipulating their function, such as luciferase from
fireflies, channelrhodopsin-2 from single-celled algae, lacZ from Escherichia
coli, or allatostatin receptor from insects. Genetic engineering technology
provides the ability to express these genes in specific cell types at specific times
in an organism’s lifespan. Thus, modern genetic engineering technology allows
scientists to stably express genetically encoded tools for the precise study of
specific neural systems and circuits.
Any gene expressed in an animal that does not normally carry this gene is
called a transgene, and any animal that carries this foreign DNA is called a
transgenic organism. The purpose of this chapter is to survey common transgenes
used in modern neuroscience research and to describe how these genes
254  Guide to Research Techniques in Neuroscience
can be restricted to specific cell types at specific times. Furthermore, we will
examine the process of creating a transgenic animal in commonly used model
organisms including mice, flies, and worms. Chapter 13 will provide additional
information on gene targeting technologies used to remove genes from a
genome or insert genes in precise locations in the genome.
TRANSGENES
There are now dozens of commonly used transgenes that can be used to answer
a variety of experimental questions. Scientists can inject these transgenes into
embryos to create transgenic lines of animals that pass on the transgene to their
offspring. Alternatively, these transgenes can be introduced directly into cells or
brain regions, as described in Chapter 11. We survey some of the major categories
of transgenes here and in Table 12.1, although this is not an exhaustive list.
Reporter Genes
A reporter gene is any gene whose protein product can visually report the location
of a particular protein, cell type, or circuit. The most common reporter genes are
GFP and its variants. When placed under the control of a cell-specific promoter,
GFP can mark the location of these specific cells so they can be used in subsequent
experiments (e.g., electrophysiology, co-localization experiments, identification of
cells in culture). In addition to GFP, there are dozens of fluorescent proteins with
different spectral properties that appear as different colors, such as red fluorescent
protein, cyan fluorescent protein, and yellow fluorescent protein.
Fluorescent proteins can be manipulated so that they tag proteins or organelles
within a cell. For example, a scientist can use recombinant DNA methods (Chapter
10) to attach GFP to a gene that encodes a protein of interest so that the location,
expression, and trafficking of the protein can be studied in real time. It is also possible
to add specific sequence tags to fluorescent proteins so that they localize to discrete
regions of the cell, such as the cell membrane. Membrane-bound fluorescent
reporters can be useful for tracing axons and dendrites in addition to cell bodies.
LacZ is another common reporter gene. Originally derived from bacteria,
lacZ encodes the enzyme β-galactosidase. This enzyme reacts with a substrate,
X-gal, to create a dark blue product. Therefore, lacZ can serve as a faithful
reporter of gene expression after histological processing with X-gal.
Genes Used to Measure Neural Activity
Chapter 7 described methods of indirectly measuring neural activity by imaging
calcium dynamics and the membrane potential. Genetically encoded
calcium indicators (GECIs), such as GCaMPs, or genetically encoded voltage
indicators (GEVIs), such as ArcLight, allow targeted observation of neural
activity in specific cell types. This specificity is an advantage over injectable
activity-sensitive dyes that label all neurons at the injection site.
Making and Using Transgenic Organisms Chapter | 12  255
TABLE 12.1  Examples of Commonly Used Transgenes
Type of
transgene Utility Examples
Reporter genes Fluorescent
proteins
Marks the location
of cells, subcellular
structures, and
organelles
Green fluorescent
protein (GFP)
and multicolored
variants
Fluorescent
proteins to
trace neural
connections
Addition of
membrane tags to
fluorescent proteins
so they are bound to
the membrane
Farnesylated-GFP,
Myristolated-GFP
Bioluminescent
proteins
Proteins that produce
light as a byproduct
of a biochemical
reaction
Luciferase
Nonluminescent
proteins
Proteins that mark
expressing neurons
after histological
processing
LacZ
Alkaline
phosphatase (AP)
Genes for
measuring
neural activity
Calcium
indicators
Sensors that change
fluorescence in
response to different
concentrations of
calcium ions
GCaMPs
Voltage sensors Sensors that change
fluorescence in
response to changes
in membrane
potential
ArcLight
Synaptic activity
sensors
Sensors that change
fluorescence in
response to changes
in synaptic vesicle
fusion
Synapto-pHluorin
Genes for
inducible
activation of
neural activity
Optogenetics Membrane-bound
channels that
depolarize cells when
stimulated by light
Channelrhodopsin-2
(ChR2)
Ligand receptors Ligand-gated ion
channels that
depolarize cells when
a ligand is added
Capsaicin receptor
(TRPV1)
DREADD (HM3Dq)
Continued
256  Guide to Research Techniques in Neuroscience
Genes Used to Manipulate Neural Activity
Neural activity can be manipulated through nongenetic methods, such as electrical
stimulation or pharmacological inhibition. However, these methods rarely
stimulate or inhibit all cells of a desired subtype, and they often affect undesired
subtypes. Transgenes that allow inducible activation or inhibition of neural
activity solve these problems by providing control over genetically targeted
cells, with no effect on cells that do not express the transgene. These transgenes
use either optically controlled or ligand-gated channels to selectively stimulate
or inhibit neural activity.
Chapter 8 described two optically controlled transgenes used to manipulate
neural activity. Channelrhodopsin-2 (ChR2), originally derived from singlecelled
algae, is a cation channel that can depolarize a neuron upon stimulation
with blue light. Because ChR2 can be placed under cell-specific promoters, it is
Type of
transgene Utility Examples
Genes for
inducible
inhibition of
neural activity
Optogenetics Membrane-bound
pumps that
hyperpolarize cells
when stimulated by
light
Halorhodopsin
(NpHR)
Archaerhodopsin
(Arch)
Ligand receptors Ligand-gated ion
channels that
hyperpolarize cells
when a ligand is
added
Allatostatin
receptor (Alstr)
DREADD (HM4Di)
Shibire mutants Temperature sensitive
mutants of the
Drosophila shibire
gene (dynamin) that
prevent synaptic
transmission at
certain temperatures
Shits
Genes for
neuronal
ablation
Toxins Kills all expressing
neurons
Ataxin
Toxin receptors When the toxin is
applied to the brain,
only expressing cells
will die
Diphtheria toxin
receptor
TABLE 12.1  Examples of Commonly Used Transgenes—cont’d
Making and Using Transgenic Organisms Chapter | 12  257
possible to stimulate specific cell types in a heterogeneous population of neurons
by delivering blue light to those neurons. The genetically encoded chloride
pump Halorhodopsin (NpHR) and the genetically encoded hydrogen pump
Archaerhodopsin (Arch) hyperpolarize neurons upon stimulation with yellow
light. Using these transgenes, it is possible to silence single action potentials or
sustained trains of neuronal spiking.
Another method of manipulating neural activity using genetically encoded
transgenes is to express a ligand-gated ion channel in the brain of an animal that
normally does not express this channel. For example, the capsaicin receptor
(TrpV1) is a ligand-gated channel that depolarizes a neuron upon exposure to
capsaicin. Flies and other invertebrates do not normally express this channel, so
capsaicin exposure will depolarize only those neurons that express the receptor.
A similar strategy can be used to inhibit activity and hyperpolarize neurons in the
presence of a ligand. For example, the insect allatostatin receptor (Alstr) is not
expressed in mammals. In the presence of the insect protein allatostatin, this receptor
inhibits neural activity. Thus, if Alstr is expressed in the mammalian brain,
addition of allatostatin will reversibly inactivate genetically targeted populations
of neurons until the ligand is washed out from the system. Synthetic G-protein
coupled receptors are also useful for modulating neural activity. Designer receptors
exclusively activated by designer drugs (DREADDs) consist of artificial
receptors for the normally inert ligand clozapine-N-oxide (CNO). In the presence
of CNO, hM3Dq will depolarize a neuron, while hM4Di will hyperpolarize a
neuron. These techniques are discussed in much more detail in Chapter 8.
Genes Used to Ablate Neurons
Lesion studies have historically been a staple of neuroscience research to investigate
the necessity of a brain region for a specific behavior or phenotype. Using
physical or pharmacological methods to ablate neurons raises concerns about the
specificity and totality of the lesions. Transgenic technology allows lesions to
be targeted to specific, genetically defined subsets of neurons. Genetic ablation
studies can be performed using the ataxin transgene, which encodes a toxin that
kills all expressing neurons. It is also possible to use a transgene that encodes a
toxin’s receptor, such as the diphtheria toxin receptor. This receptor is toxic
to neurons only when diphtheria toxin is present. Neurons that do not express
the receptor will not be sensitive to the toxin. Using a genetically encoded toxin
receptor instead of a genetically encoded toxin itself allows a scientist to ablate
neurons at any time point during an animal’s lifespan. By controlling when the
toxin is injected, the scientist controls the timing of ablation.
Genes Used to Disrupt Endogenous Gene Function
In mice, it is possible to knock out genes from the genome using gene targeting
technology, as discussed in Chapter 13. It is also possible to express transgenes
that disrupt endogenous gene or protein function. The overexpression of
258  Guide to Research Techniques in Neuroscience
dominant negative transgenic constructs directly interferes with protein function,
whereas RNA interference (RNAi) technology can be used to knock down
the expression of a gene by targeting mRNA before it is translated into a functional
protein. We will discuss dominant negatives, RNAi, and how they work
in Chapter 13.
Overexpression of Endogenous Genes
For some research questions, it is useful to produce transgenic animals that
overexpress an endogenous gene or express the gene in a cell-type that normally
does not express it. For example, a scientist wishing to perform a
gain-of-function experiment in which a gene of interest is expressed at a
much higher level than normal could create a transgenic animal that highly
overexpresses the gene. Scientists can also show that overexpression of a
gene in a knockout background can rescue the gene’s function. Alternatively,
a scientist can show that expressing a gene is sufficient to cause a
particular function. For example, he or she may hypothesize that a gene
encoding a cell-surface receptor is important during development to attract
axons from a different brain region. A transgenic mouse that expresses the
receptor in a different cell type could be used to test whether the presence
of this receptor in other cells is sufficient to cause the axons to target the
wrong region.
Now that we have surveyed some commonly used transgenes, we examine
how a scientist creates a transgenic construct and expresses foreign DNA in an
animal’s genome.
THE TRANSGENIC CONSTRUCT
The first step in producing a transgenic organism is to use recombinant DNA
technology (Chapter 10) to make a transgenic construct. This construct must
contain (1) the DNA sequence encoding the transgene and (2) the necessary
promoter sequences for the expression of the gene (Figure 12.1). Simply
inserting a gene into the genome will not cause it to be expressed: endogenous
proteins must recognize a promoter sequence to attract a transcription complex
that recruits RNA polymerase to transcribe the DNA into mRNA.
Promoter region Coding region
Transcriptional
start site
5’ 3’
FIGURE 12.1  A transgenic DNA construct.  A construct is composed of the transgene (coding
region) and promoter region. The promoter region attracts endogenous cellular transcription factors
and other proteins to drive expression of the transgene.
Making and Using Transgenic Organisms Chapter | 12  259
Promoters control the expression of genes so that they are expressed in specific
cells and tissues at specific times. For example, every cell in the human
body contains the gene encoding the enzyme tyrosine hydroxylase (TH). However,
this gene is only expressed in a subset of neurons in the brain because only
these cells have the endogenous transcription factors and other regulatory
proteins necessary to interact with the promoter for TH and activate transcription.
Therefore, a transgenic construct containing all the necessary promoter
elements for the TH gene will allow a transgene to be expressed specifically in
the subset of cells that normally transcribe TH. A scientist can express a transgene
in a different neuronal subtype by identifying a gene uniquely expressed in
those cells and taking advantage of the promoter elements for that gene.
Promoter sequences are often depicted as existing just upstream of the genes
they regulate (Figure 12.1). However, this is not necessarily the case, especially
in mammals. Promoter elements necessary to drive a gene of interest may be
located thousands or even hundreds of thousands of base pairs upstream of a
gene. They may be located in the introns within the coding sequences of a
gene or even downstream of the coding sequence of the gene. This phenomenon
restricts the promoters that can be used to drive transgene expression. In
general, it is easier to use promoters to target specific cell types in invertebrate
model organisms, as promoters in these species are often located just upstream
of the genes they regulate.
Because mammalian promoters can be difficult to isolate and place in a
transgenic construct, some laboratories make transgenic mice using BAC constructs.
BAC stands for bacterial artificial chromosome, a large 50-to 350-kb
vector that can be used to create genetic libraries and clone mammalian DNA
sequences (Chapter 10). Investigators can order a BAC that contains any gene
in the genome, flanked by 50–100 kb of genomic sequences on either side of the
gene. Thus, a transgenic construct could be produced by inserting a transgene at
the transcriptional start site of the endogenous gene in a BAC. The transgene
would then be located at the same location as the endogenous gene in relation to
promoter elements. The limitation to using this technology is that recombinant
DNA technology in BACs can be difficult due to the large size of the BAC compared
to other expression vectors, which are usually 3–10 kb in length.
Whether using small expression vectors or BACs, scientists make DNA
constructs in DNA vectors that are circular, as described in Chapter 10. However,
before injecting the transgene into cells to produce transgenic animals, the
DNA is almost always made linear by using restriction enzymes to isolate the
promoter/transgene sequence away from the vector. This helps the promoter/
transgene region integrate into the genome as a linear fragment, as well as discards
unnecessary coding sequences that may be in the vector, such as antibiotic
resistance genes.
Transgenes typically integrate into the genome in random locations.
Although there are certain regions of the genome that appear to be “hot spots”
of integration activity, scientists generally cannot control where the transgene
260  Guide to Research Techniques in Neuroscience
(A) (B)
UAS+
Promoter A
TGal4 UAS+
Promoter A
Promoter B
T
Gal4
Gal80
(C) (D)
+
Promoter A Promoter B
lox
lox
TCre +
Promoter A Promoter B
lox
lox
TCre
(E) (F)
+
Promoter A Promoter B
S
lox
lox
STO
P
TCre +
Promoter A Promoter B
S
lox
lox
STO
P
TCre-ER
(G)
+
Promoter A Promoter B
Frt
Frt
TFlp
(H)
+
Promoter A Promoter B
Frt
Frt
TFlp
(I)
+
Promoter A
TetO TtTA
(J)
+
Promoter A
TetO TrtTA
FIGURE 12.2  Binary expression systems. These systems use two or more constructs to
spatially or temporally restrict transgene expression. (A) The Gal4/UAS system. Promoter A
drives expression of the transcription factor Gal4. Gal4 binds to UAS and activates expression
of Transgene T. Therefore, T expression is indirectly controlled by Promoter A. (B) The Gal4/
Gal80/UAS system. As in (A), Promoter A drives expression of Gal4, which binds to UAS and
activates expression of Transgene T. Promoter B drives expression of Gal80, which inactivates
Gal4. Therefore, T will be expressed in cells in which Promoter A is active but not in cells
in which Promoter B is active. (C) The Cre/lox system. Promoter A drives the expression of
Cre recombinase and Promoter B drives the expression of Transgene T. Cre recognizes lox sites
and catalyzes a recombination event that removes DNA within the sites. Therefore, T will be
expressed in cells in which Promoter B is active but not in cells in which Promoter A is active.
(D) The Cre/lox system with inverted lox sites. When Cre recognizes two lox sites facing each
other, Cre rearranges the DNA between the lox sites so that it becomes “flipped” in the reverse
orientation. If a transgenic construct is produced in which the transgene is oriented in the opposite
direction, a Cre construct can be used to flip the transgene in the correct orientation. Thus,
Transgene T will be expressed in cells in which both promoters A and B are active. (E) The Cre/
lox system used to remove a stop site. Cre is used to remove a stop codon that prevents the expression
of Transgene T. Therefore, T is expressed in cells in which promoters A and B are active.
(F) Promoter A expresses Cre recombinase fused to the estrogen receptor (ER). ER sequesters
Cre in the cytoplasm until a scientist adds tamoxifen, which binds to ER and causes Cre to translocate
to the nucleus, where it can remove a stop codon preventing expression of Transgene T.
Making and Using Transgenic Organisms Chapter | 12  261
integrates unless using specific gene-targeting methods described in Chapter 13.
Because of this random integration, position effects can occur that influence the
transgene’s expression pattern. Depending on where the transgene lands in the
genome, neighboring genes and regulatory sequences can alter the levels and
pattern of expression. The number of transgene copies that integrate into the
genome can also vary the levels and pattern of transgene expression. Therefore,
transgenic organisms must be carefully evaluated to ensure that transgenes are
expressed in the desired cell types. Just because a specific promoter is used
to drive transgene expression, there is no guarantee that the transgene will be
expressed in the desired cells.
BINARY TRANSGENIC SYSTEMS
The simplest method of making a transgenic animal is to use a single DNA
construct, as just described, in which a promoter directly regulates the expression
of a transgene. An alternate approach is a binary expression system that
uses multiple constructs to further refine the spatial and temporal expression of
a transgene (Figure 12.2 and Table 12.2). To use a binary expression system, a
scientist creates two different lines of transgenic animals, each line expressing
one of the required constructs, and then mates the animals to produce offspring
that express both constructs. Alternatively, the scientist can express one construct
in a transgenic animal and deliver the other construct using other gene
delivery methods, such as viral gene delivery (Chapter 11).
These flexible expression systems have numerous advantages. The main
advantage is that they can provide control over the timing and location of transgene
expression. Another substantial advantage is the ability to mix and match
combinations of transgenes, saving time and effort in making new transgenic
constructs.
Therefore, T is expressed in cells in which Promoters A and B are active but only when
tamoxifen is added. (G) The Flp/Frt system. Promoter A drives the expression of Flippase recombinase
(Flp), and Promoter B drives the expression of Transgene T. Flp recognizes Frt sites and
catalyzes a recombination event that removes DNA within the sites. Therefore, T will be expressed
in cells in which Promoter B is active, but not in cells in which Promoter A is active. (H) The Flp/
Frt system with inverted Frt sites. When Flp recognizes two Frt sites facing each other, Flp rearranges
the DNA between the Frt sites so that it becomes “flipped” in the reverse orientation. If a
transgenic construct is produced in which the transgene is oriented in the opposite direction, a Flp
construct can be used to flip the transgene in the correct orientation. Thus, Transgene T will be
expressed in cells in which both promoters A and B are active. (I) The Tet-off system. Promoter
A drives the expression of the tetracycline transactivator (tTA) protein. tTA binds to the tetO promoter,
activating expression of Transgene T. Therefore, expression of T is indirectly controlled by
Promoter A. If a scientist applies a drug called doxycycline, tTA is reversibly inhibited. (J) The
Tet-on system. Promoter A drives the expression of the reverse tetracycline transactivator (rtTA).
rtTA is unable to bind to the tetO promoter unless doxycycline is present. Therefore, Promoter A
indirectly activates expression of Transgene T, but only when a scientist adds doxycycline.
262  Guide to Research Techniques in Neuroscience
The Gal4/UAS System
The Gal4/UAS system is a binary expression system primarily used in Drosophila,
although it has also been applied to mice and zebrafish. In one construct, a cellspecific
promoter drives the expression of the gene encoding Gal4, a transcription
factor normally expressed in yeast. In a second construct, a transgene of interest is
regulated by a promoter sequence called an upstream activation sequence (UAS).
The Gal4 protein binds to the UAS sequence and efficiently drives expression of the
transgene (Figure 12.2A). Thus, the transgene will only express in cells defined by
the promoter regulating Gal4.
Why not simply place the transgene under the control of the promoter regulating
Gal4? There are at least two reasons. First, the UAS expresses transgenes
at much higher levels than endogenous promoters, so the Gal4/UAS constructs
together allow for high levels of gene expression. Second, Gal4 lines now exist
for hundreds of genes in the Drosophila genome. There are also many constructs
in which a UAS drives a specific transgene. Therefore, the Drosophila
scientific community has created an amazingly efficient system of creating and
delivering transgenes to many different cell types (Figure 12.3).
Gal4/UAS can also be used in combination with another yeast protein,
Gal80. The Gal80 protein binds to and inhibits the activity of Gal4. Thus,
a second promoter element can drive expression of Gal80 to further restrict
the cells expressing the transgene (Figure 12.2B). Furthermore, Gal80 can be
made temperature sensitive: the Gal80ts protein is active at 19 °C but not 30 °C.
TABLE 12.2  Promoters in Binary Transgenic Systems
Binary
transgenic
system
Cell and tissue specificity
is conferred by the
promoter(s) regulating:
The gene of interest is
directly regulated by
the promoter called:
Gal4/UAS Gal4 UAS
Gal4/Gal80/UAS Gal4 and Gal80 UAS
Cre/lox Cre Any promoter can be
used…. Lox sites determine
the location of DNA
recombination
Flp/Frt Flp Any promoter can be
used…. Frt sites determine
the location of DNA
recombination
tTA/tetO tTA tetO
rtTA/tetO rtTA tetO
Making and Using Transgenic Organisms Chapter | 12  263
Therefore, it is possible to express a transgene in specific cells using the Gal4/
UAS system and to regulate the timing of transgene expression by controlling
the temperature, and thus the expression of Gal80ts.
The Cre/Lox System
The Cre/lox system is a site-specific recombination system widely used in mice.
The enzyme Cre recombinase, originally derived from the P1 bacteriophage,
recognizes specific 34 base-pair DNA sequences called lox sites. Lox sites are
added to a transgene so that they flank a sequence of DNA. This is referred to
as a “floxed” sequence, a sequence flanked by lox sites. Each lox site has an
asymmetric sequence in the middle, providing the sequence with directionality.
This directionality is important because Cre processes DNA depending on the
orientation of the lox sites:
	l	Cre excises any DNA sequences between two lox sites oriented in the same
direction Figure 12.2(C)
	l	Cre inverts any DNA sequences between two lox sites oriented in inverted
directions Figure 12.2(D)
Therefore, the Cre/lox system allows for complex rearrangement of DNA in
transgenic mice. Many transgenic lines now exist in which Cre recombinase is
regulated by cell-specific promoters. The ability to engineer various combinations
of lox sites and specific gene sequences provides unique experimental possibilities.
For example, a transgenic mouse can selectively express a transgene
Gal4 lines UAS lines
B X
C
UAS
UAS
Any combination of genetic coding sequence (1, 2, 3, etc.)
driven by cell-specific promoter (A,B,C, etc.)
UAS 1
2
3
A Gal4
Gal4
Gal4
FIGURE 12.3  The utility of the Gal4/UAS system. Scientists have created hundreds of fly
stocks in which endogenous promoters drive cell-specific expression of Gal4 transcription factors.
In turn, any transgene of interest can be placed under the control of a UAS sequence. Therefore,
a scientist can cross any combination of Gal4 line with a UAS line to drive transgene expression
in specific cell types. This system has been used extensively in the fly, as well as in Xenopus and
zebrafish model organisms.
264  Guide to Research Techniques in Neuroscience
with a “floxed stop” strategy: a “stop” codon is placed directly upstream from
a transgene, preventing the transgene’s expression. If two lox sites flank the
“stop” codon, the transgene will only be expressed in cells that express Cre to
remove the “stop” (Figure 12.2E). An alternative strategy, usually referred to
as a double-inverse orientation (DIO) or Flex construct, is the use of two
different pairs of lox sites to cause recombination that leads to Cre-dependent
expression of a transgene.
Investigators can achieve temporal control of Cre/lox technology by fusing
the gene encoding Cre recombinase with the gene encoding the estrogen
receptor (ER). The ER is normally sequestered in the cytoplasm, but it translocates
to the nucleus after binding to its ligand. The ER sequence fused to
Cre is altered so it preferentially binds to the chemical tamoxifen, an estrogen
antagonist, rather than endogenous estrogen present in an animal. In baseline
conditions, Cre-ER fusion proteins are sequestered in the cytoplasm and unable
to access lox sites in the DNA located in the nucleus. If a scientist injects tamoxifen
into a mouse, it will bind to Cre-ER, causing it to translocate to the nucleus
and recombine DNA (Figure 12.2F).
Another method for temporal and spatial control over Cre-mediated excision
is the direct introduction of Cre transgenes through gene delivery methods
as described in Chapter 11. Cre transgenes can be delivered to a floxed animal
through electroporation or viral transduction, or to cells cultured from a floxed
animal using in vitro methods of gene delivery.
The Flp/Frt System
The Flp/Frt system is a site-specific recombination system analogous to the
Cre/lox system just described. The enzyme Flippase recombinase (Flp), originally
derived from yeast, recognizes specific 34 base-pair DNA sequences
called Flippase recognition targets (Frts), excising DNA between identical
Frt sites (Figure 12.2G) and inverting DNA between inversely oriented Frt sites
(Figure 12.2H). This system is used in both flies and mice.
The Tet-off/Tet-on System
The Tet-off/Tet-on system is a tool used in the fly and mouse to temporally
control transgene expression. In the Tet-off system, a promoter drives the
expression of the tetracycline transactivator (tTA) protein. This protein binds
to another DNA sequence called the tetO operator, which regulates the expression
of a transgene. Thus, in normal conditions, the transgene expression is
regulated by the promoter that regulates tTA. However, a scientist can feed or
inject a derivative of the antibiotic tetracycline called doxycycline into these
transgenic mice. Doxycycline binds to tTA and renders it incapable of binding
to tetO. Therefore, expression of the transgene can be reversibly inhibited with
the addition of doxycycline (Figure 12.2I).
Making and Using Transgenic Organisms Chapter | 12  265
The Tet-on system works in the opposite way. A reversed tTA (rtTA) is only
capable of binding to tetO when doxycyline is present. Therefore, the addition
of doxycyline causes transcription of the transgene (Figure 12.2J). Like the Tetoff
system, the effect of doxycycline is reversible.
These systems have gained popularity for their reversibility and temporal
control, as well as for the ability to control levels of transgene activation.
Increasing the concentration of doxycycline in the Tet-off system gradually
lowers and then shuts off transgene expression, while increasing concentrations
of doxycycline in the Tet-on system gradually increases transgene expression.
MAKING TRANSGENIC ORGANISMS
After using recombinant DNA technology to produce a transgenic construct, an
investigator must deliver the construct to recently fertilized eggs so that it can
integrate into the genomes of the offspring. This section describes the process
of creating transgenic mice, flies, worms, and other animals.
Making Transgenic Mice
To make a transgenic mouse, a foreign piece of DNA is injected into the nucleus
of a newly fertilized egg, where it can randomly incorporate into the genome.
After the egg develops into a fetus and eventually a postnatal offspring, the
transgene can become stably expressed and transferred to future offspring. In
many institutions, individual investigators do not inject the transgenic construct
into eggs themselves but submit their constructs to a specialized transgenic animal
facility that has expertise in creating transgenic mice. As opposed to flies or
worms, the harvesting and injection of recently fertilized eggs is a technically
challenging process that most laboratories choose to outsource.
The transgenic construct is injected as a linear piece of DNA into the larger male
pronucleus of a recently fertilized mouse egg (Figure 12.4). A pronucleus is the
haploid nucleus of either the egg or sperm, both present in the fertilized egg prior
to their fusion. This is the stage at which insertion of foreign DNA into the genome
can occur. Because the embryo is at the one-cell stage, the incorporated gene is
replicated and can end up in nearly all of the animal’s cells in adulthood. For the
transgene to be heritable, it must be incorporated into the germline (eggs or sperm).
After several embryos have been injected with the transgene, they are subsequently
injected into a pseudopregnant mouse (Figure 12.5). The term pseudopregnant
refers to the fact that the female is not actually pregnant but has been
“tricked” into thinking she is pregnant by mating with a sterile male mouse.
This is done to produce the hormones and physiological changes necessary
to develop the embryo into a viable fetus. The mouse eventually carries the
embryos to term, and transgenic mice are born. Each transgenic facility uses
different procedures, but often 2–4 pseudopregnant females are used to produce
20–30 transgenic offspring.
266  Guide to Research Techniques in Neuroscience
These transgenic offspring are called founders, as they represent the first generation
of a new line of mice. However, not all offspring will have successfully
incorporated the transgene, and those that do may not express the transgene in
the desired cells and tissues. Therefore, once mice are born, the investigator must
detect which mice have incorporated the transgene by genotyping the animals with
a Southern blot and/or polymerase chain reaction (PCR; Chapter 10). Southern
blots are required to fully characterize the number of transgene copies and verify
FIGURE 12.4  Injection of a transgenic construct in mice.  To produce a transgenic mouse, a
scientist microinjects a transgenic construct into one of two pronucei in a recently fertilized egg.
Fertilized eggs
injected into oviduct
of pseudopregnant
female mouse
Some offspring might
properly express the
transgene, others will not
FIGURE 12.5  Injection of embryos to make transgenic mice. After injecting embryos with a
transgenic construct, these embryos are subsequently injected into the oviduct of pseudopregnant
female mice. Each female typically produces 6–10 offspring. However, only a fraction of the offspring
express the transgene.
Making and Using Transgenic Organisms Chapter | 12  267
the integrity of the transgene sequence. However, PCR of genomic tail DNA is a
rapid way to reveal if the transgene is present and is the standard method for genotyping
future offspring.Any mice that test negative for the transgene are discarded.
Mice that test positive for the transgene are mated with wild-type mice so their offspring
can be further investigated. These mice are systematically examined for the
presence of the transgene and rigorously tested to ensure that the gene is expressed
in the appropriate tissues. Because copy number and integration position can affect
the expression pattern of a transgene, multiple founder lines are characterized. The
total time necessary to produce and analyze the validity of a transgenic mouse is
usually about 6–9 months, including the time necessary for breeding, gestation,
development, and analysis of genotype and phenotype in the laboratory.
Making Transgenic Flies
The process of making a transgenic fly takes advantage of specialized, endogenous
segments of DNA called transposons (also called transposable elements)
that can move from one position to another within the genome. These
mobile genetic sequences have only modest target site selectivity within the
genome and can therefore insert themselves into random genomic locations. In
transposition, an enzyme called transposase acts on specific DNA sequences
at the end of a transposon, first disconnecting it from the flanking DNA and then
inserting it into a new target DNA site.
The most commonly used transposons in Drosophila are called P elements.
To make a transgenic fly, a DNA construct is first inserted between the two
terminal sequences of the Drosophila P element (Figure 12.6). The terminal
sequences enable the P element to integrate into Drosophila chromosomes
when the P element transposase enzyme is also present. The investigator injects
the transgenic construct into a very young fruit fly embryo along with a separate
plasmid containing the gene encoding the transposase (Figure 12.7). If this
technique is performed correctly, the injected gene enters the germ line as the
result of a transposition event.
Making Transgenic Worms
Transgenic techniques in Caenorhabditis elegans are conceptually similar to
transgenic techniques in flies. An important difference is that worms are hermaphrodites
and capable of carrying their own fertilized eggs. A transgene is
injected into the gonad of a young adult hermaphrodite at the time the embryos
are dividing. The gonad runs the length of the worm on the dorsal side, and, with
practice, an investigator can become skilled at such injections (Figure 12.8).
The transgene can either be carried as an extrachromosomal array (although
worms will not necessarily carry the gene in the next generation) or, if the worm
is subjected to gamma or ultraviolet irradiation, it can be integrated into a chromosome
to make transgene expression more stable and less variable.
268  Guide to Research Techniques in Neuroscience
To ensure that the transgenic procedure was successful, investigators often
use a co-injection marker that allows for easy identification of transgenic animals.
For example, the investigator can co-inject the transgene with another
construct encoding GFP. To avoid potential interference with the transgene
of study, the co-injection marker can be controlled by a promoter that causes
expression in a nonoverlapping, different cell type. GFP can then serve as a
positive control to verify that the transgenic manipulation was successful.
Transgenic techniques in C. elegans are good for studying gain-of-function
(sufficiency) effects, rescuing mutant lines, expressing reporter genes, and expressing
RNAi constructs. Because there are many promoter elements that allow targeting
to individual neurons, C. elegans is a powerful genetic model system used
throughout neuroscience to study the genetic basis of development and behavior.
P element
5’ 3’
Transposase
DNA ligates together P element ligates into
new DNA region
FIGURE 12.6  P elements.  The transposase enzyme splices a P element out of its genetic environment
(the genome of a transgenic construct), inserting it into a new location in the genome.
Anterior
Posterior
Trachea
FIGURE 12.7  Injection of a transgenic construct in flies.  To make transgenic flies, a scientist
microinjects a transgenic construct into the posterior end of a fresh Drosophila embryo.
Making and Using Transgenic Organisms Chapter | 12  269
Making Other Transgenic Organisms
Although mice, flies, and worms are the most frequently used genetic model
organisms, in theory, a scientist can insert a transgene into any species by
correctly injecting DNA into a recently fertilized egg. For example, some
laboratories have created transgenic rats to study behavioral tasks that are too
sophisticated for mice. Different species of fish have stably expressed transgenes
(including “GloFish,” the first transgenic organism to be sold as pets).
Cows, pigs, goats, and sheep have also expressed transgenes. A transgenic cow
has been created that can produce human protein–enriched milk, a more nutritionally
fortified product than regular bovine milk that can be provided to babies
or the elderly with special nutritional needs. Other transgenic cows produce
milk with less lactose or lower cholesterol. Products such as insulin, growth
hormone, blood anticlotting factors, and so forth can also be produced in transgenic
animals and collected through animal milk. Thus, transgenic technology
is useful not only for studying the biology of organisms, but also for producing
nutritional resources and biological reagents that can benefit the health of
humans.
CONCLUSION
This chapter has surveyed the amazing variety of transgenes useful for studying
neurons and neural circuits, as well as describing how scientists can stably
insert these genes into the genomes of a variety of model organisms. Laboratories
continually clone and create new transgenes each year that add important
tools to the genetic toolkit. For example, the DREADD andArcLight transgenes
have only been created and reported within the past few years, and now multiple
laboratories are expressing these genes in specific cell types to answer important
questions about the nervous system. The next chapter describes methods
that complement transgenic technologies by targeting genes to specific locations
in the genome. These methods can be used to create transgenic animals,
as well as to replace functional genes with noncoding genes to produce genetic
“knockouts.” These technologies further expand the neuroscientist’s toolkit of
genetic techniques and allow for genetic access to specific cell types in the
nervous system.
Anterior
Posterior
Dorsal
Ventral
Embyros
FIGURE 12.8  Injection of a transgenic construct in worms.  To make transgenic worms, a scientist
microinjects a transgenic construct into a worm’s dorsal surface at the time embryos are dividing.
270  Guide to Research Techniques in Neuroscience
SUGGESTED READING AND REFERENCES
Books
Behringer, R., Gertsenstein, M., Nagy, K., & Nagy, A. (2013). Manipulating the mouse embryo:
A laboratory manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Greenspan, R. J. (2004). Fly pushing: The theory and practice of Drosophila genetics (2nd ed.).
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hofker, M. H., & van Deursen, J. (Eds.). (2010). Transgenic mouse methods and protocols (Methods
in molecular biology). New York, NY: Humana Press.
Pinkert, C. A. (2014). Transgenic animal technology: A laboratory handbook (3rd ed.). San Diego:
Academic Press.
Review Articles
Barth, A. L. (2007). Visualizing circuits and systems using transgenic reporters of neural activity.
Current Opinion in Neurobiology, 17, 567–571.
Duffy, J. B. (2002). GAL4 system in Drosophila: a fly geneticist’s swiss army knife. Genesis, 34,
1–15.
Luo, L., Callaway, E. M., & Svoboda, K. (2008). Genetic dissection of neural circuits. Neuron, 57,
634–660.
Scanziani, M., & Hausser, M. (2009). Electrophysiology in the age of light. Nature, 461, 930–939.
Zhang, F., Aravanis, A. M., Adamantidis, A., de Lecea, L., & Deisseroth, K. (2007). Circuit-breakers:
Optical technologies for probing neural signals and systems. Nature Reviews Neuroscience, 8,
577–581.
Primary Research Articles—Interesting Examples from the Literature
Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., & Roth, B. L. (2007). Evolving the lock
to fit the key to create a family of G protein-coupled receptors potently activated by an inert
ligand. Proceedings of the National Academy of Sciences of the United States of America, 104,
5163–5168.
Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., & Deisseroth, K. (2005). Millisecond-timescale,
genetically targeted optical control of neural activity. Nature Neuroscience, 8, 1263–1268.
Conti, B., Sanchez-Alavez, M., Winsky-Sommerer, R., Morale, M. C., Lucero, J., Brownell, S.,
et al. (2006). Transgenic mice with a reduced core body temperature have an increased life
span. Science, 314, 825–828.
Garner, A. R., Rowland, D. C., Hwang, S. Y., Baumgaertel, K., Roth, B. L., Kentros, C., et al.
(2012). Generation of a synthetic memory trace. Science, 335, 1513–1516.
Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., et al. (2003).
A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.
Nature, 425, 917–925.
Liu, X., Ramirez, S., Pang, P. T., Puryear, C. B., Govindarajan, A., Deisseroth, K., et al. (2012).
Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature, 484,
381–385.
Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A., et al. (2007). Transgenic
strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature,
450, 56–62.
Marella, S., Fischler, W., Kong, P., Asgarian, S., Rueckert, E., & Scott, K. (2006). Imaging taste
responses in the fly brain reveals a functional map of taste category and behavior. Neuron, 49,
285–295.
Making and Using Transgenic Organisms Chapter | 12  271
Protocols
Berkowitz, L. A., Knight, A. L., Caldwell, G. A., & Caldwell, K. A. (2008). Generation of stable
transgenic Caenorhabditis elegans using microinjection. JoVE, 18. http://dx.doi.org/10.3791/833.
http://www.jove.com/index/Details.stp?ID5833.
Wu, J. S., & Luo, L. (2007).A protocol for mosaic analysis with a repressible cell marker (MARCM)
in Drosophila. Nature Protocols, 1, 2583–2589.
Yang, X. W., & Gong, S. (2005). An overview on the generation of BAC transgenic mice for neuroscience
research. Current Protocols in Neuroscience, Chapter 5, Unit 5.20. http://dx.doi.org/
10.1002/0471142301.ns0520s31.
Websites
Gensat Project: http://www.gensat.org/index.html.
Human Genome ed. site: http://genome.wellcome.ac.uk/doc_wtd021044.html.
The Jackson Laboratory Cre Repository: http://cre.jax.org/.
Transgenic Fly Virtual Lab: http://www.hhmi.org/biointeractive/clocks/vlab.html.
273
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00013-7
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 13
Manipulating Endogenous Genes
After Reading This Chapter, You Should be Able to:
	l	Discuss methods of manipulating genomes in specific locations
	l	Compare classical and nuclease-based gene targeting technologies
	l	Compare methods that disrupt endogenous gene products
Techniques covered:
	l	Classical gene targeting: knockouts, knockins, conditional knockouts (Cre/lox,
Flp/Frt)
	l	Nuclease-based gene targeting: meganucleases, zinc finger nucleases (ZFNs),
transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 system
	l	Disrupting gene products: RNA interference (RNAi), morpholinos, dominant
negatives
In Chapter 12, we described the process of creating a transgenic animal, an organism
that integrates a piece of foreign DNA into its genome that can pass the DNA
to future offspring. Using traditional transgenic technologies, the foreign DNA
randomly integrates somewhere in the genome on any of the animal’s chromosomes.
It is often desirable to target a DNA construct to a specific location in
the genome, either to insert genetic material in a specific locus or to disrupt the
function of an endogenous gene. Gene targeting is the process of incorporating a
DNA construct into a specific locus in an animal’s genome. In a genetic knockout
animal, gene targeting is used to replace a functional gene with a nonfunctional
sequence. In a knockin animal, gene targeting is used to insert a DNA construct
into the genome at a specific location. More complicated gene targeting strategies
allow genes to be knocked out or knocked in to specific cell types at specific times.
The purpose of this chapter is to describe methods used to deliberately target
specific regions of the genome and/or interfere with functional gene products.
These methods are useful for reverse genetics experiments in which a scientist
tests the hypothesis that a specific gene is necessary for a defined phenotype.
Gene targeting techniques have existed for about three decades, and newer
cutting-edge methods are allowing for gene targeting in a wider variety of model
organisms. In addition to using gene targeting technologies to manipulate the
genome, a scientist can deliberately disrupt the expression of a gene at different
stages of the transcription/translation process (Figure 13.1). For example,
274  Guide to Research Techniques in Neuroscience
using RNA interference (RNAi) technology, it is possible to degrade mRNA
transcripts, preventing the translation of the gene into protein and knocking
down gene expression. Using dominant negative methods, gene function can
be blocked by interfering with the protein product. Each of these methods provides
scientists with powerful tools for perturbing the expression of endogenous
genes to determine the effect on an animal’s anatomy, physiology, and behavior.
CLASSICAL GENE TARGETING METHODS
The technology necessary to target DNA constructs to specific regions of the
genome was developed in the early 1980s. The scientists who developed these
methods, Mario Capecchi, Sir Martin Evans, and Oliver Smithies, shared the
2007 Nobel Prize in Physiology or Medicine for discovering and pioneering
the procedure for introducing specific gene modifications in mice. These techniques
continue to be widely used to create knockout and knockin animals;
however, these techniques are almost exclusively used in mice. Most loss-offunction
mutations in flies and worms result from forward genetic screens
(Chapter 9). Gene targeting technology is not widely used in other mammalian
species because of the need to culture and inject embryonic stem cells during
the targeting procedure, something that turns out to be surprisingly difficult for
rats and other nonmurine species. Therefore, the classical gene targeting techniques
described below are almost always used to target genes in mice. New
methods, such as CRISPR/Cas9, allow for genome editing in theoretically any
species, including humans. These techniques are described later in the chapter.
DNA
mRNA
Protein
Translation
Transcription
Gene targeting
(knockouts,
knockins,
conditional knockouts)
RNA interference,
morpholinos
Dominant negatives
Stage of
information flow
Methods of
manipulation
FIGURE 13.1  Methods of genetically perturbing endogenous genes and proteins.  A scientist
can manipulate gene and protein function at each stage of information processing. Gene-targeting
techniques can be used to alter the genome; RNA interference and morpholino techniques can be
used to functionally block mRNA transcripts; and other techniques, such as expression of dominant
negative constructs, can block protein function.
Manipulating Endogenous Genes Chapter | 13  275
Knockout Mouse
In a knockout mouse, gene targeting is used to replace a functional gene with a nonfunctional
sequence, effectively deleting the gene from the genome. There are five
major steps in the creation of a knockout mouse: (1) using recombinant DNA technology
to create a targeting construct; (2) injecting this construct into embryonic
stem (ES) cells and selecting cells that have properly incorporated the construct into
the genome; (3) inserting successfully targeted ES cells into a blastocyst; (4) inserting
the blastocyst into a female mouse; and (5) breeding the offspring of this mouse
until a successful knockout is achieved. This lengthy process can require 1–2 years
of work and breeding time before any mice are ready to use for experiments.
Creating a Targeting Construct
The first step in making a knockout mouse is to use recombinant DNA technology
to produce an appropriate targeting construct that will replace the gene
of interest. Replacing the target gene with the genetically engineered construct
relies on the natural phenomenon of homologous recombination, in
which regions with strong sequence similarity can exchange genetic material
(Figure 13.2A). In nature, homologous recombination produces new combinations
of DNA sequences during chromosomal crossover in meiosis, resulting
in enhanced genetic variation in a population. In the lab, homologous recombination
is used to replace endogenous genomic DNA sequences with targeting
constructs containing homologous sequences of DNA (Figure 13.2B).
To use homologous recombination, a scientist designs a targeting construct
with the replacement gene flanked by long segments of genomic DNA that are
homologous to sequences in the endogenous mouse genome. These homology
arms provide targeting specificity: the region between the two homologous segments
replaces the target gene in the organism. Homologous end sequences are
at least 1 kb (kilobase) in length, usually longer. A scientist can easily identify
(A) (B)
FIGURE 13.2  Homologous recombination.  (A) Homologous recombination is a natural phenomenon
in which genetic sequences with high sequence similarity can exchange genetic material,
as occurs with homologous chromosomes during meiosis. (B) In the lab, a scientist can exploit
homologous recombination to target a transgene to a specific locus in the genome. If the transgene is
flanked by regions homologous with the locus, homologous recombination can allow the transgene
to recombine into the genome.
276  Guide to Research Techniques in Neuroscience
and choose homologous sequences surrounding a gene of interest by looking up
their sequences in a mouse genome database.
The coding region in the middle of the construct contains a mutated version
of the endogenous gene that no longer produces a functional protein product.
This is often achieved by deleting the coding region within an exon. The targeting
construct also contains a selection cassette, typically with two additional
genes: a positive selection gene placed between the homology arms and a negative
selection gene placed outside the homology arms (Figure 13.3). The positive
selection gene makes cells resistant to a drug, such as neomycin, puromycin, or
hygromycin. Only cells that have incorporated the targeting construct into their
genome will survive if a scientist exposes the cells to the drug. The negative
selection gene, often thymidine kinase or diphtheria toxin A, can be used to kill
cells that randomly integrated the targeted construct somewhere in the genome
other than the desired locus. These genes allow for quality control while selecting
properly targeted cells if and when homologous recombination takes place.
Injecting and Selecting ES Cells
After the scientist makes the targeting construct, the DNA is microinjected or
electroporated into ES cells (Chapter 11). Because culturing and delivering
DNA to ES cells is technically challenging, most scientists outsource this step
to a specialized genetic engineering facility. Technicians inject dozens or even
hundreds of cells to enhance the chances of finding a cell that has properly
incorporated the targeting construct.
When the targeting construct is delivered to ES cells, three potential outcomes
can occur: (1) it correctly recombines and integrates into the cell’s genome; (2)
it integrates with the cell’s genome but in the wrong location; or (3) it fails to
integrate into the genome at all. To select ES cells that have successfully recombined
the targeting construct at the correct location, the technician utilizes the
positive and negative selection genes in the targeting construct (Figure 13.4).
For example, a selection cassette encoding the neomycin resistance gene can be
used to ensure that the construct has integrated into the genome. After introducing
the targeting construct, ES cells are exposed to neomycin, which kills all
cells that do not express the resistance gene. Therefore, only ES cells that have
incorporated the construct survive. Including the thymidine kinase (TK) gene
in the selection cassette ensures that the targeting construct has inserted itself
in the correct location. ES cells that have incorrectly incorporated the targeting
construct into the genome will express the tk gene and die in the presence of
a chemical called gancyclovir. Gancyclovir is harmless to normal cells, but it
becomes highly toxic when phosphorylated by the tk protein product. Because
Homology HomologyReplacement Neo TK
FIGURE 13.3  A generic gene targeting construct. (A) The replacement DNA sequence is
flanked by two regions of DNA that are homologous to the genomic locus where the DNA is to be
inserted. The neomycin resistance cassette (Neo) and thymidine kinase gene (TK) are also placed in
the construct to select embryos that have properly integrated the construct into the genome.
Manipulating Endogenous Genes Chapter | 13  277
the tk gene is outside the region of homology, if homologous recombination
occurs, the gene will be lost. Therefore, neomycin and gancyclovir offer positive
and negative selection measures, respectively, to ensure that the targeting construct
has successfully inserted itself into the genome at a precise location. Statistically,
homologous recombination is an extremely rare event, and only about
1% of the ES cells will survive these positive and negative selection measures.
Injecting ES Cells into a Blastocyst
After several positive ES cells are identified, they are injected into a blastocyst.
The blastocyst is an early embryonic structure that follows the fertilization
of an egg (Figure 13.5). There are two cell types within a blastocyst: the
trophoblast, which forms the outer shell of cells around the blastocyst and later
FIGURE 13.4  Selecting embryonic stem cells. The
gene targeting construct is delivered into mouse ES cells
by microinjection or electroporation. These cells are then
exposed to the compounds neomycin and gancyclovir.
Neomycin will kill cells that do not express the neomycin
resistance gene, cells that did not incorporate the targeting
construct into the genome. Gancyclovir is harmless to cells
unless the tk gene is expressed, which can occur only if the
targeting construct integrates at the wrong genomic location.
ES cells that survive treatment with both compounds
are further tested for expression of the construct and then
injected into blastocysts.
Microinjection or
electroporation
Add neomycin
and gancyclovir
Targeting construct
Embryonic stem cells
Surviving embryonic
stem cells
Blastocyst
TrophoblastInner cell
mass
FIGURE 13.5  Injecting ES cells into a blastocyst. A blastocyst is an early embryonic stage
resembling a sphere of different cell types. The trophoblast forms the outer sphere of cells and gives
rise to the placenta. The inner cell mass contains embryonic stem cells, which give rise to all cells
and tissues in the adult organism. A scientist injects ES cells containing the targeting construct into
the inner cell mass.
278  Guide to Research Techniques in Neuroscience
develops into the placenta, and the inner cell mass, a collection of embryonic
stem cells that eventually give rise to all cells and tissues in the adult organism.
ES stem cells that have survived the positive and negative selection process are
carefully injected into the inner cell mass. The future tissues that develop from
this blastocyst will be chimeric, with some tissues derived from the inner cell
mass of the host blastocyst and some tissues derived from the injected ES cells.
Inserting a Blastocyst into a Female Mouse
The next step is to insert the blastocyst into the uterus of a pseudopregnant
female mouse (Figure 13.6A). The pseudopregnant female is not actually pregnant
but has been psychologically tricked into believing she is pregnant by mating
with a sterile male mouse. This is done so that the female’s body produces
the hormones and physiological changes necessary to carry the embryo through
to a viable fetus. After injection, the mouse carries the embryos to term and
gives birth to offspring.
Breeding Offspring Mice
Once mice are born, it is necessary to determine which mice have incorporated
the knockout construct. Often, the blastocyst is obtained from a white strain of
mice, while the ES cells are derived from a brown strain (or vice versa). This
allows the scientist to visually determine the success of the ES cell injection
into the blastocyst: if the offspring of the host mother are entirely white, then
it is likely that no injected cells successfully combined with the blastocyst. If
the offspring have both brown and white patches of fur, the knockout ES cells
were successfully incorporated into the blastocyst (Figure 13.6B). Offspring
mice will never be entirely brown because the blastocyst is composed of ES
cells in the inner cell mass prior to injection of the knockout ES cells, and both
cell types continuously divide to produce the animal’s tissues. These mice are
referred to as chimeric mice because they have a combination of tissues from
the blastocyst-derived strain and the ES cell-derived strain. Some investigators
use these chimeras to investigate the cell-autonomous role of the gene of interest
by comparing the phenotypes of mutant cells to nonmutant cells. However,
more steps must be taken to produce a full knockout.
The ultimate hope of the scientist is that the chimeric mice carry the knockout
sequence in their germ cells, the cells that undergo meiosis to produce either sperm
in males or eggs in females. Only genetic material in the germ cells (as opposed
to somatic cells) will be passed on to future offspring. Therefore, some chimeras
will be able to pass along the knockout sequence in the germline; those chimeras
that do not carry the knockout sequence in germ cells will never pass along the
knockout sequence. After visually identifying chimeric mice, the scientist breeds
them with wild-type mice to produce nonchimeric heterozygous mice with one
wild-type copy of the gene and one mutant copy (Figure 13.6C). Offspring will
either be all white (no germline transmission in the chimera) or all brown.
The final step is to breed heterozygous mice with each other to produce full
homozygous knockout animals (Figure 13.6D). This breeding scheme should
Manipulating Endogenous Genes Chapter | 13  279
result in Mendelian inheritance, with one-quarter homozygous wild-type offspring,
one-quarter homozygous knockout offspring, and one-half heterozygous
offspring. However, knocking out certain genes can lead to an embryonic lethal
phenotype, in which a homozygous knockout mouse fails to survive gestation
(A)
(B)
(C)
(D)
X
X
+/+/-
+/-
25% of progeny: +/+
50% of progeny: +/-
25% of progeny: -/FIGURE
13.6  Generating a genetically targeted mouse.  (A) A scientist injects blastocysts into
a pseudopregnant female mouse. (B) The mouse produces about 8–10 offspring, with tissues that
developed from the endogenous ES cells residing within the blastocyst and the injected ES cells.
The injected ES cells are typically removed from an animal with a brown coat color, allowing
visualization when injected into mice with a white coat color. (C) Chimeric mice are bred with
wild-type mice. Any offspring mice with a brown coat color stably express the targeting construct
and are heterozygous for the endogenous gene. (D) To produce a full knockout, heterozygous mice
are bred with each other.
280  Guide to Research Techniques in Neuroscience
due to improper development. In these cases, scientists may still be able to examine
the gene’s role in early development. If the scientist is interested in studying
the gene’s role in postnatal animals, there are some alternative approaches. First,
the scientist could compare homozygous wild-type animals with heterozygous
animals to determine if having a single mutant version of the gene exerts any
phenotypic effect. Alternatively, the scientist could employ a conditional knockout
strategy in which the gene of interest is knocked out only in a particular cell
type or at a particular time. Conditional knockouts are described later in the
chapter, as are other alternatives to genetic knockouts, including RNAi or dominant
negative strategies to disrupt gene function in a postnatal animal.
In short, it takes four generations of animals to produce the first knockout
mice of a colony: the pseudopregnant foster mothers, the chimeric offspring, the
first heterozygous offspring of the chimeras, and the final homozygous knockout
animals. Therefore, producing a knockout can require a full year or more
because of the multiple generations of mice that must be bred and maintained.
Validating a Knockout Mouse
There are many methods that can be used to validate an animal’s genotype,
and a thorough investigation uses multiple techniques (Figure 13.7).
A Southern blot (Chapter 10) is a technique used to detect the presence of
specific sequences of DNA from the genome. Samples from wild-type, heterozygous,
and knockout mice can be compared to identify the presence or
absence and the length of specific DNA sequences at the gene locus. Alternatively,
a scientist may use a polymerase chain reaction (PCR) (Chapter
10) genotyping strategy and design primers around the gene’s locus.
Well-designed PCR primers will produce different-sized PCR products for
wild-type, heterozygote, and knockout genomic DNA. Usually, a scientist
performs a Southern blot to validate a knockout and then uses PCR for routine
genotyping, as PCR is a simpler and quicker method.
PCR and Southern blotting techniques are the absolute gold standard in
assessing the genotype of an animal. A scientist may also perform complementary
tests, such as in situ hybridization and/or immunohistochemistry
(Chapter 6) to confirm that there is no gene or protein expression in the tissues
where they are naturally expressed. A western blot (Chapter 15) can also
demonstrate the absence of a protein product.
Using Knockout Mice in Experiments
A knockout mouse is a powerful tool for investigating the necessity of a gene for
biologicalprocesses,suchasdevelopmentorbehavior.However,scientistsmustbe
careful to perform the appropriate control experiments to make valid conclusions
when using this tool. Regardless of whether the scientist studies an anatomical,
behavioral, or electrophysiological phenotype, knockout mice should be directly
compared with their wild-type littermates. This strategy reduces potentially
Manipulating Endogenous Genes Chapter | 13  281
confounding differences between mice of different genetic backgrounds. Some
studies compare wild type, heterozygous, and knockout littermates to demonstrate
a dose-dependent role for the relative amount of a gene product, with a
heterozygote phenotype intermediate between wild-type and knockout.
If no phenotype is immediately discernable, scientists must consider the
possibility of a genetic compensatory mechanism. A knockout animal may
compensate for the loss of a gene by altering the expression levels or pattern of
another gene or adapting in some other way so that the resulting phenotype is
still normal. Often, the phenotypic consequence of a genetic knockout is subtle,
and more refined assays may be required to identify a difference between
knockout and wild-type animals.
Finally, if a knockout mouse does exhibit a phenotype, the scientist must be
careful about interpreting the gene’s role. For example, if knocking out a specific
(B)
(A)
Endogenous
locus
Targeting
vector
Integration of
vector in
genome
Exon 1
neo thymidine kinase
BglII BglII
BglII
BglIIBglII
4.8 kb
2.8 kb
1 2 3 4 5
4.8 kb
2.8 kb
+/- -/- +/- -/-+/+
FIGURE 13.7  Analyzing genotype after gene targeting.  (A) In the literature, scientists typically
depict a gene targeting strategy by showing the endogenous locus, the targeting vector, and
the new genomic sequence. In this example, scientists replaced the first exon of a gene with a
new genetic sequence. Note the presence of the BglII restriction enzyme sites. In the endogenous
locus, digestion of DNA with BglII results in a 2.8 kb fragment. In a mouse in which gene targeting
occurred, the fragment will be 4.8 kb in length. The small black box depicted just beneath the
endogenous locus represents a probe that can be used to analyze the size of the fragment. (B) In a
Southern blot experiment, a scientist digests the DNA with a restriction enzyme, in this case BglII,
and uses a probe to detect a specific genetic sequence. Using this method, it is possible to discriminate
between fragments that are 2.8 and 4.8 kb in length, allowing the scientist to determine the
genotype of individual mice.
282  Guide to Research Techniques in Neuroscience
gene causes an animal to lose its ability to respond to painful stimuli, it is incorrect
to immediately conclude that the gene’s function is to regulate pain. The only conclusion
that can be reached is that the gene is necessary for the animal to report a
painful stimulus. More rigorous tests must be performed to determine whether the
gene is important for tactile sensation in general, for normal development of spinal
cord neurons, for correct axonal targeting to the periphery, etc. A full understanding
of a gene’s function comes from phenotypic analysis combined with investigations
into the specific mechanisms of the gene’s contributions to the phenotype.
Knockin Mouse
A knockin mouse is a genetically engineered mouse in which a genetic sequence
is added at a specific locus in the genome. This differs from a standard transgenic
mouse in that the DNA sequence is targeted to a particular location in
the genome, rather than being randomly integrated. The procedure to make a
knockin mouse uses the same homologous recombination strategy as the procedure
just described for making a knockout mouse. However, a knockin mouse
serves a very different purpose: to add functional genomic sequences, whether
they are functional genes or promoter sequences. There are various reasons why
a scientist may choose to make a knockin mouse:
	l	To replace a functional gene with a tagged version of the same gene. The
resulting reporter fusion protein can then be used to visualize gene expression
patterns in fixed or living tissue.
	l	To replace the gene of interest with a mutated version to mimic a human disease.
For example, if a scientist hypothesizes that the cause of a human disorder
is a specific mutation in a gene, the scientist could produce a mouse model
of the disease that carries this mutation in the homologous mouse gene.
	l	To replace a regulatory region of DNA to investigate the role of a specific
promoter or repressor element in the regulation of a gene product.
As with knockout mice, scientists should compare knockin animals with
their wild-type littermates during experiments.
Conditional Knockout Mouse
It is often desirable to disrupt a gene only in a particular cell-type or at a particular
time. For example, knocking out a gene in the entire organism could be lethal
at the embryonic stage, but if the ablation is restricted to a specific tissue or
cell-type or if the ablation occurs after the animal reaches adulthood, investigators
may be able to avoid the lethality. In these scenarios, a scientist may choose
to make a conditional knockout mouse in which a gene is removed from the
genome in a precise spatial or temporal manner.
One of the most common methods of creating a conditional knockout is
by using the Cre/lox recombination system (Figure 13.8; also described in
Manipulating Endogenous Genes Chapter | 13  283
Chapter 12). Homologous recombination is used to replace an endogenous
target gene with a fully functional version of the gene flanked by a pair of
short DNA sequences called lox sites (referred to as a floxed sequence). These
lox sites do not disrupt the gene they flank, so the phenotype of floxed mice
remains normal. Lox sites are specific 34 base pair sequences recognized by a
protein called Cre recombinase (or simply Cre) that is not normally expressed
in animals. Cre recognizes and recombines lox sites, excising any floxed genetic
material. To produce conditional knockout mice, mice expressing a floxed gene
are mated with transgenic mice expressing Cre under the control of a specific
promoter. Cre catalyzes recombination between the lox sequences, excising the
target gene from the genome. Many Cre lines now exist for distinct tissue and
neural cell types. An alternative to the Cre/lox system is the Flp/Frt recombination
system. This system is similar to Cre/lox, with the enzyme flippase
recombinase (Flp) excising Flp recombinase target (Frt) sequences that flank
the genomic region of interest.
To exert temporal control over a genetic knockout, such as knocking out a
gene in adulthood, scientists may use inducible knockout systems. A popular
method is to combine the Cre/lox recombination system with the ER/tamoxifen
system. The estrogen receptor (ER) is a protein that remains in the cytoplasm
unless it binds to its chemical ligand. Cre fused to the ER remains in the cytoplasm,
where it cannot act to recombine a floxed gene. The drug tamoxifen
binds to the ER receptor, causing the fused Cre-ER protein to translocate into
X
lox
lox
Tissue specific
promoter
lox
Cre Target Gene
FIGURE 13.8  Producing conditional knockout mice with the Cre/lox system.  A scientist produces
(or obtains) a mouse in which a gene of interest is flanked by two lox sites. If this mouse is
mated with another mouse expressing Cre recombinase, Cre can recombine the genetic material
between the two lox sites and excise the gene from the genome.
284  Guide to Research Techniques in Neuroscience
the nucleus to exert its effects and excise the floxed gene. After mating an inducible
Cre line of mice with the floxed line of mice, investigators control the timing
of the knockout through the timing of tamoxifen delivery.
NUCLEASE-BASED GENE TARGETING METHODS
As mentioned above, one limitation to using classical gene targeting technologies
is that they are only effective in mice. Attempts to use similar techniques
(injecting constructs with homology arms into embryonic stem cells) in other
model organisms have proven unsuccessful or inefficient, at least partially due
to the difficulty of culturing ES cells in non-murine species. Even using mice,
the process of creating a genetic knockout or knockin can require multiple years
of laborious work. However, within the past few years, new approaches have
been developed that show outstanding promise for gene editing in a wide variety
of model organisms.
Modern gene editing tools attempt to create a double-strand break at a precise
location in the genome using a protein called a nuclease (Figure 13.9), an
enzyme that cleaves nucleic acids. The nuclease is directed to a specific target
in the genome by interacting with a guide strand of DNA or RNA. Nucleaseinduced
double-strand breaks can be repaired by two different processes that
each take advantage of endogenous cellular machinery. In nonhomologous
Genomic DNA
Nuclease-mediated
double-strand break
Homology-directed
repair (HDR)
Nonhomologous end
joining (NHEJ)
Targeting
construct
Inserted or modified
DNA sequence
Insertion or deletion mutations
FIGURE 13.9  Conceptual overview of nuclease-mediated genome targeting. A nuclease
causes a double-strand break at a specific site of the genome. Following the break, a scientist can
employ either nonhomologous end joining (NHEJ) pathways to induce a deletion or insertion or
homology-directed repair (HDR) pathways to introduce foreign DNA.
Manipulating Endogenous Genes Chapter | 13  285
end-joining (NHEJ), naturally occurring DNA break repair processes rejoin
the broken ends, often including an insertion or deletion mutation. These mutations
can lead to a frameshift mutation of a gene, introducing a premature stop
codon and effectively silencing expression. Alternatively, in a process called
homology-directed repair (HDR), a scientist introduces a DNA construct with
homology arms that have the same sequence as the broken DNA strands. Then
the naturally occurring process of homologous recombination swaps endogenous
DNA for the engineered DNA, similar to the process described above to
create a knockout or knockin mouse.
Many nuclease systems have been developed to target specific locations in
the genome:
	l	Meganucleases. Meganucleases are restriction enzymes (Chapter 10) that
recognize longer DNA sequences (typically 20–40 bp) than typical restriction
enzymes (typically 6–8 bp). Artificially engineered versions of naturally
occurring meganucleases have been developed as a tool for genome engineering.
Because specific 20–22 bp sequences typically occur only once in
the genome, a well-engineered meganuclease should target specific genomic
loci. However, despite several important advances, the engineering of meganucleases
specific to desired DNA sequences has proven challenging for most
research laboratories.
	l	Zinc finger nucleases (ZFNs). ZFNs are artificially engineered restriction
enzymes consisting of a zinc finger DNA-binding domain fused to a DNAcleavage
domain. Zing finger domains can be engineered to target specific
DNA sequences, therefore allowing for site-specific cleavage of the genome.
Unfortunately, ZFNs have proven difficult to use for many research laboratories
due to off-target effects in which ZFNs cut DNA at undesired sites, as well
as challenges with engineering the proteins for new gene targets.
	l	Transcription activator-like effector nucleases (TALENs). Like ZFNs,
TALENs are artificially engineered restriction enzymes composed of a DNAbinding
domain and a DNA-cleavage domain. The DNA-binding domain of
TALENs (called a transcription activator-like effector, or TALE) is derived
from Xanthomonas bacteria. TALEs can be engineered to target specific
DNA sequences, but often the DNA that encodes TALEs can be quite long.
Although simpler to design than ZFNs, it is still difficult to create TALEN
constructs using standard recombinant DNA technology techniques (Chapter
10) or to deliver TALEN-coding sequences to cells using common gene delivery
strategies (Chapter 11), such as viruses.
Taken together, meganucleases, ZFNs, and TALENs have proven useful for
many research groups and are still actively being optimized and improved. However,
each approach continues to manifest its own set of disadvantages that has
prevented widespread use for gene targeting among many research laboratories.
Much recent attention has focused on a nuclease system commonly referred
to as CRISPR/Cas9. CRISPR stands for “clustered regularly interspaced short
286  Guide to Research Techniques in Neuroscience
palindromic repeat” and refers to clusters of short, repeated DNA sequences normally
used by some strains of bacteria as a method of protection against foreign
nucleic acids. A “Cas” is a CRISPR associated protein, and Cas9 is a nuclease
originally derived from the bacterium Streptococcus pyogenes. In this species,
the Cas9 nuclease uses templates provided by the CRISPR sequence to cleave
foreign DNA. In the lab, the CRISPR/Cas9 system has been adapted for inducing
site-specific double strand breaks in the genomes of many model organisms.
The most common procedure utilizing CRISPR/Cas9 technology introduces
two DNA sequences into cells (Figure 13.10). One DNA sequence encodes
the Cas9 protein. The other DNA sequence codes for a guide RNA (gRNA)
that will serve as a template for genome targeting. Twenty nucleotides at the 5′
end of the gRNA direct Cas9 to a specific genomic site using standard DNA–
RNA complementary base pairing. Importantly, in the genome to be targeted,
the 20 nucleotides must lie immediately 5′ of a sequence known as a protospacer
adjacent motif (PAM), a three-nucleotide sequence of NGG (N can
be any of the four DNA bases followed by two guanine bases). Therefore,
Genomic DNA
Cas9-mediated
double-strand break
Guide RNA
(gRNA)
Cas9+
FIGURE 13.10  The CRISPR/Cas9 nuclease system.  A scientist delivers DNA to a cell encoding
the Cas9 protein and a guide RNA (gRNA). The gRNA contains a 20 bp sequence that allows
Cas9 to recognize a specific region of the genome and cause a double-strand break.
Manipulating Endogenous Genes Chapter | 13  287
a subtle limitation to using CRISPR/Cas9 gene targeting is that the break in the
genome must occur next to this PAM sequence; however, as the three-nucleotide
NGG sequence is incredibly common, this is almost never a problem. Thus,
a scientist can design and deliver a gRNA to induce Cas9-mediated genome
breaks for any genomic sequence of 20 base pairs plus the PAM.
Once the Cas9 induces a double-strand break, a scientist can use NHEJ or
HDR to cause a mutation in the genomic location or introduce foreign DNA.
This process can take place in a fertilized egg, allowing for heritable transmission
to future offspring. Because transgenic technologies are well established
for many species (Chapter 12), the CRISPR/Cas9 allows for gene targeting in
virtually any traditional and nontraditional model organism.
The CRISPR/Cas9 system has grown quickly in popularity as the nucleasebased
gene editing tool of choice and is even being explored as a potential way
to treat human genetic diseases. One reason this system has overtaken other
nuclease-based systems is its affordability and ease of use. To target specific
regions of the genome using ZFNs or TALENs, the DNA-binding domain of the
nuclease itself must be engineered, and a functional protein created. To target
specific regions of the genome using CRISPR/Cas9, a scientist only needs to
create a DNA sequence coding for a gRNA.
Now that we have described gene targeting and methods of manipulating
function at the genomic level, we survey methods of disrupting a gene by interfering
with its products—either the mRNA transcript or the translated protein.
DISRUPTING GENE PRODUCTS
In addition to disrupting gene expression by directly targeting the genome
itself, scientists can also perturb endogenous gene function by interfering with
a gene’s mRNA transcript or its translated protein product (Figure 13.1). These
techniques effectively inhibit gene function and are tractable in a wide variety
of vertebrate and nonvertebrate species.
RNA Interference (RNAi)
RNA interference (RNAi) is the process of silencing gene expression through
endogenous cellular mechanisms that degrade mRNA. Cells naturally use
RNAi to regulate normal gene expression, as well as to degrade foreign mRNA
transcripts from viruses. Scientists now use this natural process to selectively
knock down gene expression and degrade mRNA for a gene of interest. Though
knockdown efficiencies do not reach 100%, in good knockdown experiments,
only a minimal amount of mRNA remains.
The process of RNAi begins when a piece of double-stranded RNA (dsRNA)
comes in contact with an enzyme called Dicer (Figure 13.11). Dicer cleaves
dsRNA into small duplex fragments, about 23 base pairs long, called small interfering
RNA (siRNA). The siRNA-Dicer complex recruits additional cellular
288  Guide to Research Techniques in Neuroscience
proteins to form an RNA-induced silencing complex (RISC). RISC interacts
with and degrades RNA strands that have a complementary nucleotide sequence
to one of the siRNA strands, referred to as the guide strand. Thus, the RNAi process
degrades mRNA before it is translated by a ribosome into a protein product.
In theory, there are three ways to make use of the RNAi pathway to disrupt
a gene: (1) deliver a dsRNA molecule that is homologous to the mRNA transcript
to be silenced; (2) deliver synthetic siRNAs (19–23 bp) that will attract
the RISC complex to bind to the homologous mRNA transcript; and (3) deliver
short hairpin RNA (shRNA), a strand of RNA that contains complementary
dsRNA comes into
contact with Dicer
Dicer cleaves dsRNA
into siRNA
Dicer/siRNA attract
other proteins to form
RISC complex
RISC targets mRNA
with a complementary
sequence to siRNA
RISC degrades
target mRNA
FIGURE 13.11  Conceptual overview of RNAi technology. When double-stranded RNA
(dsRNA) comes into contact with the Dicer enzyme, Dicer cleaves the RNA into small fragments,
creating small interfering RNA (siRNA) molecules. The siRNA-Dicer complex recruits other proteins
to form a RISC complex. RISC targets mRNA molecules with a complementary sequence to
one of the two siRNA strands, degrading the mRNA and knocking down gene expression.
Manipulating Endogenous Genes Chapter | 13  289
sequences at either end that cause the molecule to fold onto itself, producing a
hairpin shape (Figure 13.12). Endogenous cellular RNAi machinery can then
process shRNA into small dsRNA.
The choice of which RNAi molecule to use is partially determined by the
model organism, as different animals respond with varied efficacy to these distinct
methods. Though dsRNA works well in Drosophila and C. elegans, delivering
dsRNA into mammalian cells generally causes a nonspecific interferon
stress response, making experiments difficult to interpret. siRNA can knock
down a gene of interest, but this is a transient effect, which may be suboptimal
for certain experiments. For long-term, stable gene knockdown, scientists deliver
shRNA into cells in a way that allows the shRNA to be stably integrated into
the genome. This integration can be achieved by making a transgenic organism
in which the shRNA sequence is placed under the control of a strong promoter.
There are two critical issues to consider in the design of a good RNAi experiment:
the effectiveness of the RNA probe and the use of appropriate controls. An
effective probe has the ability to knock down the expression of a specific mRNA
strand without off-target effects. Usually, a scientist designs multiple RNAi
probes against the gene of interest and compares their knockdown efficiency in
cell culture before choosing one for an actual experiment. The scientist designs
these probes to have sequences unique to the gene of interest and no other genes
(this can be done using the BLAST program described in Chapter 9). Other, additional
control probes are also necessary for a good RNAi experiment. Typical
controls include a probe with a scrambled sequence, as well as a probe against a
separate, nonessential gene to show the specificity of the RNAi effect. The most
conclusive experiments are not only repeated multiple times but demonstrate that
multiple probes against different regions of the same gene result in the same
knockdown phenotype. The gold standard of RNAi controls is a phenotypic rescue
experiment, in which a version of the knocked down gene that is not targeted
by the RNAi probe is delivered together with the RNAi probe to show that it
can restore a normal phenotype. The rescuing gene is often an orthologue from
another species with the same function but a slightly different genetic sequence.
Morpholinos
An alternative to RNAi is the use of morpholinos to block proper mRNA translation.
Morpholinos are stable, synthetic 22–25 bp antisense oligonucleotide
FIGURE 13.12  Short hairpin RNA
(shRNA). An shRNA molecule is composed
of two complementary sequences
separated by a small spacer. The molecule
naturally folds into itself, resembling a
hairpin shape.
Linear mRNA strand
shRNA
290  Guide to Research Techniques in Neuroscience
analogues designed to complement an RNA sequence. A morpholino and its
complementary mRNA will hybridize to each other, preventing a ribosome
from accessing the mRNA and translating it into a protein (Figure 13.13).
Unlike RNAi, morpholinos do not degrade their target mRNA molecules. This
is especially useful in organisms such as Xenopus frogs that do not seem to
respond to an RNAi approach. Traditionally, morpholinos have been used in
zebrafish, Xenopus, and chicks by injecting them into eggs and producing morphant
embryos. Controls used in morpholino experiments are similar to those
used for RNAi experiments.
Dominant Negatives
Dominant negative constructs encode protein variants that interfere with the
normal function of the wild-type protein. Most dominant negatives inhibit normal
protein function by competing for a substrate or binding partner without
performing the protein’s regular function.
There are a variety of methods used to design dominant negative molecules.
One common method is to engineer a mutation that incapacitates one functional
domain of a protein while leaving other regions intact to interact as normal.
Therefore, dominant negative molecules will compete with the functional
wild-type protein for endogenous binding partners. This approach is often
used for transcription factors; the DNA-binding domain is left intact, but the
(A)
mRNA
Ribosome
(B)
Morpholino
FIGURE 13.13  Morpholinos.  (A) During translation, a ribosome binds to an mRNA molecule
and translates specific nucleotide sequences into chains of amino acids. (B) A morpholino functionally
blocks the mRNA, preventing contact with a ribosome.
Manipulating Endogenous Genes Chapter | 13  291
transcriptional regulatory domain is removed. Similarly, dominant-negative
receptors may have intact extracellular ligand binding domains but may be
missing the cytoplasmic intracellular signaling domain; they compete for the
ligand but will not pass on the signal. Another common example is creating a
dominant negative subunit of an oligomeric protein (Figure 13.14). The dominant
negative subunit can bind the other subunits but prevents the proper function
of the protein.
CONCLUSION
This chapter has surveyed common methods of gene targeting and disrupting
the function of specific genes in the genome. Although manipulating genes was
once a set of classical techniques only possible in mice, newer technologies
like the CRISPR/Cas system and RNAi allow for gene targeting and loss-offunction
genetic experiments in a wide variety of model organisms. The development
of newer, efficient gene editing tools is an exciting area of research that
is sure to allow for even more experimental possibilities in the future.
SUGGESTED READING AND REFERENCES
Books
Appasani, K. (Ed.). (2011). RNA interference technology: From basic science to drug development.
Cambridge: Cambridge University Press.
Behringer, R., Gertsenstein, M., Nagy, K., & Nagy, A. (2013). Manipulating the mouse embryo:
A laboratory manual (4th ed.). New York: Cold Spring Harbor Laboratory Press.
Schepers, U. (2005). RNA interference in practice: Principles, basics, and methods for gene silencing
in C. elegans, Drosophila, and Mammals. Wenheim, Germany: Wiley-VCH.
FIGURE 13.14  Conceptual example of a dominant
negative disruption of protein function. (A) In this
example, a functional protein is composed of four identical
subunits. (B) A transgenic construct produces a
slightly mutated subunit that retains the ability to form
an oligomeric protein but blocks the protein’s function.
(A)
Protein with four functional subunits
(B)
Expression of one dominant negative
subunit causes dysfunction of entire
oligomeric protein
292  Guide to Research Techniques in Neuroscience
Review Articles
Herskowitz, I. (1987). Functional inactivation of genes by dominant negative mutations. Nature,
329, 219–222.
Hsu, P. D., Lander, E. D., & Zhang, F. (2014). Development and applications of CRISPR-Cas9 for
genome engineering. Cell, 157, 1262–1278.
Lehner, B., Fraser, A. G., & Sanderson, C. M. (2004). Technique review: how to use RNA interference.
Briefings in Functional Genomics and Proteomic, 3, 68–83.
Miller, V. M., Paulson, H. L., & Gonzalez-Alegre, P. (2005). RNA interference in neuroscience:
progress and challenges. Cellular and Molecular Neurobiology, 25, 1195–1207.
Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating and targeting
genomes. Nature Biotechnology, 32, 347–355.
Sandy, P., Ventura, A., & Jacks, T. (2005). Mammalian RNAi: a practical guide. Biotechniques, 39,
215–224.
Primary Research Articles—Interesting Examples from the Literature
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., et al. (2013). Multiplex genome
engineering using CRISPR/Cas systems. Science, 339, 819–823.
Doetschman, T., Gregg, R. G., Maeda, N., Hooper, M. L., Melton, D. W., Thompson, S., et al.
(1987). Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature,
330, 576–578.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent
and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,
391, 806–811.
Jessberger, S., Aigner, S., Clemenson, G. D., Toni, N., Lie, D. C., Karalay, O., et al. (2008). Cdk5
regulates accurate maturation of newborn granule cells in the adult hippocampus. PLoS Biol,
6, e272.
Ma, Y., Shen, B., Zhang, X., Lu, Y., Chen, W., Ma, J., et al. (2014). Heritable multiplex genetic
engineering in rats using CRISPR/Cas9. PLoS One, 9, e89413.
Mezghrani, A., Monteil, A., Watschinger, K., Sinnegger-Brauns, M. J., Barrëre, C., Bourinet, E.,
et al. (2008). A destructive interaction mechanism accounts for dominant-negative effects of
misfolded mutants of voltage-gated calcium channels. Journal of Neuroscience, 28, 4501–4511.
Ralph, G. S., Radcliffe, P. A., Day, D. M., Carthy, J. M., Leroux, M. A., Lee, D. C., et al. (2005).
Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival
in an ALS model. Nature Medicine, 11, 429–433.
Thomas, K. R., & Capecchi, M. R. (1987). Site-directed mutagenesis by gene targeting in mouse
embryo-derived stem cells. Cell, 51, 503–512.
Protocols
Birmingham, A., Anderson, E., Sullivan, K., Reynolds, A., Boese, Q., Leake, D., et al. (2007).
A protocol for designing siRNAs with high functionality and specificity. Nature Protocols, 2,
2068–2078.
Dann, C. T., & Garbers, D. L. (2008). Production of knockdown rats by lentiviral transduction of
embryos with short hairpin RNA transgenes. Methods in Molecular Biology, 450, 193–209.
Kappel, S., Matthess,Y., Kaufmann, M., & Strebhardt, K. (2007). Silencing of mammalian genes by
tetracycline-inducible shRNA expression. Nature Protocols, 2, 3257–3269.
Morozov,A. (2008). Conditional gene expression and targeting in neuroscience research. Current Protocols
in Neuroscience (Chapter 4), Unit 4.31. http://dx.doi.org/10.1002/0471142301.ns0431s44.
Manipulating Endogenous Genes Chapter | 13  293
Mortensen, R. (2007). Overview of gene targeting by homologous recombination. Current Protocols
in Neuroscience (Chapter 4), Unit 4.29. http://dx.doi.org/10.1002/0471142301.ns0429s40.
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013). Genome engineering
using the CRISPR-Cas9 system. Nature Protocols, 8, 2281–2308.
Yang, H., Wang, H., & Jaenisch, R. (2014). Generating genetically modified mice using CRISPR/
Cas-mediated genome engineering. Nature Protocols, 9, 1956–1968.
295
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00014-9
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 14
Cell Culture Techniques
After Reading This Chapter, You Should be Able to:
	l	Compare the advantages and disadvantages of using in vitro culture techniques
with in vivo techniques
	l	Describe types of cells used to examine nervous system function in vitro
	l	Describe techniques for manipulating cells in cell culture conditions
Techniques covered:
	l	Tools and reagents used in culture: equipment, media
	l	Types of cultured cells: immortalized cell lines, primary cell and tissue culture,
stem cells
	l	Manipulating cells in culture: transfection, co-culture, pharmacology, antibody
interference
Cell culture is the process by which a scientist grows and maintains cells under
carefully controlled conditions outside of a living animal. There are many reasons
why this approach is desirable. Examining the nervous system in vitro
(within glass) allows scientists to simplify the cellular environment, providing
greater control over experimental manipulations and reducing potentially
confounding interactions with other biological systems. In vitro tools and techniques
make experiments possible that would otherwise be difficult or impossible
to perform (or interpret) in intact organisms, such as performing multiple
assays in parallel with the exact same number of cells. In vitro experiments also
tend to be faster, less expensive, and require fewer animals than experiments
performed in vivo.
However, cell culture experiments also raise interesting questions: Does a
cell in a culture flask truly behave like it would inside the brain or body? What
does it mean to culture neurons, cells notorious for their intercellular communication
and neural networks, in relatively isolated conditions in which they do
not form synaptic connections? Are immortalized cell lines, cells that continue
to divide indefinitely, actually good models for cells that degenerate and perish
in the brain? Because the goal of most in vitro experiments is to generate a
hypothesis and conclusion about what occurs in vivo, it is important to design
experiments carefully and extrapolate results cautiously.
This chapter surveys approaches to studying the nervous system using
cell culture techniques. We start with a quick summary of the equipment and
296  Guide to Research Techniques in Neuroscience
reagents necessary to maintain cells outside their endogenous environments.
Then we describe three categories of cells used in culture experiments: immortalized
cell lines, primary cell cultures, and a special class of cell that has received
much attention over the past several years—stem cells. Finally, we describe
some of the methods scientists can use to perturb cells in cultured environments.
CELL CULTURE EQUIPMENT AND REAGENTS
Specialized equipment and reagents are necessary to provide cultured cells with
an environment that can support their continued growth and health outside a living
organism. Most of these supplies are used to artificially mimic the endogenous,
in vivo, cellular environment. Taking a cell out of the brain and expecting it to survive
in a cell culture dish without providing basic, life-sustaining factors would be
like catching a fish and expecting it to stay alive in a cage: unless a scientist correctly
provides the elements necessary to sustain life, the cell will quickly die. Thus, most
equipment and reagents in cell culture labs are used to continually supply cells with
oxygen, nutrients, growth factors, and other elements necessary to keep cells alive.
Other tools and reagents prevent contamination. Cells in vivo have the
benefit of an active immune system to prevent contamination from bacteria,
fungi, and other microorganisms. Cells in the brain have the added benefit of
the blood–brain barrier to further prevent contamination. In culture conditions,
cells are incredibly vulnerable, and scientists must take great care in avoiding
contamination, especially because microorganisms are ubiquitous in the environment
and can easily penetrate cell culture plates.
Equipment
Although individual laboratories study different cell lines and ask different
scientific questions, cell culture rooms tend to contain the same fundamental
pieces of equipment. This equipment includes the following:
	l	Biosafety hood. A biosafety hood or laminar flow cabinet is used to prevent
contamination by microorganisms (Figure 14.1).When not in use, these chambers
are often illuminated with UV light that helps sterilize exposed surfaces.
Just before use and throughout experiments, scientists spray all surfaces with
70% ethanol to provide further decontamination. During experiments, scientists
always keep reagents and cell culture flasks/plates covered until they are
ready for use; when exposed, a scientist should never pass their gloved hands
or other equipment over open bottles or, even worse, the cells themselves.
	l	Cell incubator. Cell incubators house and store culture flasks and plates. These
incubators maintain an appropriate temperature, humidity, and gas concentration
to mimic endogenous conditions. They are usually set at 37 °C with 5%
CO2 levels.Although 5% CO2 is not the level experienced in the body in vivo,
the carbon dioxide maintains the pH of buffers in the growth media at proper
physiological levels.
Cell Culture Techniques Chapter | 14  297
	l	Specially prepared cell culture flasks/plates. Tissue culture flasks and plates
come in many varieties depending on the needs of the investigator. Flasks
typically range in sizes of 12.5 cm2 to large, multitiered chambers with surface
areas of 1800 cm2. Multiwelled plates are often used for tissue culture
experiments, as these wells can hold an exact number of cells, and it is easy
to keep track of various experimental conditions across wells. Plates are
typically sold with 6, 12, 24, 48, 96, or 384 wells. Many cells, including
neurons, need to adhere to a substrate to grow, so tissue culture dishes often
need to be specially prepared to provide a substrate for the cells. Tissue culture
dishes or glass coverslips are often coated with attractive amino acids,
such as lysine or ornithine or extracellular matrix components like collagen
and laminin.
	l	Refrigerator. A refrigerator (properly referred to as a 4 °C incubator) maintains
cell culture media and other reagents when not in use. The growth factors
and antibiotics in culture media degrade over time, but can last for weeks if
stored at 4 °C.
	l	Water bath. A water bath is often set at 37 °C and is used to quickly warm cell
culture media and other reagents stored at 4 °C just prior to being added to cell
culture flasks/plates. If a scientist does not warm media before adding it to
cells, the cells can be shocked by the abrupt change to cold temperatures and
die.
	l	Microscope. Microscopes are used in most tissue culture rooms for routine
observation of cell culture flasks/plates to inspect the health and confluence of
cells. Confluence refers to the percentage of the surface of the bottom of the
plate covered by cells. Most cell lines should never become 100% confluent,
as cells in dense populations tend to inhibit each other’s growth, as well as
quickly drain nutrients from the culture media.
Biosafety
hood
CO2
Incubator
FIGURE 14.1  Typical cell culture room with biosafety hood and CO2 incubator.
298  Guide to Research Techniques in Neuroscience
Culture Media
Growth media is critical to cell culture experiments, supplying nutrients (amino
acids and vitamins) and a source of energy (glucose) for cells. Growth media
can vary in pH, nutrient concentration, and the presence of growth factors or
other biologically relevant components. To survive, cells must be bathed in an
isotonic fluid that has the same concentration of solute molecules as inside the
cell. The media is buffered to maintain a compatible pH (usually 7.4, though
there are some cell-specific variations). In the 5% CO2 environment of culture
incubators, a bicarbonate buffer maintains the physiological pH as well
as providing nutritional benefits to the cells. HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic
acid) is added to the culture media for extra buffering
capacity when the cells in a culture experiment require extended periods of
manipulation outside a CO2 incubator.
Serum is often added to culture media for its ability to promote survival
through undefined mixtures of growth factors, hormones, and proteins, like
platelet-derived growth factor, insulin, and transferrin. For stricter control over
the cellular environment, investigators use serum-free, chemically defined
supplements, such as N2 or B27, that contain known formulations of survival
factors. A typical growth medium in neuronal cultures is NeuroBasal, which
provides optimized amino acids and nutrients to cultured neurons, supplemented
with N2 or B27.
Adding or removing specific ingredients to or from the media can influence
cellular behavior. For example, to maintain a neural progenitor pool that continues
to divide in culture, scientists add growth factors such as epidermal growth
factor (EGF) and fibroblast growth factor (FGF) to prevent differentiation. Specific
recipes for culture media are tailored to provide optimal conditions for
promoting the health and proper physiological function of the cultured cells and
for maintaining cellular behavior.
Now that we have described some of the equipment and reagents necessary
for cell culture, we survey the different categories of cells commonly used in
cell culture experiments.
IMMORTALIZED CELL LINES
Immortalized cell lines are either tumorous cells that do not stop dividing or
cells that have been artificially manipulated to proliferate indefinitely and can,
thus, be cultured over several generations (Table 14.1). Because immortalized
cells continuously divide, they eventually fill up the dish or flask in which they
grow. By passaging (also known as splitting), scientists transfer a fraction of the
multiplying cells into new dishes to provide space for continuing proliferation.
There are many advantages to using immortalized cell lines. Because these
are standard lines used by many different labs, immortalized cells are fairly
well characterized. They are, at least theoretically, homogeneous, genetically
Cell Culture Techniques Chapter | 14  299
TABLE 14.1  Commonly Used Immortalized Cell Lines
Origin and cell type Comments
3T3 Mouse embryonic
fibroblast
Robust and easy to handle;
contact inhibited; stops
growing at very high
densities
HeLa Human epithelial cell From cervical cancer in
a human patient named
Henrietta Lacks; may
contaminate other cultured
cell lines; able to grow
in suspension (i.e., grow
without adhering to bottom
of plates)
COS (Cv-1 in Origin,
carrying Sv40 genetic
material)
Monkey kidney Efficiently transfected;
commonly used as an
expression system for
high-level, short-term
expression of proteins
293/293T/HEK-293T Human embryonic kidney Easy to transfect and
manipulate; commonly
used as an expression
system to study signaling
and recombinant proteins
MDCK (Madin-Darby
canine kidney)
Dog kidney epithelial cell Polarized with distinct
apical and basal sides, used
in studying trafficking
CHO Chinese hamster ovary Useful for stable gene
expression and high protein
yields for biochemical
assays; commonly used as
an expression system for
studying cell signaling and
recombinant proteins
S2 Drosophila
macrophage-like cells
Well-characterized
Drosophila cell line;
highly susceptible to RNAi
treatment
PC12 Rat pheochromocytoma
chromaffin cell
Neuron-like, derived from
a neuroendocrine adrenal
tumor; can differentiate into
a neuron-like cell in the
presence of nerve growth
factor
Continued
300  Guide to Research Techniques in Neuroscience
identical populations, which aid in providing consistent and reproducible
results. Immortalized cells tend to be easier to culture than cells used in primary
cultures in that they grow more robustly and do not require extraction from a living
animal. Also, because they grow quickly and continuously, it is possible to
extract large amounts of proteins for biochemical assays (Chapter 15). It is also
possible to create cell lines that continuously express a gene of interest, such as
a fluorescently tagged or mutant version of a protein.
The major disadvantage to using immortalized cells is that these cells cannot
be considered normal, in that they divide indefinitely and sometimes express
unique gene patterns not found in any cell type in vivo. Therefore, they might
not have the relevant attributes or functions of normal cells. Also, after several
passages, cell characteristics can change and become even more different from
those of a normal cell. Thus, it is important to periodically validate the characteristics
of cultured cells and not use cells that have been passaged too many
times.
Immortalized cell lines of neuronal origin can be used to study properties
unique to neurons. Scientists have used neuronal cell lines to investigate processes
that occur during differentiation in neurons, such as axon selection,
guidance, and growth. However, most neuronal immortalized cell culture
models are derived from tumors and are sometimes genomically abnormal.
One popular neuronal cell line, called PC12, is a rat pheochromocytoma
cell line derived from an adrenal gland tumor. The addition of nerve growth
factor (NGF) causes PC12 cells to reversibly differentiate into a neuronal
phenotype (Figure 14.2). These cells can synthesize dopamine, norepinephrine,
and acetylcholine. PC12 cells have been used to study molecular phenomena
associated with neuronal differentiation and have even been used
Origin and cell type Comments
Neuro-2a/N2a Mouse neuroblastoma Model system for
studying pathways
involved in neuronal
differentiation; can be
driven to differentiate by
cannabinoid and serotonin
receptor stimulation
SH-SY5Y Human neuroblastoma,
cloned from bone marrow
Dopamine beta hydroxylase
activity, acetylcholinergic,
glutamatergic,
adenosinergic; grow as
clusters of neuroblast-type
cells with short, fine neurites
TABLE 14.1  Commonly Used Immortalized Cell Lines—cont’d
Cell Culture Techniques Chapter | 14  301
in experiments to replace dopaminergic neurons in an animal model of
Parkinson’s disease. Neuroblastoma cell lines, like mouse Neuro2A, also
express neurotransmitters and have been used in electrophysiology and neurodevelopment
studies.
As useful as immortalized cell lines of neural origin can be for certain experiments,
they show obviously abnormal traits, such as the unusual combination
of neurotransmitters they produce (no normal neuron produces dopamine,
norepinephrine, and acetylcholine in the same cell!). Therefore, it is advantageous,
when possible, to use primary cultured cells—cells extracted from living
animals.
PRIMARY CELL AND TISSUE CULTURE
Primary cell and tissue culture is derived from tissue removed directly from a
living animal rather than immortalized cells that divide indefinitely (Figure 14.3).
Primary tissue culture allows scientists to directly investigate cells of interest in
a carefully controlled in vitro environment. These experiments can be especially
+NGF
FIGURE 14.2  Neuronal differentiation. Addition of growth factor to PC12 cells causes them to
differentiate into neuron-like cells that grow neurites and synthesize neurotransmitters.
Extract
brain
Section
brain
Dissect
brain
region
Culture
brain
slice
Enzymatically
dissociate
Slice cultureExplant cultureDissociated culture
FIGURE 14.3  Primary tissue cultures. After a brain is removed from an animal, it can be
cut into thin sections (usually using a vibratome) and kept alive in slice culture. Alternatively,
either a brain slice or a brain region can be finely dissected to produce an explant culture. An
explant culture can be enzymatically digested into single cells to produce a dissociated cell
culture.
302  Guide to Research Techniques in Neuroscience
advantageous for a comparison between cells derived from a wild-type animal
and cells derived from a genetically modified animal.
However, there are some disadvantages to using primary cultures
(Table 14.2). Unlike immortalized cell lines, primary cultured cells have a limited
lifetime. The age of the animal source influences the health and robustness
of the cell culture: tissue from younger, embryonic or early postnatal animals
survives better and tends to be healthier than tissue from older animals. Also, a
population of primary cells will always be more heterogeneous than a culture
of immortalized cells, no matter how careful the scientist was in extracting and
purifying the cells of interest.
There are three main categories of primary tissue culture: slice cultures,
explant cultures, and dissociated cultures.
Slice Cultures
Slice cultures maintain the structure and organization of the brain in a relatively
thick (250–400 μm) section of brain tissue cut using a vibratome (Chapter 6).
Slices provide greater access to and visibility of deep subcortical structures, like
thehippocampusandthalamus,whicharedifficulttoaccessin vivo(Figure14.3).
These slices can either be acute cultures that are used immediately (over a
period of hours) or organotypic slice cultures that are maintained over multiple
days. Acute slices are typically used for short-term electrophysiology
experiments, while organotypic cultures are used to observe structural and morphological
changes such as neuronal migration, axon outgrowth, or synapse
formation.
Slices require an air/liquid interface to properly regulate gas exchange
throughout the slice. Different protocols for culturing slices have varied success
in preserving the structure and development of the tissue to parallel that seen
in vivo.
TABLE 14.2  Immortalized Cell Lines versus Primary Cell Culture
Advantages Disadvantages
Immortalized cell lines 	l	Easier to use (grow,
transfect, etc.)
	l	Homogeneous
	l	Fairly well characterized
	l	Can create stable cell
lines expressing gene of
interest
	l	May not have the
same properties as
neurons or primary
cell type of interest
Primary cell culture 	l	Relevant cell type,
physiology, and circuitry
	l	Heterogeneous
populations with high
variability
Cell Culture Techniques Chapter | 14  303
Explant Cultures
Explant cultures consist of intact fragments of tissue (Figure 14.3). Although
they do not necessarily preserve the precise organization and orientation of the
endogenous nervous system, explants contain the same mixture of cell types.
Unlike slices, which require an air interface for proper oxygenation, explant
cultures can be submerged in the bath media. Explants are often used in coculture
assays and for studies on neurite outgrowth and neuronal migration.
Dissociated Cell Cultures
In dissociated cultures, neural tissue is separated into individual cells that
are then grown on two-dimensional coated glass coverslips or within threedimensional
substrates (Figure 14.3). After extracting an animal’s brain, specific
regions can be micro-dissected to isolate a specific neuronal subtype (e.g.,
cortical, hippocampal). This region is then mechanically or enzymatically
digested within a liquid suspension to separate individual cells from each other.
The neurons are then removed from the suspension and plated onto a substrate
on which the cells can attach and grow.
Neurons dissociated from different regions of the brain retain their initial
identities. The morphological, molecular, and physiological properties of cell
populations present in culture correspond closely to the characteristics of the
cell population present in the region of origin in a living organism. With the
proper growth factors and care, it is possible to maintain a dissociated culture
for weeks, during which time cells acquire properties of mature neurons: they
develop characteristic axons and dendrites, form synapses with one another,
and express receptors and ion channels specific to their cell types, even producing
spontaneous electrical activity. Dissociated neuronal cell cultures have been
used to study neurite outgrowth, synapse formation, and electrophysiological
parameters.
Theabilitytoprobeindividualneurons,however,comesattheexpenseoflosing
the organization and connectivity critical to in vivo functions. Another limitation
of dissociated cultures is the small quantity of cells relative to immortalized cell
lines,whichcanmakeitdifficulttoperformbiochemicalexperiments(Chapter15)
that require a high volume of starting material (e.g., millions of cells). Furthermore,
most primary cell cultures are not homogeneous. Neuronal cultures are
often mixtures of both glia and neurons that respond to different neurotransmitters,
so identifying an individual population of cells can be difficult. To minimize
heterogeneity, investigators usually attempt to dissect regions as precisely as possible
to maximize the presence of the desired cell type in culture.
Various methods have been developed to purify specific cell populations. In
a technique called immunopanning, a scientist coats the bottom of a plate with
antibodies that recognize cell-surface markers on the outside of specific cell
types. When heterogeneous populations of cells are added to the plate, the scientist
can purify the cells of interest by allowing the cells to bind to the bottom
304  Guide to Research Techniques in Neuroscience
of the plate and then wash off the undesired, unbound cells. This technique has
been used to culture oligodendrocyte precursors, retinal ganglion cells, and corticospinal
motor neurons.
STEM CELL CULTURES
A stem cell is a pluripotent cell with the capacity to generate any cell type
(e.g., neuron, muscle, blood) and an unlimited ability to renew itself. These
cells are similar to immortalized cell lines in that they are theoretically able to
propagate in culture continuously; however, they are also primary cells derived
directly from a living organism.
Stem cells are classified by their source (e.g., embryonic stem (ES) cells,
adult stem cells, induced pluripotent stem (iPS) cells), as well as the tissue they
typically generate (e.g., neural stem cells (NSCs), hematopoietic stem cells, skin
stem cells). Stem cells defined by the tissue they generate are multipotent—
able to give rise to all types of cells found in the tissue and able to continuously
self-renew. For example, NSCs can give rise to all three neural lineages—neurons,
astrocytes, and oligodendrocytes—as well as additional NSCs. Stem cells
are distinct from progenitor cells, which have a more limited capacity for selfrenewal
and may be unipotent, giving rise to a single cell type.
Stem cell culture is a form of dissociated primary culture. Therefore, stem
cells must be cultured in the presence of growth factors (EGF and basic fibroblast
growth factor (bFGF)) in specially formulated media that preserves their
multipotency and ability to self-renew. Because scientists do not yet know all
of the environmental components required to recreate in vivo conditions for
preserving stem cell function, in vitro studies may not completely capture the
effect of endogenous environmental influences.
Stem cell culture has a variety of uses in neuroscience. ES cells are examined
for their ability to differentiate into specific neuronal subtypes. NSCs can
be cultured to study the basic biology of development and aging. iPS cells can
be used to generate clones of cells from patients with neurological disorders
to characterize cellular and molecular changes in diseased neurons. While an
extensive history and theory of stem cells already exists, basic stem cell biology
is still a rapidly evolving field. Here we focus on specific applications of stem
cell culture in neuroscience and describe common in vitro techniques used to
identify stem cells.
Embryonic Stem Cells
Pluripotent embryonic stem (ES) cells can give rise to all tissues in an organism.
Derived from the inner cell mass of a blastocyst embryo, culturing ES
cells in vitro essentially traps the cells in a pluripotent state by growing them in
the presence of factors that prevent the cells from differentiating. Investigators
can reconstruct the environment of ES cells in a dish so that culture conditions
Cell Culture Techniques Chapter | 14  305
contain the specific molecules and mitogens that specify the formation of a
neuron in the developing embryo.
Using special culture conditions, ES cells are first induced to become general
neural progenitors, precursors that are committed to a neural fate. Once
they have become neural progenitors, the specific molecules known to act during
normal neuronal development are added to the culture to direct differentiation
of specific neuron subtypes. For example, to make motor neurons, cells
are exposed to high concentrations of sonic hedgehog (SHH) and retinoic acid,
morphogens that pattern spinal cord motor neurons during development. To
make midbrain dopaminergic neurons, cells are exposed to SHH and fibroblast
growth factor 8 (FGF8), morphogens that specify dopaminergic fate during
development.
Investigators monitor the progression of differentiation by examining the
culture for activation of transcription factors relevant to development and the
appearance of markers known to promote differentiation of specific neuronal
subtypes. For example, when trying to induce midbrain dopaminergic neurons,
scientists look for midbrain-specific transcription factors (Pitx3, En1, Lmx1b,
Nurr1) and later for markers of mature dopamine neurons (TH, DAT, Girk2).
This process—from the initial establishment of the ES cells to their differentiation
into specific neuronal subtypes—can take months. Also, despite attempts
to create pure populations of a specific neuronal subtype, differentiated ES cell
populations are still heterogeneous, with many other cells present in the culture.
However, the ability to differentiate ES cells into specific neuronal cell types is
useful for investigating therapies for a number of neurodegenerative disorders
such as Parkinson’s and Alzheimer’s disease.
Neural Stem Cells
Multipotent NSCs exhibit self-renewal properties and the ability to differentiate
into all neural subtypes. They can be extracted from regions of embryonic or
adult brains, where they normally divide and give rise to neurons or glia. The
embryonic brain contains many NSCs, while adult brains have far fewer NSCs.
Furthermore, the ability of adult NSCs to produce neurons decreases with age.
Investigators typically culture adult NSCs from the subgranular zone of the
dentate gyrus in the hippocampus and the subventricular zone in the wall of the
lateral ventricles. After dissection, the region containing NSCs is dissociated
and can be moderately purified through centrifugation. NSCs can also be grown
and passaged in special media that promote NSC survival. By removing growth
factors from the media, NSCs can be differentiated into neurons, astrocytes, and
oligodendrocytes.
It is difficult to distinguish neural progenitors from NSCs, and scientists
often confuse the terms. Both types of cells proliferate and express common
sets of molecular markers (e.g., nestin, Pax6). Therefore, functional assays must
also be used to determine the identity of cultured cells. Two defining features of
306  Guide to Research Techniques in Neuroscience
NSC cultures that can be tested to confirm cellular identity are (1) the ability to
give rise to all three types of the neural lineage (multipotency) and (2) the ability
to propagate more NSCs (self-renewal).
Self-renewal is often examined by performing primary neurosphere and
secondary neurosphere assays. When NSCs are cultured in nonadherent conditions
in the presence of the growth factors EGF and bFGF, they give rise
to neurospheres, balls of dividing cells (Figure 14.4). However, neurospheres
form as a general result of dividing neural precursors, including progenitor cells
that do not continuously self-renew. Therefore, while this assay is frequently
used, it does not definitively distinguish between stem and progenitor cells.
The secondary neurosphere assay involves culturing cells from established neurospheres
to see if the cells generated by the first neurosphere are able to continue
proliferating, an indicator of self-renewal. These assays can be difficult
to quantify, as cells and spheres can each fuse, so a sphere may not necessarily
form from a single NSC. Also, neurospheres are species-specific; rat and human
NSCs do not form neurospheres as often as mouse NSCs.
Induced Pluripotent Stem Cells
iPS cells are differentiated cells that scientists have reverted back to a stem cell
state. A variety of strategies have been employed to carry out this feat. One
common strategy is to use viruses to introduce specific transcription factors
into differentiated somatic cells, usually fibroblasts. The presence of these transcription
factors seems to allow these cells to exhibit stem cell behaviors. Other
strategies employ a combination of viral delivery and chemical manipulations
to coax somatic cells into a pluripotent state.
Once iPS cells are generated, they are examined for characteristics
of “stemness” by comparing them to ES cells in various ways. Scientists
FIGURE 14.4  Neurospheres. Dividing neural progenitor and stem cells form spheres in culture.
Courtesy of Dr Victoria Rafalski.
Cell Culture Techniques Chapter | 14  307
typically examine transcription factor profiles, measuring the presence
of markers such as Oct4, Nanog, Sox2, AP, SSEA4, and TRA-1-80 using
immunohistochemistry (Chapter 6) and reverse transcription polymerase
chain reaction techniques (Chapter 10). The potential for these iPS cells to
differentiate is measured by driving cells into specific lineages using established
protocols. The gold standard of determining whether iPS cells are like
stem cells is determining whether these cells retain the ability to generate
chimeric mice (Chapter 13).
In neuroscience, the ability to reprogram fully differentiated cells into an ESlike
state opens up the possibility of modeling human disease in vitro, directly
from patient-derived cells. These cells could reveal the cellular and molecular
pathogenesis of diseases such as Parkinson’s and autism spectrum disorders.
iPS cells may also be used for therapeutic purposes: for example, generating
new neural cell types for transplantation from a patient’s own somatic cells.
Work has also progressed in bypassing the iPS cell stage to directly reprogram
somatic cells into functional neurons.
MANIPULATING CELLS IN CULTURE
One of the major advantages of studying the nervous system in vitro is the
ability to control the cellular environment. In addition to other methods of neuromodulation
(Chapter 8), scientists can manipulate neurons in culture using
several methods.
Transfection and Infection
Transfection and infection are the processes of delivering DNA to cells using
nonviral and viral methods, respectively. One of the major benefits of cell culture
systems, especially dissociated cultures, is that they are very amenable to
both DNA delivery methods. Therefore, it is usually easy to introduce recombinant
DNA molecules into cultured neurons to change gene/protein expression
and alter cellular physiology. Transfection and infection procedures are
discussed in much greater detail in Chapter 11.
Co-Culture Systems
Co-culturing allows a variety of cell types to be cultured together to examine
the effect of one culture system on another (Figure 14.5). This procedure is useful
when examining the effect of one type of tissue on another, one region of the
brain on another, or how a particular secreted molecule leads to changes in neural
development or physiology. For example, co-cultures of different regions of spinal
cord explants initially revealed differing effects on the ability to attract or repel
neurite outgrowth. Biochemical purification from explants in co-culture experiments
led to the identification of specific molecules that could then be introduced
308  Guide to Research Techniques in Neuroscience
into immortalized cell lines to express and secrete these molecular guidance cues.
Transfected cell lines could then be co-cultured with spinal cord explant cultures
to examine the neurite outgrowth response of the spinal cord neurons to specific
guidance cues. Such approaches have deciphered a large variety of chemoattractants
and chemorepellants that have since been validated in vivo.
Pharmacology
Because of the unparalleled access to the medium in cell culture conditions,
it is relatively easy to add pharmacological agents to culture media to affect
proteins, ion channels, and receptors of interest. See Chapter 8 for a detailed
description of pharmacological techniques.
Antibody Interference
Antibodies are molecules that bind with high affinity to antigens, usually parts
of a protein. In nature, these antibodies function as part of the immune system.
In the laboratory, antibodies can be used in many different assays to study protein
expression and binding partners (Chapter 15). If the antigen of an antibody
is accessible in living cells, it is also sometimes possible to block protein function
by applying antibodies against the protein of interest. These antibodies bind
the protein at a site critical for its normal function, thus inhibiting function
in vitro or in vivo.
CONCLUSION
The ability to grow cells and tissues outside of a living organism allows
scientists much greater access and control over the cellular environment
compared with in vivo conditions. Therefore, scientists can investigate the
cellular and molecular mechanisms of nervous system function with great
detail. The tools and techniques of in vitro culture techniques can provide
many informative results, but ultimately this knowledge must be tested
in the intact nervous system to fully understand how a biological process
works in a living organism.
FIGURE 14.5  The use of co-cultures to
study guidance cues. In this example, midbrain
explants are placed in culture medium. COS cells
(top) transfected with a control plasmid (A) do
not influence the direction of axon outgrowth
from the explants. However, COS cells transfected
with netrin-1 (B) sufficiently attract axons.
Courtesy of Dr Jie Li and Dr Mary Hynes.
Control Netrin
Cell Culture Techniques Chapter | 14  309
SUGGESTED READING AND REFERENCES
Books
Barres, B. A., & Stevens, B. (Eds.). (2013). Purifying and culturing neural cells: A laboratory
manual. New York: Cold Spring Harbor Laboratory Press.
Butler, M. (2004). Animal cell culture and technology (2nd ed.). London: BIOS Scientific Publishers.
Freshney, R. I. (2005). Culture of animal cells: A manual of basic techniques (5th ed.). Hoboken, NJ:
Wiley-Liss.
Martin, B. M. (1994). Tissue culture techniques: An introduction. Boston: Birkhäuser.
Skloot, R. (2011). The immortal life of henrietta lacks. New York: Broadway Books.
Review Articles
Blau, H. M., Brazelton, T. R., & Weimann, J. M. (2001). The evolving concept of a stem cell: entity
or function? Cell, 105, 829–841.
Jaenisch, R., & Young, R. (2008). Stem cells, the molecular circuitry of pluripotency and nuclear
reprogramming. Cell, 132, 567–582.
Seaberg, R. M., & van der Kooy, D. (2003). Stem and progenitor cells: the premature desertion of
rigorous definitions. Trends in Neurosciences, 26, 125–131.
Primary Research Articles—Interesting Examples from the Literature
Barberi, T., Klivenyi, P., Calingasan, N.Y., Lee, H., Kawamata, H., Loonam, K., et al. (2003). Neural
subtype specification of fertilization and nuclear transfer embryonic stem cells and application
in parkinsonian mice. Nature Biotechnology, 21, 1200–1207.
Dotti, C. G., & Banker, G. A. (1987). Experimentally induced alteration in the polarity of developing
neurons. Nature, 330, 254–256.
Dugas, J. C., Mandemakers, W., Rogers, M., Ibrahim, A., Daneman, R., & Barres, B. A. (2008).
A novel purification method for CNS projection neurons leads to the identification of brain
vascular cells as a source of trophic support for corticospinal motor neurons. Journal of Neuroscience,
28, 8294–8305.
Foo, L. C., Allen, N. J., Bushong, E. A., Ventura, P. B., Chung, W. S., Zhou, L., et al. (2011). Development
of a method for the purification and culture of rodent astrocytes. Neuron, 71, 799–811.
Pan, P. Y., Tian, J. H., & Sheng, Z. H. (2009). Snapin facilitates the synchronization of synaptic
vesicle fusion. Neuron, 61, 412–424.
Rafalski, V. A., Ho, P. P., Brett, J. O., Ucar, D., Dugas, J. C., Pollina, E. A., et al. (2013). Expansion
of oligodendrocyte progenitor cells following SIRT1 inactivation in the adult brain. Nature Cell
Biology, 15, 614–624.
Raineteau, O., Rietschin, L., Gradwohl, G., Guillemot, F., & Gahwiler, B. H. (2004). Neurogenesis
in hippocampal slice cultures. Molecular and Cell Neuroscience, 26, 241–250.
Soldner, F., Hockemeyer, D., Beard, C., Gao, Q., Bell, G. W., Cook, E. G., et al. (2009). Parkinson’s
disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell,
136, 964–977.
Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell, 126, 663–676.
Tessier-Lavigne, M., Placzek, M., Lumsden, A. G., Dodd, J., & Jessell, T. M. (1988). Chemotropic
guidance of developing axons in the mammalian central nervous system. Nature, 336, 775–778.
Vierbuchen, T., Ostermeier, A., Pang, Z. P., Kokubu,Y., Sudhof, T. C., & Wernig, M. (2010). Direct
converstion of fibroblasts to functional neurons by defined factors. Nature, 463, 1035–1041.
Watanabe, K., Kamiya, D., Nishiyama, A., Katayama, T., Nozaki, S., Kawasaki, H., et al. (2005).
Directed differentiation of telencephalic precursors from embryonic stem cells. Nature Neuroscience,
8, 288–296.
310  Guide to Research Techniques in Neuroscience
Protocols
Current protocols in neuroscience. Chapter 3: Cellular and developmental neuroscience. (2006).
Hoboken, NJ: John Wiley & Sons, Inc.
Loring, J. F., Wesselschmidt, R. L., & Schwartz, P. H. (Eds.). (2007). Human stem cell manual:
A laboratory guide. Amsterdam: Academic Press.
Poindron, P., Piguet, P., & Förster, E. (Eds.). (2005). New methods for culturing cells from nervous
tissues. Basel: Karger.
Zigova, T., Sanberg, P. R., & Sanchez-Ramos, J. R. (Eds.). (2002). Neural stem cells: Methods and
protocols. Totowa, NJ: Humana Press.
Websites
American type culture collection: http://www.atcc.org.
European collection of cell cultures: http://www.hpacultures.org.uk/aboutus/ecacc.jsp.
Life technologies protocols: http://www.lifetechnologies.com/us/en/home/references/protocols.html.
311
Guide to Research Techniques in Neuroscience. http://dx.doi.org/10.1016/B978-0-12-800511-8.00015-0
Copyright © 2015 Elsevier Inc. All rights reserved.
Chapter 15
Biochemical Assays and
Intracellular Signaling
After Reading This Chapter, You Should be Able to:
	l	Discuss why studying proteins is important to neuroscience
	l	Explain why antibodies are used to study proteins and how they are produced
	l	Describe common biochemical assays used to investigate intracellular signaling
Techniques covered:
	l	Antibody production: making monoclonal and polyclonal antibodies
	l	Techniques used to purify proteins: affinity chromatography, immunoprecipitation
assay
	l	Techniques used to measure the amount and location of proteins within cells and
tissue: western blot (immunoblot), enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), immunohistochemistry (IHC), immunoelectron microscopy,
reporter proteins
	l	Techniques used to investigate protein–protein interactions: co-immunoprecipitation,
protein affinity chromatography, yeast two-hybrid assay
	l	Techniques used to investigate posttranslational modifications: posttranslational
modification assays (kinase assay), posttranslational modification-specific antibodies
	l	Techniques used to investigate protein–DNA interactions: electrophoretic shift
assay (EMSA), chromatin immunoprecipitation assay (ChIP), ChIP on chip assay,
luciferase assay
Proteins are molecular machines responsible for virtually all of the structural
and functional properties of cells. Structural proteins maintain a cell’s shape,
size, and durability. Transmembrane proteins serve as receptors for hormones
and neurotransmitters, allowing the cell to receive information from the extracellular
environment. Other proteins act as downstream messengers from cell
surface receptors and mediate the response to an extracellular cue. Some proteins
serve as enzymes that facilitate the metabolic and physiological needs
of the cell. Finally, some proteins regulate the transcription and expression of
genes that code for other proteins to further affect a cell’s physiology. Because
of the important role that proteins play in neural structure and function, anyone
interested in how the brain works should appreciate the techniques used by
scientists for their investigation.
312  Guide to Research Techniques in Neuroscience
Proteins interact with each other and other biomolecules much like neurons
interact in functional circuits. Instead of acting in isolation, they communicate
with upstream signaling molecules and subsequently affect downstream proteins
and biochemical substrates. Intracellular signaling (or signal transduction)
is a term used to describe the biochemical pathways that affect a cell’s
physiology and metabolism. Often, these pathways begin with the activation of
a cell surface receptor, which leads to a series of biochemical events that culminate
in a change in gene expression in the nucleus. Just as an organism senses
a change in the environment and uses neural circuits to produce an appropriate
response, an individual neuron senses a change in its environment and uses
intracellular signaling pathways to change its transcriptional, physiological, and
metabolic properties (Figure 15.1).
The purpose of this chapter is to survey the methods used to study the
important roles that proteins play within neurons. We will begin with a general
description of intracellular signaling events and why they are useful for understanding
the properties of neurons. Next, we will describe two fundamental
methods used to study proteins: producing and using antibodies, and purifying
proteins of interest. The bulk of the chapter then surveys a variety of methods
that answer useful questions about intracellular signaling events: (1) whether a
protein is expressed in a particular population of cells and how much is present;
(2) whether a protein interacts with another protein; (3) what types of posttranslational
modifications can occur for a protein and which proteins cause these
modifications; and (4) whether a protein is capable of interacting with DNA and
regulating gene expression.
Environment Response
Environment Response
FIGURE 15.1  A metaphor for intracellular signaling. Just as the brain senses changes in the
environment and uses neural circuitry to execute an appropriate behavioral response, a neuron
senses changes in its extracellular environment and uses intracellular signaling to produce changes
in metabolism and gene expression.
Biochemical Assays and Intracellular Signaling Chapter | 15  313
INTRODUCTION TO SIGNAL TRANSDUCTION
AND INTRACELLULAR SIGNALING
The study of signal transduction pathways is fundamental to several subfields
of neuroscience including clinical neuroscience, learning and memory,
regulation of homeostasis, and neural development. For example, many
researchers have investigated the signaling pathways responsible for axon
guidance and synapse formation. Extracellular guidance molecules bind
to membrane receptors, signaling which direction the neuron should grow.
Intracellular signaling cascades then provide the metabolic and structural
changes necessary for the axon to extend in the correct direction. When an
axon reaches its postsynaptic target, other extracellular cues shut off this
signaling cascade and new cascades develop to strengthen synaptic connections.
Clearly, an understanding of the biochemical events within a neuron is
necessary to truly understand the nature of axon guidance, as well as other
vital neuronal functions.
An example of a well-known cell signaling pathway is the JAK–STAT path-
way(Figure15.2).Variousextracellularproteinssuchascytokinesandgrowth
factors bind to specific transmembrane receptors. Some of these receptors
bind to proteins called Janus Kinases (JAKs). Protein kinases are proteins
that, when activated, have the ability to add a phosphate group to other proteins.
When a cytokine binds to its receptor, JAKs transfer a phosphate group
to the receptor. This phosphate group causes a conformational change in the
three-dimensional structure of the receptor that allows other proteins to bind
called Signal Transducers and Activators of Transcription (STATs). When
STAT proteins bind to the receptor, JAKs add an additional phosphate group
to the STAT proteins, causing them to dissociate from the receptor and bind
to each other. These new STAT dimers translocate to the nucleus and activate
specific target genes.
The JAK–STAT pathway is one of several pathways that play a prominent
role in the development and functional physiology of the nervous system. Several
elements of this pathway are consistent with other signaling pathways: an
extracellular cue binds to a membrane protein, causing an intracellular signaling
cascade that eventually affects the expression of genes. These genes code for
other proteins or genetic regulatory sequences that allow the cell to adapt to its
environment.
FUNDAMENTAL TOOLS USED TO STUDY PROTEINS
This chapter describes many techniques that can be used to answer specific
questions about intracellular signaling. Many of these techniques routinely
make use of two fundamental biochemical tools that are essential for
studying proteins: antibodies and protein purification techniques. Therefore,
before discussing any specific assays, we first examine these fundamental
tools.
314  Guide to Research Techniques in Neuroscience
Making and Using Antibodies
Antibodies are proteins used by an organism’s immune system to detect foreign
proteins, called antigens. An antibody recognizes a specific region of an antigen,
called the epitope. Antibodies are extremely useful to scientists because
they bind specific proteins with high affinity. For example, an antibody can be
tagged with a marker to bind and indicate the presence of a protein in a sample.
Alternatively, an antibody can be attached to a column or plate in order to collect
and purify a protein of interest.
Antibodies for research are classified as either monoclonal or polyclonal
antibodies. Monoclonal antibodies are collections of antibodies that recognize
the same epitope (Figure 15.3). They are derived from single cell lines, usually
cells obtained from mice. Once produced, these cell lines provide a renewable
source of antibodies and all batches of antibody will be identical. Because
they are directed against the same epitope, these antibodies may not detect
Ligand
Intracellular
Extracellular
Ligand binds
to its receptor
on the cell
membrane
P P
P P
P
P
PP
Nucleus
Target genes
Promoter
Binding of ligand to receptor
causes phosphorylation
and dimerization of STAT
proteins
Dimerized STAT translocates
to the nucleus and binds to the
promoter of target genes to
activate transcription
STAT proteins are eventually
dephosphorylated and
exported to the cytoplasm
STAT proteins
JAK proteins
FIGURE 15.2  The JAK–STAT signaling pathway. Some receptors for ligands (e.g., cytokines
and growth factors) interact with Janus Kinase (JAK) proteins. When a ligand binds to the receptor,
JAKs add a phosphate group to the intracellular domain of the receptor. These phosphate groups
attract Signal Transducers and Activators of Transcription (STAT) proteins to the receptor. JAKs
add a phosphate group to STAT proteins, causing them to dissociate from the receptor and bind
to each other. These STAT dimers then translocate to the nucleus, where they can affect gene
transcription.
Biochemical Assays and Intracellular Signaling Chapter | 15  315
homologous epitopes across species. In contrast, polyclonal antibodies are a
collection of antibodies that may not be completely homogeneous, recognizing
different epitopes of the same antigen (Figure 15.3). Therefore, these antibodies
may detect homologous epitopes across different species. The physical accessibility
of an epitope of a protein varies; therefore, polyclonal antibodies may
be better able to bind to a target protein because a heterogeneous collection of
antibodies increases the likelihood of one or more epitopes being accessible.
However, polyclonal antibodies are prone to batch-to-batch variability, so the
quality may not be consistent over time. These antibodies are commonly produced
in rats, rabbits, chickens, goats, sheep, and donkeys.
The procedures for producing monoclonal and polyclonal antibodies are
described in Figures 15.4 and 15.5, respectively. A great variety of antibodies are
commercially available and range in cost from $50 to as much as $1000 ($200–300
is common). If an antibody for a protein is not commercially available, individual
labs may produce their own antibodies, but usually this work is contracted to an
antibody-production service that produces custom antibodies on demand. If an
antibody is difficult to produce, scientists can use recombinant DNA techniques
(Chapter 10) to produce proteins tagged with a short amino acid sequence that can
be easily detected by commercially available antibodies (Box 15.1).
Several biochemical techniques depend on the use of antibodies (Table 15.1).A
specific antibody may be useful for one, several, or (rarely) all of these techniques,
depending on the particular binding affinity of the antibody–protein complex. For
example, an antibody that works well for immunohistochemistry (IHC) may work
very poorly for chromatin immunoprecipitation (ChIP). Even within IHC, the antibody
may work much better for labeling proteins in cell culture than labeling proteins
in tissue sections. Therefore, the only way to know if an antibody is useful for
a particular technique is to try it. Often, commercial manufacturers indicate which
biochemical techniques an antibody is best suited for based on internal testing.
While antibody-based techniques are overwhelmingly used in neuroscience
research to identify and report the presence of proteins, antibodies cannot
always be generated to recognize a target molecule of interest. Recently,
Monoclonal antibody Polyclonal antibody
FIGURE 15.3  Monoclonal versus polyclonal antibodies. A monoclonal antibody recognizes a
single epitope of an antigen. A polyclonal antibody is a mixture of several antibodies that recognize
different epitopes of an antigen.
316  Guide to Research Techniques in Neuroscience
scientists have developed an alternative to antibodies called aptamers.Aptamers
are nucleic acid or peptide-based molecules that are engineered to bind to a
specific target molecule (a chemical compound, protein, nucleic acid sequence,
or even cell types or entire organisms). To create aptamers, a lab (or a scientific
supply company) exposes the target molecule to a library of random DNA,
RNA, and/or protein molecules to determine which molecules have the highest
affinity. Aptamers have proven useful for identifying and labeling substances
within the brain, such as amyloid plaques in murine and human brains. Because
Inject antigen into mice
Fuse cells to produce hybridomas
Culture myeloma
tumor cells
Extract immunogenic B cells
from spleen
Culture hybridomas and
select for cells that express
antibodies
Clone and expand colony
of antibody-producing cells
to mass-produce antibody
FIGURE 15.4  Producing a monoclonal antibody. A scientist injects an antigen into an animal,
usually a mouse. The mouse’s immune system naturally produces antibodies as a response. After
several days, the scientist extracts immunogenic B cells that produce antibodies from the spleen.
These cells are fused with tumor cells that divide indefinitely. After culturing these cells and selecting
for hybrids that continuously produce an antibody of interest, a scientist can mass produce a
monoclonal antibody.
Biochemical Assays and Intracellular Signaling Chapter | 15  317
Inject animal with antigen
and allow time for B cells to
produce antibodies
Remove blood
Centrifuge to
collect blood serum
Purify antibodies
on a column containing
the antigen
FIGURE 15.5  Producing a polyclonal antibody. A scientist injects an antigen into an animal,
often multiple times over the course of weeks. The animal’s immune system produces many antibodies
against the antigen as a response. The scientist then collects blood, separates the serum from
the red blood cells, and purifies the serum using affinity chromatography.
BOX 15.1  Creating and Using Tagged Proteins
Most of the techniques used to detect specific proteins rely on antibodies.
However, antibodies do not exist for all native proteins. Furthermore, producing
good antibodies can sometimes be difficult and expensive, and the antibodies are
not guaranteed to be effective for all techniques. Therefore, many scientists use
standard recombinant DNA techniques (Chapter 10) to add short amino acid tags
to a protein of interest. The genetic sequence for the protein of interest can be
inserted into a plasmid vector containing the sequence for one of these tags to
create a fusion protein. The scientist can then use antibodies against the tag to
examine or purify the tagged protein. Common tags include c-Myc, His (histidine),
FLAG, GST (glutathione-S-transferase), and HA (hemagglutinin). FLAG and GST
tags are often used for western blot and IP assays, while c-Myc and HA tags are
often used in immunohistochemistry. These peptide tags have a variety of origins:
the polyhistidine (His)-tag was developed commercially for protein purification,
while HA is derived from human influenza hemagglutinin, a viral gene, which may
explain the ability to easily produce antibodies to this peptide sequence.
318  Guide to Research Techniques in Neuroscience
aptamers are able to isolate molecules in vivo, they also have therapeutic potential
in the treatment of neurodegenerative diseases and cancer.
Purifying Proteins
The ability to purify a protein of interest is fundamental to biochemical research.
Purifying proteins allows an investigator to generate antibodies, determine a
protein’s sequence, and determine a protein’s binding partners. Two common
TABLE 15.1  Techniques that Depend on Protein-Specific Antibodies
Technique Role of antibody in technique
Western blot Antibody binds to a protein that has been run
on a polyacrylamide gel to mark the presence,
approximate size, and relative quantity of the
protein in a sample.
Enyzme-linked
immunosorbent assay (ELISA)
Antibody binds to a protein to measure the quantity
of the protein in a sample.
Radioimmunoassay Antibody binds to radioactive proteins of known
concentration. A sample containing nonradioactive
proteins is added to compete with the radioactive
proteins, allowing a scientist to determine the
protein concentration in a sample.
Immunohistochemistry Antibody binds to a protein to show spatial
expression in tissue or cells.
Immunoprecipitation Antibody is bound to small beads. A sample is
mixed with the beads, so the antibody binds and
purifies a protein. The protein sample is then eluted
off the beads.
Co-immunoprecipitation Antibody is bound to small beads. A sample is
mixed with the beads, so the antibody binds and
purifies a protein. After the protein is eluted, the
investigator uses a separate antibody in conjunction
with a western blot or ELISA experiment to detect
the presence of any secondary proteins that may
interact with the immunoprecipitated protein.
Chromatin
immunoprecipitation
Antibody is bound to small beads in a column. A
sample is run through the column, so the antibody
binds and purifies a protein. After the protein is
eluted, the investigator uses PCR to determine the
presence of any bound DNA sequences.
Antibody interference Antibody binds to a protein, preventing the proper
function of the protein.
Biochemical Assays and Intracellular Signaling Chapter | 15  319
methods of purifying proteins include chromatography and immunoprecipitation
(IP).
Chromatography
Chromatography allows a mixture of proteins (and other biomolecules) in a
solution to be separated on a column. In this procedure, a scientist runs the solution
through a column containing a porous matrix (Figure 15.6). The proteins
pass through the column differently depending on how they interact with the
matrix, and therefore, proteins can be differentially collected as they flow out
the bottom of the column.
Within the matrix, proteins can be separated on the basis of many different
properties. For example, in gel-filtration chromatography, proteins descend
through a column based on their size. In ion-exchange chromatography, proteins
are separated based on their charge. A more specific way to purify a protein,
affinity chromatography, uses small molecules fixed to the porous matrix to
bind to proteins that may have a high affinity, such as a substrate binding to an
enzyme. After the solution is run through the column and the protein of interest is
bound to the matrix, the proteins are eluted from the column by changing the salt
concentration or pH and the purified sample is acquired in a small collection tube.
Immunoprecipitation
Immunoprecipitation (IP) uses antibodies to purify a protein of interest out of
solution (Figure 15.7). Specific antibodies are first bound to a mixture of beads.
When a solution is mixed with these antibody-conjugated beads, the antibody
FIGURE 15.6  Chromatography. A biological
sample is run through a column composed of a porous
matrix. Different columns have different properties
and separate proteins based on properties such as their
size, charge, and binding partners.
Unpurified sample
Purification column
Collection tube
320  Guide to Research Techniques in Neuroscience
will bind to its epitope, precipitating the protein out of solution. After the initial
solution is washed away, a scientist can elute the protein off the beads by removing
the antibodies, usually by changing the salt concentration or pH of the solution.
This elution product can be further purified, although this can also reduce
the yield of the final product.
Taken together, the ability to purify proteins based on their binding partners,
charge, size, and other properties, as well as the ability to use antibodies to
purify proteins out of solutions, allow scientists to perform many cell signaling
assays to investigate proteins in the nervous system.
INVESTIGATING PROTEIN EXPRESSION
A fundamental question in any cell signaling experiment is whether a particular protein
is expressed in a population of cells and, if so, how much of the protein is present.
There are a variety of techniques a scientist may use to answer these questions,
some more quantitative and others with better spatial resolution. A quick comparison
of the strengths and limitations of each technique is presented in Table 15.2.
Western Blot
The western blot (WB) (also known as an immunoblot) is the most commonly
used method to measure the expression of a protein in a tissue or cell sample
(Figure 15.8). Before performing a WB, scientists extract proteins from freshly
dissected brain tissue or cultured cells using a simple protocol that lyses the
cells and removes DNA contaminants. The sample is then processed using what
is known as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
(PAGE), or simply SDS-PAGE. SDS is a negatively charged detergent that
(A) (B) (C) (D) (E)
FIGURE 15.7  Immunoprecipitation. (A) Before an immunoprecipitation procedure begins, a
protein of interest exists in a heterogeneous sample with many other proteins. (B) A scientist adds
antibodies that recognize the protein to small beads and then adds the beads to the sample. (C) The
antibodies bind to the proteins, causing them to adhere to the beads and precipitate out of solution.
(D) The heterogeneous sample is washed away from the beads, and (E) the proteins can be removed
from the beads by changing the salinity or pH of the solution.
Biochemical Assays and Intracellular Signaling Chapter | 15  321
binds to hydrophobic regions of the protein, allowing it to become soluble. The
detergent also causes the protein to take on a net negative charge, regardless of
its amino acid composition. Therefore, the protein will migrate toward a positive
electrode when voltage is applied during electrophoresis.
The scientist loads the protein sample onto a polyacrylamide gel
(Figure 15.8A). In the presence of an electric field, the gel acts as a physical
filter so that larger proteins travel relatively slowly and smaller proteins travel
relatively quickly. This allows the investigator to separate a heterogeneous
mixture of proteins into a spectrum of proteins with different molecular weights.
At this stage of the WB process, the investigator can visualize the protein
bands on the polyacrylamide gel using a stain such as Coomassie blue. This
dye will stain all of the proteins in each lane of the gel. The result can appear
like a smear, with some prominent bands representing highly expressed proteins
in the sample. A scientist might perform this step to demonstrate that the total
amount of protein in each lane is the same.
After the protein sample is run on a gel, the final step in a WB procedure is
to identify the presence of a specific protein. Because the gel is relatively thick
and extremely delicate, a scientist first transfers the proteins onto a thin, nitrocellulose
membrane (Figure 15.8B). This procedure, referred to as the blotting
process, uses current to transfer the proteins from the gel onto the membrane.
Once the proteins have transferred to the membrane, the investigator can detect
the presence of a particular protein by incubating the membrane in a solution
TABLE 15.2	 Methods of Measuring Protein Expression
Technique Description
Western blot Allows for the detection of a protein in a sample, with
measurements of relative concentration between samples.
Enzyme-linked
immunosorbent assay
Allows for measurement of protein concentration in a
sample. More sensitive than a western blot. Can quantify
specific amount of protein, rather than determine the
relative amount of protein (as in a western blot).
Radioimmunoassay Precise measurement of protein concentration in samples
in which the protein may be very dilute.
Immunohistochemistry Characterizes the spatial distribution of the expression of a
protein in cells and tissues. Not very quantitative.
Immunoelectron
microscopy
Characterizes the spatial distribution of the expression of a
protein within cells at a very high resolution.
Reporter proteins Characterizes the spatial distribution of the expression of
a protein without the need to use immunohistochemistry.
Can be performed in live cells or tissues.
322  Guide to Research Techniques in Neuroscience
with an antibody specific to the protein (Figure 15.8C). Then the membrane is
incubated in a solution with a secondary antibody that recognizes the primary
antibody; this secondary antibody is coupled to a radioactive isotope, an easily
detectable enzyme, or a fluorescent dye. The labeled antibody marks the presence
of the protein as a discrete band within a lane of the gel. The scientist can
compare the location of the protein on the blot to a standardized protein ladder
that highlights specific molecular weights in kilodaltons.
Therefore, the final result of a WB experiment is a depiction of the band
of protein, along with its molecular weight and necessary controls (Figure
15.9). The most important control in a WB is a loading control, a measurement
of a separate protein in the sample to ensure that each lane contains the
same quantity of total protein. For example, an antibody to β-actin or GAPDH
(glyceraldehyde 3-phosphate dehydrogenase), proteins expressed in all cells,
is often used to ensure that each lane contains the same total amount of protein,
allowing a scientist to compare the intensity of bands in different samples
Sample wells
Cathode (-)
Anode (+)
Polyacrylamide gel
Protein samples
added to gel
Proteins migrate
down gel and are
separated by size
Cathode (-)
Anode (+)
Gel
Nitrocellulose
Absorbent
paper
Proteins transferred
from gel to
nitrocellulose
Nitrocellulose
Secondary antibody
Primary antibody
Bound protein
Marker
Protein of interest
detected and visualized
using antibodies
(C)
(B)
(A)
FIGURE 15.8  The western blot. (A) Protein samples are run on an SDS-PAGE gel, which separates
proteins on the basis of size. (B) The proteins within the gel are transferred to a thin nitrocellulose
membrane. The gel and membrane are stacked and an electric field causes the proteins
to migrate from the gel to the membrane. (C) To detect a specific protein of interest, a scientist
incubates the nitrocellulose membrane in a primary antibody, and then a secondary antibody that
recognizes the primary antibody. The secondary antibody contains a marker that can be used to
visualize the protein on the membrane.
Biochemical Assays and Intracellular Signaling Chapter | 15  323
to determine relative protein amounts. When characterizing a new antibody, a
scientist should perform a negative control with a sample in which the specific
protein is absent, as well as a positive control with a sample in which the specific
protein is definitely present. These controls allow the scientist to verify
that the antibody is specific and capable of reliably indicating the presence of
the protein of interest.
An investigator may wish to know not only which cells express a protein of
interest, but also where, within those cells, that protein is localized. In this case,
the investigator can use a process called cell fractionation to divide a sample
into its cellular components before a WB experiment begins (Figure 15.10).
Individual organelles within a cell vary in their weight and can be pulled out of
solution through a series of centrifugation steps. Tissue is first dissociated into a
liquid homogenate and then centrifuged through a series of spins, each increasing
in time and force. After each step, the pellet containing specific cellular components
is collected. Finally, the scientist can run each sample in a separate lane
of a WB experiment to determine which fraction contains a particular protein.
Enzyme-Linked Immunosorbent Assay
An enzyme-linked immunosorbent assay (ELISA) is an alternative to a WB,
used to measure the expression of a protein, especially a protein that may be
expressed in extremely low quantities. A traditional WB requires milligrams of
protein, whereas an ELISA is able to quantify nanograms.
Antibody to
protein of interest
Antibody to 2nd
protein of interest
Loading control
Protein samples
FIGURE 15.9  How data are often presented in a western blot experiment. Western blot data are
often presented as blots on gels. These dark blots represent signal detected from an antibody. The darker
the band, the more protein was present in the sample. Each row usually represents detection from a
specific antibody.A loading control is included to show that each well contains the same amount of starting
protein. Usually, this is an antibody against β-actin or glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). A reasonable conclusion from this mock figure would be that the first protein of interest is
detected in samples 1, 3, and 4, but not sample 2. Furthermore, a second protein of interest is detected in
samples 1 and 2, but not 3 or 4.
324  Guide to Research Techniques in Neuroscience
A scientist binds an antibody to the bottom surface of a plate (Figure 15.11).
A sample is added to the plate, and any protein present that can bind to the antibody
becomes adhered to the surface. The sample is washed away, but the bound protein
remains.A secondary detecting antibody that is linked to an enzyme is then applied
to the plate, such that the protein of interest is sandwiched between both antibodies.
A chemical substrate is added to the sample that is converted by the enzyme into
a chromogenic or fluorescent signal. Finally, the scientist uses an optical device to
measure the signal and determine the quantity of protein present.
Radioimmunoassay
A radioimmunoassay (RIA) is a highly sensitive method of measuring very
low concentrations of proteins. Samples are usually derived from blood, extracellular
fluid, or cerebrospinal fluid.
To perform an RIA reaction (Figure 15.12A), a scientist first labels a
known quantity of protein with a radioisotope, usually an isotope of iodine
(125I or 131I), as iodine readily attaches to the amino acid tyrosine. The radiolabeled
protein is then mixed with an antibody specific for that protein. This
antibody–protein mixture is divided into several samples. To the first sample,
the scientist adds a known amount of unlabeled, nonradioactive protein.
This protein competes for antibody binding sites with the radioactive form of
the protein. In the subsequent samples, the scientist continually increases the
concentration of nonradioactive protein, increasing the amount of displaced
radioactive protein. Finally, the scientist removes all proteins bound to antibodies
from the samples using a secondary antibody so that only unbound proteins
remain. The radioactivity of each sample is measured. From these data, a scientist
can create a standard binding curve reflecting the ratio of bound to unbound
protein versus the concentration of nonradioactive protein (Figure 15.12B).
To measure the concentration of a protein from a biological sample, the
scientist runs this unknown quantity of protein in parallel with the known preceding
quantities. Once the scientist identifies the ratio of bound to unbound
Biological
sample
800 g
10 min 15 min 60 min 2 h
20,000 g 100,000 g 150,000 g
Pellet contains
nuclei, cell debris
Pellet contains
mitochondria
Pellet contains
membrane and
vesicles
Pellet contains
ribosomes
FIGURE 15.10  Cell fractionation. A biological sample can be centrifuged at successively higher
speeds to collect specific organelles within cells.
Biochemical Assays and Intracellular Signaling Chapter | 15  325
protein, it is possible to use the standard binding curve to deduce the concentration
of the unknown sample (Figure 15.12C).
A RIA is sensitive and can measure extremely small protein concentrations,
such as the concentration of neuropeptides in the extracellular fluid of brain tissue.
However, this technique requires specialized equipment and is relatively expensive.
It also requires safety precautions due to the use of radioactivity.
Immunohistochemistry
IHC was previously described in Chapter 6 as a method to visualize the expression
of proteins in cells or brain sections. This technique can be used to determine
the spatial expression pattern of a protein, a relative advantage over the
WB, ELISA, and RIA techniques described previously. IHC is not a very good
technique for quantifying the amount of protein present in a sample, though it
FIGURE 15.11  Enyzme-linked immunosorbent
assay. A scientist coats the
bottom of a plate with antibodies specific
to a protein of interest and then adds a biological
sample in solution. The proteins
bind to the antibodies, and the sample is
washed off the plate. Other antibodies
conjugated to a marker are added to the
plate, allowing a scientist to quantify the
amount of protein in the sample.
Add solution with
heterogeneous mixture
of proteins
Wash solution
off plate
Add additional
layer of antibodies
with marker
Add antibodies to
bottom of plate
326  Guide to Research Techniques in Neuroscience
is possible to compare the relative signal from two different samples to show a
relative difference in expression.
Immunoelectron Microscopy
Immunoelectron microscopy (IEM) is an extension of IHC used in combination
with electron microscopy to visualize protein expression within subcellular
structures. Like a cell fractionation procedure followed by a WB, IEM can be
(A)
(B) (C)
Standard binding curve
%ofbound
radioactiveprotein
Concentration of
nonradioactive protein
Determination of concentration
of unknown value
%ofbound
radioactiveprotein
Concentration of
nonradioactive protein
Solutions with mixtures of radioactive and nonradioactive proteins
Radioactive protein Nonradioactive protein
FIGURE 15.12  A radioimmunoassay. (A) A scientist mixes different concentrations of
nonradioactive and radioactive versions of a protein in a series of reaction tubes with an antibody
against the protein. The nonradioactive and radioactive proteins compete to bind with the antibody.
The scientist then collects the antibodies and bound proteins using a secondary antibody
so that only unbound proteins remain. (B) By measuring the radioactivity of each sample, the
scientist can produce a standard binding curve, comparing the amount of radioactive protein
bound to an antibody with the concentration of nonradioactive protein added to the sample. (C)
This binding curve can be used to determine the unknown concentration of a protein from an
experimental sample.
Biochemical Assays and Intracellular Signaling Chapter | 15  327
used to show that a protein is localized in a particular subcellular compartment.
A cell fractionation experiment is more quantitative, whereas IEM is more spatially
precise. Therefore, these techniques can be used in combination to provide
strong evidence about protein distribution within a cell.
Reporter Proteins
To visualize proteins without the need for IHC, scientists can use recombinant
DNA technology to attach a reporter protein, such as green fluorescent
protein, to proteins of interest (Chapter 6). Tagging a protein of interest
with a reporter protein is more time-consuming than IHC, as various recombinant
DNA constructs, gene delivery strategies, and/or transgenic strategies
must be employed before an experiment can take place. However, this
method overcomes some of the limitations of IHC. For example, reporter
genes allow an investigator to determine the expression and location of a
protein in living cells.
INVESTIGATING PROTEIN–PROTEIN INTERACTIONS
Almost all proteins function within the cell by interacting and/or forming a complex
with other proteins. As the term intracellular signaling implies, proteins
communicate with other proteins within the cell in a chain of events that serve
as the basis of normal cellular physiology and metabolism. Therefore, a scientist
might ask whether a specific protein interacts with another protein of interest.
Alternatively, a scientist might want to know the identity of all proteins that interact
with a protein of interest. The following techniques address these questions.
Co-Immunoprecipitation
One of the simplest methods of determining if a protein physically interacts
with another protein is by performing a co-immunoprecipitation (Co-IP)
assay. This technique is an extension of the IP assay described previously
(Figure 15.7). A scientist lyses cells to extract proteins and then mixes the
solution with beads linked to antibodies. An antibody directed toward one
of the proteins in the complex immunoprecipitates the protein out of solution.
Any proteins that are tightly associated with this protein will also precipitate
out of solution and be released in the elution process (Figure 15.13).
The eluted proteins can be separated and identified using a WB protocol
with antibodies for the immunoprecipitated protein and potential binding
partner (Figure 15.14). If the identities of the potential binding partners
are unknown, they may be determined using a technique called mass
spectrometry (Box 15.2). Note that Co-IP does not show if a protein directly
interacts with another protein; the two proteins may exist in a complex, indirectly
bound with intermediary proteins.
328  Guide to Research Techniques in Neuroscience
Protein Affinity Chromatography
A scientist can use protein affinity chromatography to isolate and identify proteins
that physically interact with each other. To capture the interacting proteins,
a target molecule is attached to polymer beads within a column. These target
molecules have specific interactions with the protein of interest that will capture
IP: Protein A
WB: Protein A
IP: Protein A
WB: Protein B
Protein B
1 2 3 4
Used to ensure that the starting sample had an
equal amount of Protein B
This is a western blot against Protein B, after an IP
experiment with an antibody against Protein A
This is a western blot against Protein A after an IP
experiment with an antibody against Protein A
Protein samples
FIGURE 15.14  How data are often presented in a co-immunoprecipitation experiment.
Co-immunoprecipitation experiments are often followed by western blots. Just as in a
western blot, the dark blots represent signal detected from an antibody. The darker the band,
the more protein was present in the sample. A co-IP figure must report the antibody used to
immunoprecipitate a protein as well as the antibody used to detect the protein in the western
blot. A reasonable conclusion from this mock figure would be that Protein B interacts with
Protein A in samples 1 and 3, but not in samples 2 and 4. This result is not because Protein B is
absent from samples 2 and 4, as the bottom gel shows presence of this protein in all four initial
samples (before the IP took place).
(A) (B) (C) (D) (E)
FIGURE 15.13  Co-immunoprecipitation. (A) Before an experiment begins, a protein of interest
exists in a heterogeneous sample bound to other proteins. (B) A scientist adds antibodies that
recognize the protein to small beads and then adds the beads to the sample. (C) The antibodies bind
to the proteins, causing them to adhere to the beads and precipitate out of the solution. Any proteins
that interact with the protein will also adhere to the beads. (D) The heterogeneous sample is washed
away from the beads, and (E) the proteins can be removed from the beads by changing the salinity
or pH of the solution. At this point, the scientist can use other methods, such as a western blot, to
identify the interacting proteins.
Biochemical Assays and Intracellular Signaling Chapter | 15  329
BOX 15.2  Mass Spectrometry
Mass spectrometry (MS) is a technique used to identify and sequence proteins by
breaking them apart and determining the mass to charge ratio of their fragments.
Automated programs compare the calculated mass of protein and peptide fragments
from a sample with the masses computed from protein databases. Thus, MS
can identify components of an unknown mixture.
In de novo sequencing, MS can determine the amino acid sequence of an
unknown peptide. By assembling many overlapping peptides, the whole sequence
of a protein may be determined, though this is rarely done in practice. MS is often
used to identify posttranslational modifications (PTMs), such as phosphorylation
or acetylation, and the residues on which they reside. This technique is extremely
sensitive and requires little material (subpicomole amounts).
Determining the masses of proteins and peptides requires the sample to be in
the gas phase, and two techniques have been developed to do this: matrix-assisted
laser desorption ionization (MALDI) and electrospray ionization (ESI). In MALDI,
peptides are mixed with a UV-absorbing organic acid and dried on a metal slide.
The sample is then blasted with a UV laser; the matrix absorbs the laser light, causing
it to go into the gas phase carrying the sample (often peptides), which become
charged. In ESI, the sample is in an acidic solution that charges the sample. The
charged sample travels through a capillary into a heated area where solvent molecules
are lost due to the heat and the increasing charge on the droplets: as the
charge density becomes higher and higher, the droplets explode, becoming finer
and finer until only single molecules remain.
Once the molecules enter a gas phase, their mass-to-charge ratio can be measured.
Many different types of detectors exist for this purpose; commonly coupled
with MALDI is a time of flight (TOF) detector. In such an instrument, ionized
peptides are accelerated in an electrical field and fly toward a detector. The time
it takes each peptide to reach the detector is determined by its mass and charge,
with large peptides moving slowly and highly charged peptides moving relatively
quickly. Other detectors use electrical or magnetic fields to selectively filter or
detect different mass to charge ratios.
Many molecules can have the same mass-to-charge ratio despite having very
different chemical structures. For example, the peptides PEPTIDE and TIDEPEP are
indistinguishable by their mass alone. This situation is common and occurs with
proteins, nucleic acids, and organic molecules. To distinguish these types of molecules,
they may be fragmented and the masses of their constituent components
measured; with peptides, this usually involves smashing them with gas molecules
such as argon or nitrogen, to cleave the peptide bonds. The result of this collision
induced dissociation (CID) is overlapping fragments that reveal the original
sequence of the peptide. In the preceding example, observing EPEP or PEPT would
distinguish between the two possibilities. Ideally, collisions create a virtual ladder
of fragments, each differing by only one amino acid. In practice, a perfect ladder is
rarely observed, but enough detail is present to match a peptide to known protein
sequences.
MS is a core tool in the field of proteomics, the systematic characterization of
the complete set of proteins in a system at a particular time, allowing for insight
Continued
330  Guide to Research Techniques in Neuroscience
the protein as it passes through the column. They can be ligands for a receptor,
substrates for an enzyme, or antibodies for an antigen. Cellular proteins are
washed through the column, and the proteins that interact with the target attach
to the matrix, while all other proteins pass through. The proteins that interact
with the target are eluted off the beads, and their identity can be determined
using a WB. Just as in the co-IP assay, if the identities of the interacting proteins
are unknown, they can be determined using mass spectrometry (Box 15.2). Also
similar to a co-IP assay, this technique does not show if the two proteins directly
interact or exist in a larger protein complex.
Yeast Two-Hybrid Assay
A yeast two-hybrid assay investigates protein–protein interactions by exploiting
a transcription system normally used by yeast cells. In a normal yeast cell,
a transcription factor called Gal4 binds to a promoter region called an upstream
activating sequence (UAS). Gal4 is composed of a binding domain (BD), which
binds to the UAS, as well as an activation domain (AD), which initiates transcription
of a target gene.
In a yeast two-hybrid assay, a scientist uses recombinant DNA technology
to divide the Gal4 protein into these two separate domains (Figure
15.15A). The Gal4 binding domain is fused to a protein of interest thought
to interact with another protein. This protein can be considered the bait in
that its role in the experiment is to attract other binding partners. The scientist
fuses the Gal4 activation domain to a potential binding partner. This
binding partner can be considered prey in that it could potentially bind with
the bait. If the two proteins do not interact, the BD and AD fragments will
be physically separated, and no transcription will occur. However, if the
two proteins are able to bind, the Gal4 BD and AD fragments will become
physically close enough to cause transcription of the downstream coding
sequence (Figure 15.15B; Box 15.3). If the gene adjacent to the UAS is a
reporter gene, such as lacZ, the scientist will be able to identify the interaction
between the two proteins.
A yeast two-hybrid assay is useful to test hypotheses that two proteins interact,
but this assay can also be used as a screen to identify potential binding
partners for a protein of interest (Figure 15.16). For example, a scientist can
identify unknown “prey” for a protein of interest by attaching the activation
domain to a large mixture of DNA fragments from a cDNA library (Chapter 10).
into signaling networks and protein interactions. Proteomics involves the analysis
of many proteins at the same time—for example, identifying variations in protein
expression in the brains of patients with Alzheimer’s disease compared to healthy
brains. MS (along with bioinformatics tools to manage the large amounts of data)
allows for the profiling of these thousands of different proteins.
BOX 15.2  Mass Spectrometry—cont’d
Biochemical Assays and Intracellular Signaling Chapter | 15  331
Individual ligation products are then introduced into yeast cells containing the
target protein. If one of the members of the library encodes a protein that interacts
with the target protein, the two Gal4 fragments will interact and activate
transcription of the reporter. The scientist can then identify the yeast colony
expressing the reporter, purify and sequence the DNA attached to the activation
domain, and determine the identity of the binding protein. While this assay
shows that the proteins can directly interact, it does not show that they interact
in vivo or even that they can interact in mammalian cells.
INVESTIGATING POSTTRANSLATIONAL MODIFICATIONS
A posttranslational modification (PTM) is a biochemical modification that
occurs to one or more amino acids on a protein after the protein has been translated
by a ribosome. One of the most commonly studied PTMs is the addition
Promoter Reporter
gene
(A)
(B)
Expression of
reporter gene
DNA-binding
domain
Transcriptional
activation domain
Target protein Binding partner
FIGURE 15.15  Yeast two-hybrid assay. (A) A scientist uses recombinant DNA technology to
fuse the DNA binding domain of Gal4 to a protein of interest and the transcriptional activation
domain of Gal4 to a potential binding partner. These constructs are introduced into yeast cells. If the
two proteins do not interact, the DNA binding domain and transcriptional activation domain of Gal4
do not interact. However, (B) if the two proteins do interact, then the Gal4 complex is complete and
can activate transcription of a reporter gene.
332  Guide to Research Techniques in Neuroscience
Recombined cDNA library clones
tagged to transcriptional
activation domain of Gal4
Recombined protein of interest
tagged to DNA-binding domain
of Gal4
Randomly transform
yeast cells
Yeast cells on plate
Yeast cells on plate
Wait 1-3 days
for transcription
to occur
Examine cells
for presence of
reporter protein
FIGURE 15.16  Using a yeast two-hybrid assay as a screen. Recombinant DNA technology is used
to fuse thousands of genetic sequences from a cDNA library to the Gal4 transcriptional activator. These
constructs, as well as a construct in which a protein of interest is fused to the Gal4 DNA binding domain,
are randomly introduced into yeast cells. In some yeast cells, a target protein will interact with the protein
of interest, and the Gal4 will activate expression of a reporter gene. A scientist can visualize the expression
of the reporter by examining the color of cell colonies grown on a plate. To determine the identity of
the protein binding partner, the scientist can extract and sequence the DNA from these colonies.
Biochemical Assays and Intracellular Signaling Chapter | 15  333
BOX 15.3  Walkthrough of an Intracellular Signaling Experiment
Let us say that you work in a laboratory that studies a specific aspect of neural
development: how a neuron’s axon correctly extends to its postsynaptic target. The
tip of an axon is referred to as the axonal growth cone, and it typically reaches its
destination by responding to extracellular guidance cues produced by other cells.
There are three general steps that occur to guide the growth cone: (1) an extracellular
guidance cue is secreted to guide the axon’s position; (2) a receptor on the
axon’s surface recognizes that cue and transduces it into an intracellular signaling
cascade that affects other proteins within the developing neuron; and (3) various
downstream organelles receive and respond to the signal, including the cytoskeleton
that must be reorganized to move the growth cone toward the site where the
initial cue was detected. If you know the identity of the receptor and the extracellular
cue, you might be interested in figuring out how the receptor translates the
cue into a response. What is the intracellular signaling pathway that leads to the
cytoskeletal changes necessary for the growth cone to turn toward the cue? Let
us focus on identifying the next step of signaling that occurs after the receptor is
activated: the activation of a second protein that binds to the intracellular domain
of the receptor.
There are multiple approaches for finding proteins that interact with the receptor.
For example, you could immunoprecipitate the receptor and then perform
mass spectrometry to identify any other proteins that co-precipitated. However,
this approach would not tell you if the two proteins interacted directly or were
simply part of a larger complex of proteins. Another approach would be to perform
a yeast two-hybrid assay. This technique is used to identify protein-binding
partners that directly interact with the receptor. To perform this assay, you would
begin by genetically engineering a construct that attaches the receptor’s intracellular
domain to the DNA-binding domain of a transcriptional activator, such as
Gal4, to act as the bait. Then you would create or use a cDNA library to attach different
cDNA molecules to the activation domain of Gal4 to act as the prey. Finally,
you would introduce the bait and prey constructs into yeast cells that express a
reporter gene, often lacZ, under the control of a promoter that is recognized by the
transcriptional activator (UAS for the Gal4 activator). Thus, the bait will bind to the
promoter but cannot initiate transcription of the reporter until a specific prey with
the activation domain interacts with the bait to create the full activator. Then, the
reporter gene will be transcribed and can be detected.
To detect yeast cells that express lacZ, you could grow thousands of cells on an
agar plate that also contains X-gal, the substrate that causes a visible blue byproduct
to form in those cells. The final step would be to pick blue yeast colonies off
the plate, extract and sequence the DNA of the prey, and identify the protein that
interacts with the receptor based on the cDNA sequence. This screen allows you
to conclude that the prey you found can interact with the intracellular domain
of the receptor. Most likely, you will have discovered a number of positive hits
to sequence and should confirm the interaction’s specificity to determine which
would be a good candidate for a follow-up.
This yeast two-hybrid experiment is a good start, but you could learn even
more about the receptor–protein interaction by attaching different functional
Continued
334  Guide to Research Techniques in Neuroscience
or removal of a phosphate group, PO4 (Figure 15.17). Other common PTMs
are listed in Table 15.3. These modifications often change the ultrastructural
and functional properties of a protein, causing substantial downstream signaling
effects. For example, the phosphorylation state of a protein often indicates
whether it is functional or inactive.
There are multiple questions a scientist may ask about a protein of interest
regarding PTMs. For example, a scientist might want to know if a protein of interest
receives a PTM.This question can be answered using mass spectrometry (Box 15.2)
regions of the receptor’s intracellular end to use as bait. This would tell you with
finer resolution exactly what part of the receptor is interacting with the prey or
allow you to capture only prey that will interact with a specific region—say, the
kinase domain—or a particular region you know to be functionally important. To
convince yourself and other scientists of the interaction between the receptor and
intracellular protein, you could perform additional, complementary experiments.
For example, co-immunoprecipitation, as just described, is often used to verify
yeast two-hybrid results. You could also use protein affinity chromatography. In
cell signaling studies, there is no such thing as too much evidence; figures often
contain several different methods to validate the same result.
Now that you have a candidate (or several candidates, based on the results of
the yeast two-hybrid experiments), you should follow up with other experiments to
make sure that your candidate protein makes sense as a protein that mediates the
intracellular response to a guidance cue. One question you might ask right away
is whether the candidate protein is expressed in the right place (the axon growth
cone) and at the right time (the embryonic or postnatal period during which the
axon grows toward its target). If the interacting protein is not present in axonal
growth cones or at a time when axons are growing out, the interaction may not be
relevant to the growth cone turning behavior you’re interested in, or the interaction
may not actually occur. To examine protein expression, you could use immunohistochemistry
to stain for the expression of the protein in histological preparations
of the growth cone at different developmental time points. You could also collect
tissue samples containing the growth cones from different time points and perform
western blots to examine the expression of the protein at each time point.
If you have determined that a protein binds to the receptor, is expressed in
the growth cone, and is expressed during the developmental period in which the
growth cone migrates toward its target, you have laid the foundation for many
future experiments. For example, you could ask whether the interaction between
the receptor and the protein causes the protein to be phosphorylated. You could
use techniques described in other chapters, such as RNA interference (RNAi—
Chapter 13), to investigate the consequence of knocking down the expression of
the intracellular protein. There are many future experiments that can and should be
performed, and the more complementary lines of evidence you collect, the more
you will know, and the better your study will be received.
BOX 15.3  Walkthrough of an Intracellular Signaling Experiment—cont’d
Biochemical Assays and Intracellular Signaling Chapter | 15  335
to analyze the molecular weights of protein fragments, identifying fragments with a
PTM. Other questions include whether a specific enzyme is responsible for modifying
a protein of interest or how the PTM changes over time following a stimulus.
These questions can be answered using the following techniques.
ATP
P
Protein kinase
ADP
FIGURE 15.17  Phosphorylation. A protein kinase adds a phosphate group from ATP to a
substrate protein.
TABLE 15.3  Common Posttranslational Modifications (most of these
posttranslational modifications can be reversed by opposing enzymes)
Classification Chemical modification
Type of enzyme
mediating the
modification
Phosphorylation Addition of a phosphate group (-PO4) to
a residue of serine, tyrosine, threonine,
or histidine
Protein kinase
Acetylation Addition of an acetyl group (-COCH3) at
the N-terminus of a protein or a lysine
residue
Acetyltransferase
Methylation Addition of a methyl group (-CH3) at a
residue of lysine or arginine
Methyltransferase
Glycosylation The addition of a glycosyl group to a
residue of asparagine, hydroxylysine,
serine, or threonine, resulting in a
glycoprotein
(Multiple types of
enzymes mediate
this reaction)
Sulfation Addition of a sulfate group (SO4) to a
tyrosine residue
Tyrosylprotein
sulfotransferase
Farnesylation Addition of a farnesyl group (C15H26O)
to the C-terminus of a protein. Usually
causes the protein to anchor to the cell
membrane
Farnesyltransferase
Ubiquitination Addition of a ubiquitin protein to a
lysine residue. Usually targets proteins
for proteasomal degradation.
Ubiquitin ligase
336  Guide to Research Techniques in Neuroscience
PTM-Specific Assays
PTM-specific assays are designed to test the hypothesis that a particular protein
mediates the PTM of another protein. Each PTM has its own biochemical assay.
Here, we describe a common type of assay: a kinase assay (Figure 15.18).
A kinase assay is used to determine whether one protein is capable of phosphorylating
another protein. As mentioned previously, protein kinases are
enzymes that catalyze the covalent transfer of phosphate from adenosine triphosphate
(ATP) to a substrate protein. A scientist can incorporate a radioactive label,
such as [32P] orthophosphate, into molecules of ATP. This radioactive ATP acts
as the phosphate donor for the protein kinase substrate. To perform this assay,
the scientist combines the hypothetical protein kinase, the substrate protein, and
radioactive ATP into a reaction tube. Next, the scientist isolates the substrate protein
using immunoprecipitation and visualizes the presence of 32P using autoradiography
(Figure 15.19).
FIGURE 15.18  A kinase assay. To determine if a protein kinase is able to
phosphorylate a protein, a scientist mixes the kinase, the protein, and radioactive
ATP into a reaction tube. Fifteen minutes later, the scientist immunoprecipitates
the protein and runs samples on a gel to detect for the presence
of radioactivity.
Substrate protein
Protein kinase
32P] ATP
+
+
32P
WB with antibody
against substrate
protein
1 2 3 4
Protein samples
FIGURE 15.19  How data are often presented in a kinase assay. The 32P will only be present
if the protein kinase is able to phosphorylate the substrate protein. To ensure that the amount
of substrate protein was equal in each sample, a scientist typically performs a western blot
using an antibody against the substrate protein. Alternatively, a scientist could stain the gel with
Coomassie blue. A reasonable conclusion from this mock figure would be that the protein kinase
under investigation was able to phosphorylate a protein in sample 2 but not samples 1, 3, or 4.
Biochemical Assays and Intracellular Signaling Chapter | 15  337
Kinase assays are easily the most common PTM assay represented in the
literature, as at least 2% of the genes in the mammalian genome code for protein
kinases. However, there are many different PTM-specific assays: for example,
methyltransferase or acetyltransferase assays are used to determine whether a
protein is capable of adding a methyl or acetyl group, respectively, to a target
protein.
PTM-Specific Antibodies
It is often possible to produce an antibody to a protein of interest that is
directed against a site on the protein that receives a PTM (Figure 15.20).
Such an antibody binds to its substrate protein only when the PTM is present.
PTM-specific antibodies can be used in any of the previously described
antibody-based techniques to assay the extent of modifications, or with spatially
sensitive techniques such as IHC or IEM to determine the localization of
modified proteins. Such analysis is especially informative when dealing with
dynamic changes in protein localization and activation. By exposing a given
sample to various molecular signals, such as growth factors, a scientist can
assay any changes in localization as well as changes in activity corresponding
to the modifications.
INVESTIGATING PROTEIN–DNA INTERACTIONS
The final step in many intracellular signaling pathways is the activation of a
protein that directly affects gene transcription. Scientists often ask whether
a specific protein of interest is able to bind to DNA and affect the expression
of a specific target gene. Three techniques are commonly used to examine
interactions between proteins and DNA: the electrophoretic mobility shift
assay, the ChIP assay, and the luciferase assay. Because these techniques
answer slightly different questions about protein–DNA interactions, scientists
often use them in concert to strengthen the evidence that a protein interacts
with DNA and affects the expression of target genes.
P
FIGURE 15.20  A posttranslational modification-specific antibody. The antibody binds to an
epitope on a protein that is phosphorylated at a specific amino acid residue, but not when the phosphate
group is not present.
338  Guide to Research Techniques in Neuroscience
Electrophoretic Mobility Shift Assay
An electrophoretic mobility shift assay (EMSA, also known as a gel shift
assay) is used to determine if a protein is able to directly interact with a short,
specific sequence of DNA. Before the experiment begins, the investigator
hybridizes two complementary DNA strands (about 30–40 bp in length) and
labels the strands with a radioactive probe. The investigator also purifies the
protein that is hypothesized to interact with the strand of DNA.
During the experiment, the radiolabeled DNA is run in several lanes of a
polyacrylamide gel (Figure 15.21A). The speed at which the DNA molecules
Cathode (-)
Anode (-)
DNAmigration
1 2 6543
1 2 6543
(A)
(B)
FIGURE 15.21  Electrophoretic mobility shift assay. (A) To determine if a protein is capable of
interacting with a short sequence of double-stranded DNA, a scientist radioactively labels short DNA
sequences, mixes these sequences with purified proteins, and runs the mixture on a gel. Any DNA
sequences that interact with a protein will migrate much slower on the gel. Lane 1 represents DNA alone.
Lane 2 represents DNA with an interacting protein. Lane 3 represents a scrambled control, in which
the nucleotide sequence is jumbled. Lane 4 represents a reaction in which a sufficient amount of nonradiolabeled
DNA is added to the mixture, taking a high percentage of the interactions with the binding
protein, allowing the radiolabelled DNA band to be shifted back to normal. Lane 5 represents a reaction
that includes an antibody to the protein. The antibody–protein interaction increases the macromolecular
complex, “supershifting” the DNA so it moves more slowly down the gel. Lane 6 represents a reaction
in which a protein is included that is known not to bind to DNA, such as a membrane-bound protein. (B)
How data are often presented in an EMSA reaction, corresponding to the treatments described above.
The dark gradient at the bottom represents the unbound, radioactive DNA strands that have migrated to
the bottom of the gel.
Biochemical Assays and Intracellular Signaling Chapter | 15  339
move from one side of the gel to the other during electrophoresis is determined
by their size. In one lane, the DNA is run by itself. In another lane, the DNA is
run with the purified protein. If the protein interacts with the DNA strand, the
size of the DNA–protein complex will be greater than the DNA strand alone, and
therefore, the band of DNA will be shifted upward on the gel (Figure 15.21B).
Several controls are necessary for a proper EMSA experiment: (1) in addition
to the DNA sequence of interest, the investigator should run a slightly
scrambled DNA sequence to ensure that the DNA–protein interaction is specific
to the exact sequence. (2) The investigator should perform a competitive
binding assay in which nonradiolabeled DNA is added to the reactions with
the radiolabeled DNA. If there is sufficient nonradiolabeled DNA, the amount
of protein bound to the radiolabeled DNA will decrease, and the size of the
radiolabeled DNA band will be shifted back to normal. (3) An antibody to the
purified protein should be added to a lane with the protein and labeled DNA.
This will further increase the size of the DNA–protein complex and create a
“supershifted” band, demonstrating that it is indeed the protein of interest that
is causing the shift in the first place. (4) The investigator could run a nonspecific
protein known not to interact with DNA, such as a membrane-bound protein,
to ensure that there is nothing “sticky” about the DNA strand in the EMSA
reaction.
One of the strengths of an EMSA assay is that it demonstrates that a protein
can directly bind to a sequence of DNA. One of the limitations is that all
reactions take place in a test tube. Therefore, the only conclusions that can be
reached using this assay is that a protein is capable of binding DNA, but not that
it actually does so in a living cell. For this conclusion, it is necessary to perform
a ChIP assay.
Chromatin Immunoprecipitation
ChIP is used to determine if a protein interacts with a specific region of DNA.
The advantage of ChIP over an EMSA assay is that the starting material comes
from living cells, most often cultured cells, though fresh tissue is sometimes
used. However, unlike an EMSA, it is impossible to determine if a protein interacts
with a sequence of DNA directly or if it is part of a greater macromolecular
complex.
The fundamental concept behind a ChIP assay is that before the experiment
begins, a protein of interest is physically bound to DNA or is part of a
DNA–protein complex in living cells (Figure 15.22). The scientist begins the
experiment by quickly fixing the cells in a diluted formaldehyde solution so that
DNA–protein complexes are cross-linked and remain intact. Next, the DNA is
sonicated and sheared into approximately 600–1000 bp fragments. If the crosslinking
is adequate, the proteins will still be bound to sonicated DNA. At this
stage, the scientist immunoprecipitates the DNA–protein complexes using an
antibody against the protein of interest.
340  Guide to Research Techniques in Neuroscience
Aftertheimmunoprecipitationstep,thepurifiedproteinsarereversecross-linked
so that the DNA fragments can be purified. If the scientist has a hypothesis about
the identity of the bound DNA sequence, he or she can then determine the identity
of the DNA fragments by performing a polymerase chain reaction (PCR) reaction
(Chapter 10) with primers for a specific region of DNA. The final result of a ChIP
assay is usually a photograph of an agarose gel that reveals the end product of PCR
reactions (Figure 15.23). Alternatively, quantitative real time PCR (qRT-PCR) can
also be performed to better quantify the amount of PCR product in each lane.
What if a scientist does not have a hypothesis as to which DNA sequences
are bound to a protein of interest? It is possible to screen all DNA sequences
in the genomes of commonly used organisms by performing a ChIP on chip
Genetic sequence A
IgG Protein X Input
Genetic sequence B
FIGURE 15.23  How data are often presented in a ChIP assay. The bands represent PCR products,
i.e., DNA run on an agarose gel. The labels at top are the immunoprecipitating antibodies. IgG
is a random immunoglobulin and should not show any signal. The input lane represents the solution
before running the immunoprecipitation, so all DNA should be present. A reasonable conclusion
from this mock figure would be that Protein X interacts with DNA sequence A but not sequence B.
1. Crosslink protein to DNA
2. Sonicate to shear DNA
3. Immunoprecipitate with antibody
to protein
4. Reverse crosslinks and
purify DNA
5. Run PCR using primers for
DNA sequence of interest
FIGURE 15.22  Chromatin immunoprecipitation.
Biochemical Assays and Intracellular Signaling Chapter | 15  341
assay. In this assay, the DNA sequences obtained at the end of a ChIP experiment
are run on a DNA microarray (Chapter 9) to determine all of the DNA
sequences bound to the immunoprecipitated protein.
Several positive and negative controls are necessary for a proper ChIP
experiment.An important positive control is an input lane containing PCR products
from DNA obtained after sonication but prior to immunoprecipitation. This
reaction should result in a strong signal and ensures that the DNA of interest
was properly obtained in the first place. Another important positive control is an
immunoprecipitation reaction with an antibody for a protein known to bind to
any region of DNA, such as a histone protein. ChIP results using a histone antibody
should, therefore, produce a strong DNA signal. Negative controls include
performing an immunoprecipitation with nonspecific antibodies (random IgG is
usually used) and using primers that amplify nonspecific DNA regions.
Luciferase Assay
A luciferase assay is used to determine if a protein can activate or repress
the expression of a target gene. Unlike the ChIP or EMSA assays, which only
assess the ability of a protein to interact with a region of DNA, a luciferase assay
is able to establish a functional connection between the presence of the protein
and the amount of gene product that is produced. This assay is unable to determine
whether the protein directly interacts with DNA itself; the protein could
indirectly affect transcription by activating or repressing a separate protein or
protein complex that affects transcription.
Luciferase is an enzyme used for bioluminescence by various organisms in
nature, most famously the firefly. The scientist produces a construct in which the
regulatory region of a target gene is fused with the DNA coding sequence for luciferase
(Figure 15.24). A separate DNA construct encodes the protein hypothesized
Promoter region Luciferase reporter
5’ 3’
?
Hypothetical
transcriptional
activator
FIGURE 15.24  A luciferase assay. To determine if a protein is able to activate (or suppress) transcription
of a gene of interest, a scientist uses recombinant DNA technology to produce a construct
in which the gene’s promoter is placed adjacent to a luciferase reporter gene. This construct, as well
as a construct coding for the protein, is introduced into cultured cells. If the protein is able to activate
transcription, the cell will produce the luciferase reporter. The amount of luciferase produced can be
quantified using a luminometer.
342  Guide to Research Techniques in Neuroscience
to affect transcription. The scientist transfects a cell culture system, such as HEK
293T cells, with both constructs. If the protein is able to upregulate transcription of
the target gene, the cells will express luciferase. If the protein downregulates transcription,
the cells will express less luciferase than normal. The scientist examines
the expression of luciferase about 2–3 days after the initial cell transfection. The
cells are lysed, and the cell contents are placed in a reaction tube. If the proper substrate
is added, luciferase will catalyze a reaction that produces light. This light is
detected with a luminometer, a device that precisely quantifies how much light is
produced in each reaction tube. The amount of light produced provides a quantitative
measure of the effect of the protein on expression of the target gene.
Due to variability in conditions within each well in a cell culture plate, a
proper luciferase assay experiment should repeat each condition at least in triplicate.
The scientist should compare luciferase expression between conditions
in which (1) no protein is present, (2) protein is present, and (3) the regulatory
sequence regulating luciferase expression is mutated so that the protein
is no longer able to bind. If a scientist knows enough about the structure of a
protein such that it is possible to mutate the protein’s DNA-binding domain or
enhance the ability of the protein to affect transcription, such conditions would
also strengthen the conclusions of a luciferase assay experiment.
CONCLUSION
The purpose of this chapter was to survey common methods used to investigate
protein expression and signaling. Although these methods are numerous,
they essentially fall into four categories: (1) determining whether a protein is
expressed in a particular population of cells and how much; (2) determining
whether a protein interacts with other proteins; (3) determining the types of
PTMs a protein can receive and which proteins cause these modifications; and
(4) determining whether a protein is capable of interacting with DNA and/or
regulating gene expression. Anyone outside the fields of biochemistry and cell
signaling should not be intimidated by the huge variety of techniques. Focusing
on the experimental questions rather than the methods can clarify the results and
conclusions of an intracellular signaling study.
SUGGESTED READING AND REFERENCES
Books
Berg, J. M., Tymoczko, J. L., & Stryer, L. (2010). Biochemistry (7th ed.). NewYork: W. H. Freeman.
Fields, R. D. (2008). Beyond the synapse: Cell-cell signaling in synaptic plasticity. Cambridge:
Cambridge University Press.
Moss, T., & Leblanc, B. (2009). Protein interactions: Principles and protocols (3rd ed.).
New York: Humana Press.
Wilson, K., & Walker, J. M. (2010). Principles and techniques of biochemistry and molecular
biology (7th ed.). New York: Cambridge University Press.
Biochemical Assays and Intracellular Signaling Chapter | 15  343
Review Articles
Araujo, S. J., & Tear, G. (2003). Axon guidance mechanisms and molecules: lessons from invertebrates.
Nature Review Neuroscience, 4, 910–922.
Grant, S. G. N., & Blackstock, W. P. (2001). Proteomics in neuroscience: from protein to network.
Journal of Neuroscience, 21(21), 8315–8318.
Greer, P. L., & Greenberg, M. E. (2008). From synapse to nucleus: calcium-dependent gene transcription
in the control of synapse development and function. Neuron, 59, 846–860.
Guan, K. L., & Rao, Y. (2003). Signalling mechanisms mediating neuronal responses to guidance
cues. Nature Review Neuroscience, 4, 941–956.
Tronson, N. C., & Taylor, J. R. (2007). Molecular mechanisms of memory reconsolidation. Nature
Review Neuroscience, 8, 262–275.
Primary Research Articles—Interesting Examples from the Literature
Cowan, C. W., Shao, Y. R., Sahin, M., Shamah, S. M., Lin, M. Z., Greer, P. L., et al. (2005). Vav
family GEFs link activated Ephs to endocytosis and axon guidance. Neuron, 46, 205–217.
Flavell, S. W., Cowan, C. W., Kim, T. K., Greer, P. L., Lin, Y., Paradis, S., et al. (2006). Activitydependent
regulation of MEF2 transcription factors suppresses excitatory synapse number.
Science, 311, 1008–1012.
Kim, M. S., Pak, Y. K., Jang, P. G., Namkoong, C., Choi, Y. S., Won, J. C., et al. (2006). Role of
hypothalamic Foxo1 in the regulation of food intake and energy homeostasis. Nature Neuroscience,
9, 901–906.
Kondo, M., Takei, Y., & Hirokawa, N. (2012). Motor protein KIF1A is essential for hippocampal
synaptogenesis and learning enhancement in an enriched environment. Neuron, 73, 743–757.
Koyano, F., Okatsu, K., Kosako, H., Tamura, Y., Go, E., et al. (2014). Ubiquitin is phosphorylated
by PINK1 to activate parkin. Nature, 510, 162–166.
Soden, M. E., Jones, G. L., Sanford, C. A., Chung, A. S., Guler, A. D., Chavkin, C., et al. (2013).
Disruption of dopamine neuron activity pattern regulation through selective expression of
a human KCNN3 mutation. Neuron, 80, 997–1009.
Visel, A., Blow, M. J., Li, Z., Zhang, T., Akiyama, J. A., Holt, A., et al. (2009). ChIP-seq accurately
predicts tissue-specific activity of enhancers. Nature, 457, 854–858.
Protocols
Aparicio, O., Geisberg, J. V., Sekinger, E., Yang, A., Moqtaderi, Z., & Struhl, K. (2005).
Chromatin immunoprecipitation for determining the association of proteins with specific
genomic sequences in vivo. Current Protocols in Molecular Biology (Chapter 21), Unit 21.3,
http://dx.doi.org/10.1002/0471142727.mb2103s69.
Golemis, E. A., Serebriiskii, I., Gyuris, J., & Brent, R. (2011). Interaction trap/two-hybrid system
to identify interacting proteins. Current Protocols in Neuroscience (Chapter 4), Unit 4.4,
http://dx.doi.org/10.1002/0471142301.ns0404s55.
Harlow, E., & Lane, D. (1999). Using antibodies: A laboratory manual. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press.
Pandey, A., Andersen, J. S., & Mann, M. (2000). Use of mass spectrometry to study signaling pathways.
Science’s STKE, 2000, PL1.
Walker, J. M. (2002). The protein protocols Handbook (2nd ed.). Totowa, NJ: Humana Press.
Websites
Resource for information on protein methods – expression, purification, antibody production:
http://www.piercenet.com/method/protein-methods-library
345
Glossary
Ablation  Permanent destruction of a cell or tissue, creation of a lesion.
Absolute recovery  In microdialysis, the total amount of substance collected in the
dialysate over a set amount of time.
Action potential  A rapid, all-or-none, transient depolarization of the cell membrane
of a neuron.
Acute culture  A primary cell or tissue culture used for short-term experiments,
usually lasting less than a day. For example, a slice culture for patch clamping
experiments.
Adeno-associated virus (AAV)  A type of virus used as a DNA delivery tool that
infects dividing and postmitotic cells. The DNA integrates into the nucleus,
permitting long-term gene expression.
Adenovirus  A type of virus used as a DNA delivery tool that infects dividing and
postmitotic cells. The DNA does not integrate into the genome, so expression is
often transient, lasting weeks or months.
Aequorin  A chemiluminescent protein derived from the jellyfish Aequorea victoria
that emits blue light when bound to calcium. This protein has been useful for
imaging calcium activity in living cells.
Affinity chromatography  A form of chromatography that uses small molecules
fixed to a porous column to bind to proteins with a high affinity, such as a substrate
binding to an enzyme.
Agonist  A compound that can bind and activate an endogenous receptor, thus
mimicking an endogenous ligand.
Allatostatin receptor  A ligand-gated receptor found in insects. In the presence of
the insect hormone allatostatin, the receptor causes hyperpolarization of cells. Thus,
this receptor can be used as a genetically encoded tool to inhibit neural activity.
Amino acid  A category of molecule consisting of amine and carboxyl functional groups,
as well as a variable side chain. Used as the monomeric building blocks of proteins.
Amperometry  A form of voltammetry that uses an electrode to detect neurochemicals
in living tissue. Unlike in other voltammetry techniques, the electrode is held at a
specific, constant voltage.
Amplifier  A device used in electrophysiology recordings to amplify the relatively
weak electrical signals derived from a recording electrode.
Anhedonia  The loss of desire to pursue an activity that is normally enjoyable.
346 Glossary
Anisotropic  Describes diffusion of substances that is not equal in all directions
but instead tends to move randomly along a single axis. The opposite of
isotropic.
Antagonist  A compound that can bind to a receptor but does not cause activation.
Therefore, it prevents the biological activity of an endogenous ligand.
Anterograde tracer  A chemical probe that labels efferent axon tracts from the cell
body to the presynaptic terminals.
Antibody  Specialized protein used by an organism’s immune system to detect and
bind to foreign proteins. Used by neuroscientists to identify or purify proteins of
interest.
Antigen  A foreign substance recognized by an antibody.
Aptamer  Engineered nucleic acid or peptide-based molecule that binds to a specific
target molecule. Used as an alternative to antibodies.
Archaerhodopsin (Arch)  A genetically encoded transmembrane pump that
hyperpolarizes neurons upon stimulation with yellow light.
ArcLight  A genetically encoded voltage indicator.
Array tomography  A technique that combines immunohistochemistry techniques
with ultrathin, serial sections. This technique improves spatial resolution of
traditional immunohistochemistry experiments and allows scientists to create
highly detailed three-dimensional images of protein expression.
Artificial chromosome  A large DNA molecule that contains the telomeric,
centromeric, and replication origin sequences necessary for replication and
preservation in yeast, bacteria, or phage. Useful to neuroscientists as a vector
for cloning relatively large DNA fragments (50–350 kb).
Ataxia  A neurological condition exhibited as a gross lack of coordination of muscle
movements.
Ataxin  A genetically encoded toxin that can be used to ablate specific neurons of
interest.
Autoclave  A large device used to sterilize tools, glassware, and reagents.
Autofluorescence  The natural ability of some structures to fluoresce, even without
the addition of exogenous fluorescent tags or dyes.
Autoradiography  A technique that uses the pattern of decay emissions produced
from a radioactive substance to form an image on an X-ray film. Used in
neuroscience to detect radioactive probes.
Avidin  A small molecule with an extremely high affinity for another molecule called
Biotin.
Axial  An imaginary plane that divides the brain and body into superior and inferior
sections (top to bottom).
BAC  See Bacterial artificial chromosome.
BAC construct/transgenic  A recombinant DNA or recombineering construct that
uses a bacterial artificial chromosome as the vector. Useful for creating transgenic
animals with large, endogenous sequences of DNA.
Glossary  347
Bacterial artificial chromosome (BAC)  A large (50–350 kb) DNA vector capable
of replicating in bacteria. Useful for manipulating large sequences of DNA,
such as in the generation of transgenic or knockout constructs.
Bacteriophage  Any one of a number of viruses that infect bacteria. Sometimes simply
referred to as phage.
Barnes maze  A maze used to test spatial learning and memory in rodents. The
animal must learn the location of a drop box on a large circular table using spatial
cues placed around the maze.
Base pair  Describes a single nucleotide–nucleotide complementary match between
the two strands of DNA. These base pairs are always hydrogen bonds between
adenine–thymine or cytosine–guanine bases.
Basic Local Alignment Search Tool (BLAST)  A free, public access database used
to identify and compare DNA and protein sequences.
Basophilic stain  A histological stain used to visualize cell bodies.
Between-subjects study  A study that compares two different groups of human subjects
or animals. For instance, in functional imaging experiments, a between-subjects study
may test the differences of old versus young, or male versus female participants.
Bimolecular fluorescence complementation (BiFC)  A technique used to visualize
the interaction between two proteins. Each protein is fused to half of a fluorescent
protein. When the proteins interact, the two halves of the fluorescent protein can
fold together to emit light and report the interaction.
Binary expression system  An approach that uses two or more constructs to express
a transgene in cells. Used to refine the spatial or temporal properties of transgene
expression.
Biolistics  A method of DNA delivery that uses a gene gun to physically shoot DNAcoated
bullets into cells.
Biotin  A small molecule with an extremely high affinity for another molecule called
avidin. Used to tag molecular probes so that future histochemical processing with
avidin-bound probes can enhance a signal.
BLAST  See Basic Local Alignment Search Tool.
Blastocyst  An early embryonic structure that follows the fertilization of an egg.
Bleed-through  A phenomenon in fluorescent microscopy that occurs when the signal
of one fluorophore appears when a scientist uses a filter designed to detect the
signal of a separate fluorophore.
Blocked design  A strategy for presenting stimuli to human subjects in functional
imaging experiments. Stimuli of the same category are presented together in
blocks. An alternative to an event-related design.
Blood oxygen level-dependent (BOLD) effect  The phenomenon in which the brain
microvasculature increases the flow of oxygen-rich blood to neurons following an
increase of neural activity.
Blunt ends  Ends of fragments of DNA cut with restriction enzymes that result in no
5’ or 3’ overhangs; both strands of DNA terminate at the same complementary base
pair. Compare with sticky ends.
348 Glossary
BOLD effect  See Blood Oxygen Level-Dependent effect.
Brain atlas  A tool that depicts brain anatomy in three-dimensional coordinates.
BrdU  Bromodeoxyuridine, a synthetic analog of the DNA base thymidine, commonly
used to indicate cell proliferation and neurogenesis.
Bregma  A landmark on the surface of the skull, defined as the intersection between
the sagittal and coronal sutures.
Brightfield microscopy  A type of light microscopy in which white light passes
directly through or is reflected off a specimen.
Brodmann’s areas  Divisions of the cortex defined by the cytoarchitecture of the
cortical layers. Developed by Korbinian Brodmann in the early twentieth century
and still used today.
Calcium phosphate transfection  A method of delivering DNA into cells by using
chemical reactions that form calcium phosphate precipitates.
Canine Adenovirus (CAV)  A viral vector with retrograde transport properties.
Cannula  A narrow, cylindrical tube that can be implanted on the surface of the skull
for long-term access to deep brain structures in a living animal.
Capsaicin receptor  A genetically encoded, ligand-gated ion channel that depolarizes
neurons in response to the capsaicin molecule. Can be used by neuroscientists to
stimulate genetically defined populations of neurons.
Cas9  See CRISPR/Cas9.
Case study  A natural event that demonstrates something important about the nervous
system.
cDNA  See Complementary DNA.
cDNA library  A collection of hundreds or thousands of DNA plasmids, each containing
a unique cDNA molecule. Used to collect and store DNA sequences that
were actively expressed in a sample prior to exposure with the reverse transcriptase
enzyme.
cFos  See Fos.
Cell-attached recording  A patch-clamp technique in which a glass micropipette
forms a gigaseal with the membrane of a cell, but the membrane is not punctured.
Cell culture  An in vitro approach in which a scientist grows and maintains cells
under carefully controlled conditions outside of a living animal.
Cell fractionation  A process in which a scientist centrifuges a sample multiple
times at various speeds to divide the sample into different cellular components and
organelles.
Cell signaling  See Intracellular signaling.
Central dogma of molecular biology  The overarching model of information flow in
cells, briefly stated as “DNA codes for molecules of RNA, and molecules of RNA
code for proteins.”
Cerebral angiogram  A structural brain imaging technique that uses X-rays and
radio-opaque dyes to produce images of the brain’s vasculature.
Glossary  349
Channelrhodopsin-2 (ChR2)  A genetically encoded cation channel capable of
depolarizing a neuron with millisecond-timescale resolution in response to
stimulation with blue light.
Chemical gene delivery  The process by which a scientist uses a chemical reaction to
deliver DNA into cells.
Chemical mutagenesis  The use of chemical agents, such as EMS or ENU, to
mutagenize hundreds or thousands of eggs/larvae for the purpose of performing a
forward genetic screen.
Chemosensory jump assay  A behavioral test in flies that exploits the tendency of
flies to exhibit a startle response and jump when encountering a novel odor.
Chimeric mice  Mice composed of patches of tissues from different genetic sources.
An intermediate step in the procedure to create knockout or knockin mice.
ChIP on chip assay  An assay in which chromatin immunoprecipitation is followed
up with a microarray to determine the identity of all genetic sequences bound to a
particular protein in vitro or in vivo.
ChR2  See Channelrhodopsin-2.
Chromatin immunoprecipitation (ChIP)  A technique used to determine whether a
sequence of DNA physically interacts with a specific protein, usually a transcription
factor or histone protein.
Chromatography  A category of biochemistry techniques used to separate proteins
on a column based on their size, charge, substrates, or other properties.
Chromogenic label  A label on a probe that allows a scientist to perform a simple
biochemical reaction that produces a colored byproduct. This byproduct can be
visualized using simple light microscopy.
Chronic mild stress  A series of mildly stressful events (periods of constant light,
tilting an animal’s cage, wetting the animal’s bedding, etc.) that is used to create
rodent models of depression and anxiety.
Circadian rhythms  The regular, roughly 24-h cycle of stereotyped biochemical,
physiological, and behavioral processes.
CLARITY  A method of making extracted tissue (such as a brain or other organ)
transparent to visualize structures in an intact system.
Classical conditioning  A paradigm of animal learning in which a scientist couples
an initially neutral stimulus, such as a tone or light, with a salient stimulus, such as
food or an electric shock. An animal eventually associates the neutral stimulus with
the salient stimulus.
Cloning vector  A DNA vector capable of storing DNA sequences for recombinant
DNA techniques but lacking the necessary noncoding sequences that allow the
DNA to be expressed in a host cell.
Co-culture system  An in vitro culture system in which two different cultured cell
types or explants are cultured together in the same chamber, allowing a scientist to
examine the effect of one type of cell or tissue on another.
Co-immunoprecipitation (Co-Ip)  A method that determines whether a protein
interacts with another protein.
350 Glossary
Codon  A series of three nucleotides that code for a specific amino acid during translation.
Cognitive neuroscience  The field of neuroscience dedicated to elucidating the neural
basis of thought and perception.
Cohesive ends  See Sticky ends.
Colorimetric label  See Chromogenic label.
Competent cell  A bacterial cell transiently capable of receiving DNA sequences
through its cell wall.
Complementary DNA (cDNA)  A fragment of DNA produced from an RNA
template following a reaction with the enzyme reverse transcriptase.
Complementary strands  Refers to two strands of DNA that have complementary
base pairs, allowing the two strands to hybridize into a double-stranded molecule.
Complementation test  A test used to determine if a mutant animal with an abnormal
phenotype has a mutation in the same gene as a different mutant animal with the
same abnormal phenotype.
Compound microscope  A microscope in which two or more lenses are used in
concert to enhance the magnification power.
Computerized tomography (CT)—also called computerized axial tomography
(CAT)  A structural imaging technique in which several X-rays are taken from
multiple orientations around a single plane to produce an image of the brain.
Condenser  The part of a microscope that focuses light from a light source onto a specimen.
Conditional knockout  An animal in which a gene is knocked out of the genome using
gene targeting measures, but only in a specific cell type, and/or at a specific time.
Conditioned place preference/aversion  Rodent behavioral assay demonstrating an
association between a specific environment and a positively reinforcing stimulus
(preference) or negatively reinforcing stimulus (aversion).
Conditioned stimulus (CS)  In classical conditioning, the neutral stimulus that
eventually becomes associated with a salient, unconditioned stimulus (US).
Confluence  The relative amount of space that a growing population of cultured cells
takes up on the surface of a cell culture flask/plate.
Confocal microscope  A specialized fluorescent microscope that can produce clear
images of structures within specimens by selectively exciting and collecting
emitted light from single points on the specimen.
Construct  A recombinant DNA sequence capable of coding for a functional protein
when properly expressed in host cells.
Construct validity  Refers to a state in which an animal model and human model
have the same underlying genetic or cellular mechanism that may result in a certain
behavior, such as a transgenic mouse engineered to have the same mutation that
causes Alzheimer’s disease in humans.
Contextual fear conditioning  A form of classical conditioning in which an animal
associates a neutral stimulus with an aversive stimulus, such as a foot shock, and
the animal subsequently displays a fearful behavior, such as freezing, when placed
in the training chamber, even in the absence of the aversive stimulus.
Glossary  351
Continuous reinforcement  In operant conditioning, a paradigm in which an animal
performs a single response to receive a rewarding stimulus. Compare with fixed
ratio reinforcement or progressive ratio reinforcement.
Coomassie blue  A blue dye used to stain proteins in SDS-PAGE gels.
Coronal plane  A neuroanatomical plane that divides the brain into anterior and
posterior sections (from ear to ear).
Cosmid  A large (30–50 kb) DNA plasmid capable of replicating in bacteria. Useful
for engineering very large DNA constructs.
Countercurrent apparatus  A device used to fractionate a population of flies based
on their behavioral responses to a sensory stimulus.
Cranial window  An implant or thinning of a rodent’s skull that allows for direct
observation of fluorescent neurons in vivo.
Craniotomy  A procedure used to remove a small section of the skull during a
surgical procedure.
Cre/lox system  A binary expression system used to control the spatial expression of
a transgene.
Cre recombinase  A protein derived from bacteriophage that recognizes lox sites.
Cre excises any DNA sequences between two lox sites oriented in the same
direction and inverts any DNA sequences between two lox sites oriented in
inverted directions.
CRISPR/Cas9  A nuclease system used to introduce double-strand breaks at precise
genomic locations.
Cross-linking fixative  A chemical fixative that creates covalent chemical bonds
between proteins in tissue and cells.
Cross-talk  See Bleed-through.
Cryostat  A device used to freeze and cut tissue specimens into 5–50 μm sections.
Cued fear conditioning  A form of classical conditioning in which an animal associates
a neutral stimulus with an aversive stimulus, such as a foot shock, and then
displays freezing behaviors in the presence of the formerly neutral stimulus, even in
the absence of foot shock.
Current (I)  The rate of flow of electrical charge over time.
Current clamp  An electrophysiological technique in which a scientist injects current
into a cell and measures the change of voltage over time.
Cyclic voltammogram  The end result of a fast-scan cyclic voltammetry experiment
that plots the amount of measured current versus the applied voltage. These
data are useful for detecting the presence of various neurochemicals in neural
tissue.
Cyclotron  A large machine that produces positron-emitting isotopes, such as the kind
used in positron emission tomography (PET) experiments.
Cytokine  Extracellular signaling molecule used in intercellular communication.
DAPI  A fluorescent stain used to label nuclei acids (and therefore also cell nuclei).
352 Glossary
Darkfield microscopy  A form of light microscopy that uses an oblique light source
to illuminate a specimen from the side so that only scattered light enters the
objective lens. This causes some organelles to stand out from a dark background.
Defensive marble burying assay  A behavioral paradigm used to assay anxiety in
a rodent. Marbles are placed in an animal’s cage, and a scientist measures how
many marbles an animal buries in the cage bedding over a fixed amount of time.
Animals that are more anxious typically bury more marbles than nonanxious
animals.
Dehydrating fixative  A histological fixative that disrupts lipids and reduces the
solubility of protein molecules, precipitating them out of the cytoplasmic and
extracellular solutions.
Dependent variable  The variable in an experiment that is measured and dependent
upon the value of an independent variable.
Depolarization  A decrease in the absolute value of a cell’s membrane potential.
Descriptive science  The act of observing properties of the nervous system without
experimental manipulation.
Designer receptor exclusively activated by a designer drug (DREADD)  An
artificially engineered membrane-bound receptor capable of stimulating or
inhibiting neural activity upon binding to a normally inert ligand.
Dicer  An enzyme that recognizes double-stranded RNA and cleaves it into fragments
about 23 bp long (siRNA). This siRNA-dicer complex then recruits additional
cellular proteins to form an RNA-induced silencing complex (RISC), which
mediates the phenomenon of RNA interference (RNAi).
Differential interference contrast (DIC) microscopy  A type of brightfield microscopy
that uses optical modifications within the microscope to exaggerate changes in
the light-scattering properties of cellular structures, producing a three-dimensional,
textured appearance.
Diffuse optical imaging (DOI)  A type of noninvasive optical imaging method in
which light is reflected off neural structures through the scalp.
Diffusion  The passive transport of molecules from a region of higher concentration to
a region of lower concentration caused by random molecular interaction.
Diffusion MRI  A technique used to image fiber tracts in the brain by measuring the
anisotropic diffusion of water molecules along fiber bundles.
Diffusion tensor imaging (DTI)  A diffusion MRI method that measures the
anisotropic diffusion of water molecules along fiber tracts in the brain to image
connections between different brain regions.
Digoxigenin  A steroid molecule frequently used to tag molecular probes in histology
experiments.
Diphtheria toxin receptor  A genetically encoded receptor that will kill an expressing
cell in the presence of the diphtheria toxin, which is normally harmless to
rodent neurons.
Dipstick assay  See Olfactory avoidance assay.
Glossary  353
Direct IHC  A category of immunohistochemistry experiments in which the primary
antibody is directly conjugated to a fluorescent or chromogenic label. This typically
produces a weaker signal than indirect IHC.
Dissecting microscope  See Stereomicroscope.
Dissociated cell culture  A primary cell culture method in which tissue removed
directly from an animal is separated into individual cells.
DNA ladder  A sample of various DNA fragments of known sizes, run on an agarose gel
next to experimental DNA samples to determine the size of fragments in the samples.
DNA library  A comprehensive collection of hundreds or thousands of cloned DNA
fragments from a biological sample, stored in expression vectors or artificial chro-
mosomes.
DNA vector  A nucleic acid storage system that can hold an isolated DNA fragment
of interest.
Dominant negative  The twisted ladder shape of a double-stranded DNA molecule.
Double helix  A mutated protein that interferes with the function of the wild-type
protein.
Double inverse orientation (DIO) construct  A recombinant DNA construct that allows
Cre-dependent expression of a transgene due to the presence of two pairs of lox sites.
Doxycycline  A drug that binds to the tetracycline transactivator protein (tTA) or
reversed transactivator protein (rtTA) in Tet-off/Tet-on transgenic animals. This
drug allows scientists to gain inducible control of the Tet-off/Tet-on system,
allowing temporal control of transgene activity.
DREADD  See Designer receptor exclusively activated by a designer drug.
Dynamic clamp  Electrophysiological method that uses computer simulation to
model artificial membrane or synaptic conductances in live cells.
Electrode  A metal or glass probe used to measure neural activity from biological tissue.
EEG  See Electroencephalography.
Electroencephalography (EEG)  A noninvasive technique used to measure the gross
electrical activity of the brain.
Electrolytic lesion  A physical lesion in the nervous system caused by injection of current.
Electromyogram  A technique used to record the electrical potential generated by
muscle cells and, therefore, to record motor activity.
Electron microscope (EM)  A form of microscopy that uses a beam of electrons
rather than a beam of photons to image a specimen, greatly enhancing the resolving
power.
Electron microscope tomography  See Electron tomography.
Electron tomography (ET)  A procedure used to collect and combine several transmission
electron microscope images from the same specimen, allowing scientists to
image the three-dimensional ultrastructure of organelles and macromolecules.
Electrophoretic mobility shift assay (EMSA)  A technique used to determine if a
protein is capable of directly binding to a short sequence of DNA.
354 Glossary
Electrophysiology  The branch of neuroscience that explores the electrical activity
of living neurons and investigates the molecular and cellular processes that govern
their signaling.
Electroporation  The process of using an electric field to deliver DNA constructs into
cells.
Electrospray ionization (ESI)  A technique used to cause a sample to enter a gaseous
phase for mass spectrometry experiments.
Elevated plus maze  A behavioral assay used to assay anxiety in rodents.
Embedding  The process of surrounding a brain or tissue section with a substance
that infiltrates and forms a hard shell around the tissue.
Embryonic lethal  A phenotype caused by a transgenic or knockout construct that
causes the death of an animal during gestation.
Embryonic stem (ES) cell  A pluripotent cell capable of giving rise to any tissue in
an organism.
Emission filter  A filter on a fluorescent microscope that blocks extraneous wavelengths
of light, including the light used to illuminate the specimen, but allows
emitted light wavelengths to pass through to a detector.
Ensembl  A public genomic database, useful for looking up genomic sequences and
genetic information for a variety of organisms.
Enzyme-linked immunosorbent assay (ELISA)  A technique used to quantify the
amount of protein in a sample.
Enzymatic histochemistry  A histochemical procedure that utilizes an endogenous
protein’s activity to generate a visible signal.
Epifluorescent microscopy  Standard fluorescent microscopy. Specimens labeled
with fluorescent probes are illuminated by light of the excitation wavelength. The
specimen is then viewed using a second filter that is opaque to the excitation wavelength
but transmits the longer wavelength of the emitted light.
Epitope  The specific region of an antigen to which an antibody binds.
ER/tamoxifen system  A system used in genetic engineering experiments to allow
inducible gene activity/suppression at specific time points. The estrogen receptor
(ER) is sequestered in the cytoplasm. In the presence of tamoxifen, ER translocates
to the nucleus. In genetic engineering experiments, ER can be fused to other useful
proteins, such as Cre recombinase, to allow for inducible activation of Cre activity
when a scientist adds tamoxifen.
Estrogen receptor (ER)  An endogenous receptor that resides in the cytoplasm until
binding with its ligand, at which point it translocates to the nucleus.
Ethology  The study of natural animal behaviors.
Event-related design  A strategy for presenting stimuli to human subjects in functional
imaging experiments in which stimuli are presented as isolated, individual events of
short duration. An alternative to a blocked design.
Event-related field  A distinct, stereotyped waveform in magnetoencephalography
(MEG) that corresponds to a specific sensory, cognitive, or motor event.
Glossary  355
Event-related potential  A distinct, stereotyped waveform in electroencephalography
(EEG) that corresponds to a specific sensory, cognitive, or motor event.
Excitation filter  A filter on a fluorescent microscope that allows only light in a specific
range of wavelengths to pass through to an objective to illuminate a specimen.
Excitatory postsynaptic potential (EPSP)  A localized potential change in a small
region of a neuron that causes the membrane potential to depolarize, bringing the
membrane potential closer to the threshold to generate an action potential.
Exon  A sequence of an RNA molecule that remains in the final mRNA sequence
after splicing occurs.
Explant culture  A primary cell culture method in which intact chunks of tissue are
removed from a living organism and cultured in an in vitro environment.
Expression vector  A DNA vector containing all of the necessary noncoding sequences
necessary for a host cell to express a coding DNA sequence as a protein.
Extracellular recording  An electrophysiological recording in which the recording
electrode is placed outside a neuron in the extracellular environment.
Eye coil  A wire loop implanted around the outer circumference of a monkey’s eye.
Used to ensure that the monkey correctly fixes its gaze during a visual task.
Face validity  A concept that establishes the validity of an animal model by demonstrating
that the animal’s behavior is similar to the analogous human behavior.
Fast-scan cyclic voltammetry  A technique used to measure neurochemicals in
neural tissue.
Feeding acceptance assay  A Drosophila assay that examines taste preferences.
Slightly starved flies are offered a choice of appetitive, aversive, or neutral stimuli,
each dyed a different color. The amount of each stimulus can be scored by
examining the color of the fly’s stomach.
Fiber stain  A histological stain used to mark the presence of fiber tracts in the
nervous system.
Fixation  The process of using chemical methods to preserve, stabilize, and strengthen
a biological specimen for subsequent histological procedures and microscopic
analysis.
Fixed ratio reinforcement  In operant conditioning, a paradigm in which an animal
must perform a set number of responses to receive a rewarding stimulus. Compare
with continuous reinforcement or progressive ratio reinforcement.
Five choice serial reaction time task  A rodent behavioral assay used to study
attention and impulsivity.
Flat skull position  The placement of an animal on a stereotaxic frame such that the
top of the skull is perfectly level, at the same height along the rostral–­caudal axis.
Flex construct  A recombinant DNA construct that allows Cre-dependent expression
of a transgene due to the presence of two pairs of lox sites.
Flight simulators  Specialized chambers that suspend a fly from a thin pin and
project visual stimuli on a screen, depicting the external, visual environment. Used
to determine the effect of visual stimuli on flying behavior.
356 Glossary
Flippase recognition target (Frt)  A 34 bp sequence recognized by the flippase
recombinase (Flp) enzyme.
Flippase recombinase (Flp)  A protein that recognizes flippase recognition target (Frt)
sites. Flp excises any DNA sequences between Frt sites oriented in the same direction,
and inverts any DNA sequences between two Frt sites oriented in inverted directions.
Floxed  Term used to describe any DNA sequence flanked by two lox sites.
Flp/Frt system  A binary expression system used to control the spatial expression of
a transgene.
Fluorescence recovery after photobleaching (FRAP)  A protein visualization
technique in which a scientist uses strong laser illumination to bleach fluorescently
tagged proteins in a particular region of a cell and then measures the time course of
fluorescence that returns to the bleached region.
Fluorescence resonance energy transfer (FRET)  A technique used to visualize if
and when two proteins interact.
Fluorescent in situ hybridization (FISH)  A technique used to examine the location
of specific DNA sequences within chromosomes, commonly used to detect chromosomal
abnormalities.
Fluorodeoxyglucose (FDG)  A radioactive form of glucose used to image functional
brain activity in positron emission tomography (PET) experiments.
Fluorophore  A molecule that has the ability to absorb light at a specific wavelength
and then emit light at a different, typically longer wavelength.
FM dyes  Lipophilic dyes that fluoresce when bound to a membrane. They are
particularly useful for reporting the exocytosis and endocytosis events that occur
during synaptic vesicle recycling and can stain nerve terminals in an activitydependent
manner.
Footprint pattern assay  A behavioral assay used to examine a rodent’s motor
coordination and balance by observing its footprint patterns.
Forced swim test  A behavioral experiment used to assay depression by placing
an animal in a narrow chamber of water and measuring how long it takes for the
animal to stop trying to escape.
Formalin assay  An assay used to examine an animal’s sensitivity to noxious chemical
stimuli by observing its response to a small injection of the noxious chemical formalin.
Förster resonance energy transfer (FRET)  See Fluorescence resonance energy
transfer.
Forward genetics  A method used to identify genes that contribute to a phenotype.
Scientists generate random mutations in eggs/larvae and then examine the phenotypes
of the offspring. Contrast with Reverse genetics.
Fos  A transcription factor used as an immediate early gene, i.e., an indirect marker of
neural activity.
Founder  Individuals in the first generation of a transgenic or genetically targeted animal.
Fractional intensity change  The change in fluorescent signal during calcium or voltage
imaging compared to baseline fluorescence. Denoted as ΔF/F or ΔI/I.
Glossary  357
Freezing microtome  A microtome with a cooling unit that allows a tissue sample to
be frozen during sectioning.
Functional brain imaging  Techniques used to image neural activity in the central
nervous system.
Functional connectivity  In whole-brain imaging, brain regions that demonstrate
temporally correlated fluctuations in BOLD activity.
Functional magnetic resonance imaging (fMRI)  A technology that uses the
principles of magnetic resonance imaging (MRI) to indirectly measure neural
activity in the nervous system over time.
Gain of function experiment  An experiment in which an independent variable
(e.g. neural activity or gene expression) is increased to determine its effect on a
dependent variable.
Gal4  A transcription factor derived from yeast that binds to a specialized upstream
activation sequence (UAS) to activate the transcription of a gene.
Gal4/UAS system  A binary expression system used to control the spatial expression
of a transgene. Widely used with invertebrate and zebrafish model organisms.
Gal80  A protein derived from yeast that inhibits Gal4 transcription factors.
GCaMP  A genetically encoded calcium indicator.
Gel electrophoresis  The process of using an electrical field to force DNA through an
agarose gel. Used to isolate, identify, and characterize the properties of DNA fragments.
Gel-filtration chromatography  A chromatography technique that separates proteins
on the basis of their size.
Geller–Seifter conflict test  A behavioral paradigm that assays anxiety in rodents. A
rodent is trained to press a lever to receive a food reward. In subsequent experiments,
the lever is paired with an aversive foot shock. Anxious animals tend to
press the lever significantly less to receive food than nonanxious animals.
Gel shift assay  See Electrophoretic mobility shift assay.
Gene  The fundamental molecular unit of heredity, a segment of DNA that codes for a
functional protein.
Gene gun  A device used in biolistic experiments to physically shoot DNA-coated
bullets onto tissue specimens.
Gene targeting  The process of incorporating a DNA construct into a specific locus in
an animal’s genome.
Genetically encoded calcium indicator (GECI)  A genetically encoded transgene
used to indirectly measure neural activity by imaging calcium dynamics.
Genetically encoded voltage indicator (GEVI)  A genetically encoded transgene
used to visually measure membrane potential.
Genome  The complete set of genes or genetic material present in a cell or organism.
Genomic DNA library  A collection of thousands of plasmids that each contain
fragments of an animal’s genome.
Genotype  The genetic constitution of an animal.
358 Glossary
Germ cells  Cells that pass on genetic information to an organism’s offspring (sperm
in males and eggs in females).
GFP  See Green fluorescent protein.
Go/no-go task  A form of operant conditioning in which a rodent is trained to produce
a response in the presence of a “go” cue yet withhold a response in the presence
of a separate “no-go” cue. Used to test attention and impulsivity.
Golgi stain  A classic histological stain used to label a small subset of neurons in their
entirety.
Graded potential  See Localized potential.
Green fluorescent protein (GFP)  A protein derived from jellyfish, capable of
absorbing wavelengths of blue light and emitting wavelengths of green light. Ubiquitously
used in the biosciences as a reporter protein.
Growth factors  Proteins produced by certain cells as cell communication molecules
that stimulate the growth and differentiation of other cells.
Guide RNA  An RNA molecule that serves as a template for genomic targeting in the
CRISPR/Cas9 system.
Gyri  Ridges on the cerebral cortex (plural for “gyrus”).
Halorhodopsin (NpHR)  A genetically encoded transmembrane pump that
hyperpolarizes neurons upon stimulation with yellow light.
Hanging wire assay  A behavioral assay used to measure neuromuscular deficits in rodents.
Hargreaves assay  A behavioral assay used to measure pain in rodents by aiming a
high-intensity beam of light at the hind paw and measuring the time it takes for the
rodent to withdraw its paw.
Hemoglobin  The protein that transports oxygen throughout the bloodstream.
Herpes simplex virus  A neurotropic virus that can be used to deliver DNA to some
cell types and also serve as a multisynaptic tracer.
Heterologous expression systems  A dissociated cell culture system that can easily
be transfected with DNA.
HM3Dq  A DREADD transgene capable of stimulating neurons upon binding the
normally inert ligand clozapine-n-oxide (CNO).
HM4Di  A DREADD transgene capable of inhibiting neurons upon binding the
normally inert ligand clozapine-n-oxide (CNO).
Holding potential  The set voltage that is held constant in a voltage clamp experiment.
Homologous recombination  A natural phenomenon in which DNA regions with
strong sequence similarity can exchange genetic material.
Homology arms  Regions of homology that flank a gene targeting construct to exploit
the phenomenon of homologous recombination in the production of a knockout or
knockin mouse.
Homology-directed repair (HDR)  A method of inserting DNA into the genome
following nuclease-mediated double-strand break repair. A scientist introduces a DNA
construct with homology arms that have the same sequence as the broken DNA strands.
Glossary  359
Horizontal plane  A neuroanatomical plane that divides the brain into superior and
inferior sections such that the brain is complete from the anterior-posterior and leftto-right
planes.
Hot plate assay  A behavioral paradigm used to assay nociception in rodent models. A
rodent is enclosed on a heated plate and observed for licking and paw withdrawal.
Hyperpolarization  Any change in the membrane potential of a neuron that causes a
net increase in the absolute value of the membrane potential.
IACUC  See Institutional Animal Care and Use Committee.
I/V curve  A plot of the voltage across a cell membrane compared with the current
that flows through the ion channels of the membrane.
Ibotenic Acid  A pharmacological agent used to irreversibly ablate neurons when
directly injected into the nervous system.
Immediate early gene (IEG)  A gene that tends to be actively transcribed during
periods of high neural activity.
Immersion  The process of fixing small brains or even entire animals by immersing
them in fixative solutions.
Immortalized cell line  A cell line manipulated so that it will continually divide and
multiply.
Immunoblot  See Western blot.
Immunocytochemistry (ICC)  A histological technique that uses antibodies to stain
proteins in cells.
Immunoelectron microscopy  A combination of immunohistochemistry and
electron microscopy used to visualize protein expression within subcellular
compartments.
Immunofluorescence (IF)  A histological technique that uses antibodies to stain
proteins with fluorescent reagents.
Immunohistochemistry (IHC)  A histological technique that uses antibodies to stain
proteins in tissues.
Immunopanning  A technique used to purify certain cell types. A scientist coats the
bottom of a plate with antibodies that recognize cell surface markers on the outside
of specific cells. When heterogeneous populations of cells are added to the plate,
the scientist can purify the cells of interest by allowing the cells to bind to the
bottom of the plate and then wash off the undesired, unbound cells.
Immunoprecipitation (IP)  A protein purification method that uses antibodies to
purify specific proteins out of solution.
In silico screen  A method of identifying genes or proteins of interest by searching
public genome and bioinformatics databases.
In situ hybridization (ISH)  A histological method used to label the location of
mRNA transcripts.
In vitro  Any process that takes place in a controlled environment outside a living
organism, such as a cell culture dish or test tube.
360 Glossary
In vivo  Any process that takes place in a whole, living organism.
Independent variable  The experimental variable that is intentionally manipulated by
the researcher and is hypothesized to cause a change in the dependent variable.
Index of refraction  A measure of the relative speed of light in a specific medium,
such as air, water, or oils.
Indirect IHC  A method of performing an immunohistochemistry experiment in
which a primary antibody binds to an antigen and then a secondary antibody binds
to the primary antibody. The secondary antibody is conjugated to a fluorescent or
chromogenic tag that allows for subsequent visualization.
Induced pluripotent stem (iPS) cell  A somatic stem cell that has been manipulated
into reverting back to a pluripotent stem cell state, capable of giving rise to multiple
cell types.
Infection  The process of delivering DNA into cells using viruses.
Inhibitory postsynaptic potential (IPSP)  A localized potential change in a small
region of a neuron that causes the membrane potential to hyperpolarize, bringing
the membrane potential farther from the threshold to generate an action potential.
Inner cell mass  A group of cells within a blastocyst that give rise to all cells and tissues
in an organism.
Insertional mutagenesis  The process of causing genetic mutations by insertion of
mobile genetic elements called transposons into the genomes of offspring. These
transposons insert at random locations in the genome, occasionally disrupting an
endogenous gene.
Inside-out recording  A patch clamp recording method in which a tiny patch of the
membrane is isolated and oriented such that the intracellular surface is exposed to
the medium and the extracellular surface resides on the inside of the glass pipette.
Institutional Animal Care and Use Committee (IACUC)  An academic institution’s
internal review committee that approves and inspects all procedures using
vertebrate animals.
Institutional Review Board (IRB)  An academic institution’s internal review
committee that approves and inspects all procedures that use human subjects.
Intracellular recording  An electrophysiology recording technique in which an
electrode is gently placed inside a cell.
Intracellular signaling  The biochemical pathways that take place within a cell that
affect the cell’s physiology and metabolism.
Intracerebroventricular (i.c.v.) injection  The injection of a solution directly into
the ventricular system of the brain.
Intraperitoneal (i.p.) injection  The injection of a solution into the peritoneum (body
cavity) of an animal.
Intron  A sequence of an RNA molecule that is cut out of a final mRNA sequence
during RNA splicing.
Inverted microscope  A microscope in which the objective lens is placed beneath the
sample and the light source and condenser are placed above the sample.
Glossary  361
Ion-exchange chromatography  A chromatography technique that separates proteins
based on their charge.
Irradiation mutagenesis  The use of high-intensity UV light to mutagenize
hundreds or thousands of eggs/larvae for the purpose of performing a forward
genetic screen.
Isofluorane  A gaseous anesthetic agent used in mammals.
Isotropic  The diffusion of a substance in all directions within a medium. The
opposite of anisotropic.
Ketamine  A chemical anesthetic agent that acts by inhibiting NMDA and HCN1 ion
channels. Often used in concert with Xylazine.
Kinase assay  An assay used to determine if a protein kinase is capable of
phosphorylating a substrate protein or protein fragment.
Knockdown  The disruption of mRNA transcript expression, therefore causing a
decrease in functional protein products.
Knockin  The use of gene targeting procedures to add a functional gene or gene
sequence to a specific location in a genome.
Knockout  The use of gene targeting procedures to remove a functional DNA
sequence from the genome, thus knocking out a gene of interest.
LacZ  A bacterial gene that encodes the enzyme β-galactosidase. This gene is often
used as a reporter gene, as β-galactosidase can react with the substrate X-gal to
produce a dark blue byproduct.
Lambda  Landmark on the surface of the skull, defined as the intersection between
the lines of best fit through the sagittal and lambdoid sutures.
Learned helplessness  A paradigm used to create rodent models of depression. A
scientist exposes an animal to aversive stimuli at random intervals. Theoretically,
this treatment creates a condition in which the animal experiences a lack of control,
causing it to show symptoms of behavioral despair.
Lectins  Proteins that exhibit extremely high binding affinities for sugars, many of
which can be used as transsynaptic neural tracers.
Lentivirus  A retrovirus that can be used to deliver transgenes to both
dividing and postmitotic cells and integrate these sequences in the genome of
the host cell.
Lesion  A physical cut or destruction of biological tissue.
Linkage analysis  A strategy to map a gene’s location in the genome by identifying
the position of the novel gene in relation to the location of known genes.
Lipofection  A chemical transfection method that uses a lipid complex to deliver
DNA to cells.
Liposomes  Tiny vesicular structures composed of a sphere of phospholipids that have
the same composition as a plasma membrane.
Local field potential  An electrophysiological measurement of the sum of all
dendritic synaptic activity within a volume of tissue.
362 Glossary
Localized potential  A local change in membrane potential caused by the activity of
individual ion channels. These potentials include excitatory postsynaptic potentials
(EPSPs) and inhibitory postsynaptic potentials (IPSPs).
Loss of function experiment  An experiment in which an independent variable (e.g.
neural activity or gene expression) is decreased or lost to determine its effect on a
dependent variable.
Lox site  A 34 bp DNA sequence recognized by the Cre recombinase enzyme.
Luciferase  A genetically encoded bioluminescent protein cloned from various
species of fireflies.
Luciferase assay  A technique used to determine whether a protein is able to interact
with a genetic regulatory sequence and activate the transcription of a gene.
Magnetic resonance imaging (MRI)  A non-invasive structural imaging technique
that utilizes the magnetic properties of different tissues to produce highly detailed
structural images of the brain and body.
Magnetoencephalography (MEG)  A functional imaging technique that measures
changes in magnetic fields on the surface of the scalp produced by changes in
underlying patterns of neural electrical activity.
Magnification  A critical parameter in microscopy that measures how much larger the
sample appears compared to its actual size.
Mass spectrometry  A technique used to identify proteins and/or peptide sequences
by determining the mass to charge ratio of proteins, peptides, and their fragments.
Match to sample task  A rodent behavioral assay that uses operant conditioning to
test working memory.
Matrix-assisted laser desorption ionization (MALDI)  A mass spectrometry
method of vaporizing proteins into the gas phase by blasting them with a UV laser
so that their mass to charge ratio can be measured.
Meganuclease  A restriction enzyme that recognizes longer DNA sequences
(typically 20–40 bp) than typical restriction enzymes (typically 6–8 bp).
Membrane potential  The voltage across the cell membrane generated by differential
ionic charges on the intracellular versus extracellular sides of the membrane.
Microarray  A tool used to measure the expression of thousands of genes between
one or more tissue samples. It consists of a grid (array) of known nucleic acid
sequences spotted on a slide so that cDNA from a biological sample can be
hybridized with the sequences to detect whether any of the known sequences
are present in the sample.
Microdialysis  A method to sample neurochemicals from brain extracellular fluid
based on the diffusion of chemicals from the extracellular fluid into a microdialysis
probe’s semipermeable membrane.
Microelectrode  A small (micrometer scale) metal or glass probe used to measure
electrical signals from cells or tissue.
Microinjection  The process of injecting small volumes of a solution (usually
containing DNA) into cells with a thin needle.
Glossary  363
Microiontophoresis  The process of using a small electrical current to infuse
substances out of a glass electrode into the brain.
Microscope  An instrument that manipulates the trajectory of light rays so that small
objects appear larger.
Microstimulation  The process of sending electrical current through an electrode to
induce changes in membrane potential.
Microtome  A piece of equipment with a sharp knife to cut medium-thickness
(25–100 μm) sections of tissue.
Midsagittal  A view or section of the brain that perfectly divides the left and right
hemispheres.
Mixed design  A whole brain imaging task paradigm that uses elements of both
blocked and event-related designs.
MNI template  A standardized three-dimensional coordinate map of the human brain
produced by the Montreal Neurological Institute. Based on the average of hundreds
of individual MRI brain scans and matched to landmarks in Talairach space.
Molecular cloning  The process of identifying, isolating, and making copies of a
particular DNA fragment through recombinant DNA technology.
Monoclonal antibody  An antibody made by a single clone of cells that only
recognizes a single epitope, or specific region of a protein. Contrast with
polyclonal antibody.
Morpholinos  Stable, synthetic 22–25 bp antisense oligonucleotide analogues that complement
an RNA sequence to block proper mRNA translation as an alternative to RNAi.
Morris water maze  A spatial learning and memory task for rodents that involves
a hidden platform submerged in opaque water whose location can be recognized
through distant visual cues.
MRI  See Magnetic resonance imaging.
Multielectrode array (MEA)  A group of individual electrodes or tetrodes arranged
into one electrical recording unit to record extracellular activity from multiple cells
at the same time.
Multiple cloning site  A region in a DNA vector with a variety of recognition
sequences for different restriction enzymes that make it simple to insert a foreign
DNA sequence into the vector.
Multipotent  The ability of some cells to give rise to all types of cells found in a
particular tissue—for example, the ability of a neural stem cell to give rise to
neurons, astrocytes, and oligodendrocytes.
Muscimol  A GABA receptor agonist used to reversibly inhibit neural activity.
Myelin  The fatty insulating material surrounding axons produced by glial cells that
provide insulation to neurons.
Near-infrared spectroscopy (NIRS)  A noninvasive, optical brain imaging method
that records neural activity-induced changes in the reflectance properties of nearinfrared
light as it is reflected off the scalp.
Necessity experiment  See Loss of function experiment.
364 Glossary
Neural progenitor  A dividing cell with a limited ability to give rise to another
dividing cell but that has the ability to generate either a neuron, astrocyte, or
oligodendrocyte.
Neural stem cell (NSC)  A multipotent, self-renewing cell that divides and gives rise
to neurons, astrocytes, or oligodendrocytes, as well as other neural stem cells.
Neurosphere  A ball of cells generated from a dividing neural progenitor or neural
stem cell that indicates an ability to proliferate.
Neurosphere assay  See Primary neurosphere assay.
Neuroethology  The study of the neural basis of an animal’s natural behaviors.
Neuron doctrine  The theory, advocated by Ramón y Cajal, that discrete neurons are
the basic structural and functional units of the nervous system.
Next-generation sequencing (NGS)  Methods of identifying DNA base pair
sequences quickly, with use of fewer reagents, and less expensively than the
standard Sanger sequencing method. Enables large-scale genome exploration.
Nissl stain  A basophilic stain that highlights RNA (the Nissl substance) in cells.
Cresyl violet is a commonly used Nissl stain.
Nociception  The detection of a noxious stimulus, typically perceived as pain.
Nomarski microscopy  See Differential interference contrast (DIC) microscopy.
Nonhomologous end-joining  A naturally occurring DNA break repair process that
rejoins double-strand breaks, occasionally by introducing an insertion or deletion
mutation.
Nonmatch to sample task  A rodent assay that uses operant conditioning to test
working memory.
Nonratiometric dye  An organic chemical whose fluorescence intensity varies directly
with its ability to bind a substance such as calcium. Contrast with ratiometric dyes.
Northern blot  A nucleic acid hybridization technique used to detect the presence and
relative concentration of mRNA in a sample.
Novel object recognition  A learning and memory task in which a rodent will spend
more time exploring an object that has not previously been presented, demonstrating
memory for the previously presented object.
NpHR  See Halorhodopsin.
Nuclease  An enzyme that cleaves nucleic acids.
Nucleotide  A molecule composed of a phosphate group, a nitrogenous base (adenine,
thymine, cytosine, guanine, or uracil), and a sugar molecule (2′-deoxyribose for DNA,
ribose for RNA) that combines with other nucleotides to form a DNA or RNA polymer.
Numerical aperture (NA)  A measure of the light-collecting ability of a microscope
objective lens. The NA depends on the angle by which light enters the objective and
the medium light must pass through. Higher NA objectives collect more light and
consequently have better resolving power.
Objective lens  The lens on a microscope that gathers and focuses light from the
specimen in a microscope. This lens is typically placed adjacent to the specimen.
Glossary  365
Ocular lens  The lens, typically 10× magnification, located in the eyepiece of a
compound microscope.
Ohm’s law  The relationship between voltage (V), current (I), and resistance (R) in an
electrical circuit, expressed as V = I × R.
Olfactory avoidance assay  A Drosophila chemosensory assay in which a scientist
presents an odor on a stick in a fly chamber and measures the distance a fly
maintains from the odor. Also called a dipstick assay.
Olfactory jump response  See Chemosensory jump assay.
Open field test  A rodent assay used to measure exploratory behavior in a large open
chamber. More anxious animals stay near the walls of the chamber and do not
explore the center area.
Operant conditioning  The use of consequences (a positive reward or negative
punishment) in response to a specific behavior in an attempt to influence that behavior.
For example, a rodent learns to press a lever to receive a pleasurable food reward.
Optical imaging  A brain imaging technique that measures changes in light reflectance
from the surface of the brain due to changes in blood flow and metabolism
caused by neural activity.
Optodes  See Optrodes.
Optogenetics  An approach to stimulating and inhibiting neurons by expressing
genetically encoded neuronal actuators capable of depolarizing or hyperpolarizing
neurons in response to light.
Optrodes  Either (1) A recording device that records changes in light reflectance
in optical brain imaging techniques, or (2) the combination of an optic fiber and
microelectrode in an optogenetic experiment.
Organotypic slice culture  A slice of brain tissue kept in culture conditions for multiple
days to weeks. Such cultures require an air/liquid interface to properly regulate
gas exchange throughout the slice.
Oscilloscope  An instrument that displays the membrane potential over time.
Outside-out recording  A patch-clamp technique that exposes the extracellular side
of an excised membrane patch to the bath solution and the intracellular side to the
inside of the recording pipette.
P element  A transposable genetic element used in Drosophila that is able to change
locations in the genome when the P element transposase enzyme is also present.
Packaging cells  An immortalized cell line that is transfected with the necessary
recombinant DNA sequences to make viral particles. The endogenous cell machinery
produces virus, which is eventually harvested in the extracellular media.
Partial agonist  A compound that can bind and activate an endogenous receptor, but
not to the maximal extent of a full agonist or endogenous ligand.
Particle-mediated gene transfer  The process of introducing DNA into cells by physically
shooting DNA-coated pellets through the cell membrane. Also called biolistics.
Passaging  The process of transferring a fraction of cells into a new container to
provide space for the cells to continue to divide.
366 Glossary
Patch-clamp techniques  A set of electrophysiological methods in which a glass
electrode forms a tight seal with a patch of membrane, allowing the study of single
or multiple ion channels.
Pavlovian conditioning  See Classical conditioning.
PCR  See Polymerase chain reaction.
Perforated patch  A category of patch-clamp technique in which a chemical is added
to the recording pipette to cause small holes to form in the membrane. This is
useful for making the contents of the glass pipette continuous with the cell but
without the disadvantage of cytoplasmic contents leaking into the pipette.
Perfusion  A method used to circulate a chemical fixative through the cardiovascular
system so that the brain is thoroughly preserved.
Peri-stimulus time histogram (PSTH)  A graph of the number of action potentials
recorded with respect to the time a stimulus was presented.
PET  See Positron emission tomography.
Phage  See Bacteriophage.
Pharmacogenetics  A method of manipulating neural activity by expressing genetically
encoded ion channels and receptors not usually found in the species of interest.
Phase-contrast microscopy  An optical method of enhancing contrast in a light
microscope to amplify small differences in the index of refraction of different
cellular structures.
Phenotype  An observable trait or set of traits in an animal.
Photoactivation  In the context of photoactivatable fluorescent proteins, a change in
the fluorescence properties of a fluorophore, usually causing it to fluoresce more or
less.
Photobleaching  The phenomenon in which fluorescence intensity emitted from a
fluorophore decreases over time as it is continuously exposed to light.
Photoconversion  The process of delivering a pulse of light to a fluorophore whose
emission spectra change from one color to another.
Phototaxis  The tendency of an organism to move toward light and away from dark.
Phototoxicity  The phenomenon in which illumination leads to the death of cells
expressing a fluorophore, typically because illumination of the fluorophore
generates free radicals.
Physical gene delivery  A method of introducing foreign DNA into cells by physically
disrupting the cell’s membrane. Includes microinjection, electroporation, and particlemediated
gene transfer (biolistics).
Plasmids  Independently replicating accessory chromosomes in bacteria that exist as
circles of DNA. Commonly used as vectors for molecular cloning.
Pluripotent  The ability of a cell to generate any type of cell, including more
pluripotent cells.
Polyclonal antibody  A collection of antibodies that each recognizes a distinct
epitope, or region, of the same antigen. Contrast with monoclonal antibody.
Glossary  367
Polymerase chain reaction (PCR)  A biochemical reaction that uses controlled
heating and cooling in the presence of DNA synthesizing enzymes to exponentially
amplify a small DNA fragment.
Porsolt test  See Forced swim test.
Position effects  The phenomenon of transgenes having different expression patterns
depending on where the transgene randomly integrates in the genome.
Positron  An antimatter counterpart of an electron. When a positron contacts an
electron, an annihilation event occurs that generates gamma photons.
Positron emission tomography (PET)  A brain imaging technique in which positronemitting
isotopes generate annihilation events that can be detected by a gamma
detector. Depending on the positron-emitting isotope, PET can detect neural
activity or the location of specific receptors in the brain.
Positron-emitting isotope  An unstable version of a molecule that emits positrons as
it decays, allowing it to be detected in positron emission tomography (PET).
Posttranslational modification (PTM)  A modification to a protein, usually
the addition or subtraction of a functional chemical group, after it has been
translated by a ribosome. For example, the addition or removal of a phosphate
group.
Potential  See Membrane potential.
Precess  The circular movement of the axis of a spinning body around another axis
due to a torque. Describes the circular movement of a proton.
Predictive validity  An aspect of an animal model’s validity that is fulfilled if
treatments used in human patients have the same effect on the animal model. For
example, rodent models of depression have predictive validity if human antidepressants
relieve behavioral measures of depression in rodents.
Prepulse inhibition (PPI)  The attenuation of a startle response caused by the
presentation of a weaker sensory stimulus before the presentation of the startlecausing
stimulus.
Primary antibody  The antibody that recognizes and binds to a specific antigen.
Primary cell culture  The culture of cells extracted directly from a tissue of interest.
Can be in the form of dissociated cells, explants, or slices.
Primary neurosphere assay  An assay of cultured cells for the ability to form
neurospheres to examine whether they are proliferative. Cannot distinguish
between progenitor cells and stem cells. Compare to secondary neurosphere
assay.
Primer  A short, single-stranded oligonucleotide used in PCR to hybridize to a
template strand in the region flanking the region to be amplified.
Proboscis extension response (PER)  A reflex in Drosophila that can be used to test
the responses of specific gustatory receptors. A scientist applies taste ligands to
gustatory receptor neurons on the leg or proboscis and examines proboscis
extension.
Progenitor  A cell that divides, but with a limited capacity for self-renewal.
368 Glossary
Progressive ratio reinforcement  In operant conditioning, a paradigm in which an
animal must perform an action more and more (such as pressing a lever an increasing
number of times) to receive a rewarding stimulus. Compare with continuous
reinforcement or fixed ratio reinforcement.
Promoter  A regulatory region of DNA that controls the spatial and temporal
expression of a gene under its control.
Pronucleus  One of the nuclei present in a recently fertilized egg—either the male
sperm nucleus or the female egg nucleus.
Protein  A biological macromolecule composed of one or more sequences of amino
acids. Proteins serve as the structural and enzymatic molecules that regulate
virtually every process within cells.
Protein kinase  A protein that adds a phosphate group to another protein.
Proteomics  The systematic, large-scale study of the complete set of proteins in
a system (such as a cell or tissue) at a particular time, making use of mass
spectrometry and bioinformatics to identify proteins and potential functional
interactions.
Protospacer adjacent motif (PAM)  A three-nucleotide sequence of NGG that must
lie immediately 3′ of the genomic sequence to be targeted in CRISPR/
Cas9-mediated double-strand breaks.
Pseudopregnant mouse  A female mouse that has been mated with a sterile male
mouse so she produces the proper hormones to act as a surrogate mother.
Pulse chase labeling  A process in which a labeled probe (the pulse) is briefly
injected into animals or added to cultured cells, then washed away and replaced by
unlabeled molecules (the chase). By following the changes in localization of the
labeled probe, different protein trafficking pathways can be observed.
qRT-PCR  See Quantitative real time PCR.
Quantitative real time PCR (qRT-PCR)  A method to both amplify and quantify a
specific DNA fragment (especially a cDNA fragment produced from mRNA).
Quantum dots  Semiconductor nanocrystals that can be used as bright fluorophores.
Radial arm maze  A behavioral test used to assay spatial learning and memory in
rodents. It utilizes a maze consisting of an array of arms radiating out from a
central starting point, with only some arms containing food. A rodent is trained to
traverse down only one arm and not others.
Radiofrequency (RF) pulse  A pulse of electromagnetic energy used in MRI to
excite hydrogen protons in a human (or sometimes, an animal) subject. After the
RF pulse is switched off, the protons relax, generating the MR signal used to form
images of the brain.
Radioimmunoassay (RIA)  A sensitive method for measuring extremely low concentrations
of proteins.
Raster plot  A graph that displays each action potential (or membrane potential over
a threshold) as a dot or tick mark in relation to a certain time period. Often, the
Y-axis represents categories of stimuli, while the X-axis represents time.
Glossary  369
Ratiometric dye  An organic molecule that is either excited at or emits at a slightly
different wavelength when bound to a substance, such as calcium. Therefore, the
ratio of fluorescence intensity at either the excitation or emission wavelengths
reveals changes in the concentration of the substance.
Recognition site  See Restriction site.
Recombineering  A method of creating recombinant DNA molecules using homologous
recombination to avoid restriction enzyme digests and ligation reactions.
Reduction  One of the guidelines for animal welfare stating that scientists must use
the minimum number of animals required to obtain statistically significant data.
Refinement  One of the guidelines for animal welfare stating that scientists must
minimize distress and pain and enhance animal well-being.
Refractive index  See Index of refraction.
Region-of-interest (ROI) analysis  Method of brain imaging data analysis that divides
the brain into discrete regions that can be compared for significant differences
in signal intensity.
Relative recovery  In microdialysis, the relative concentration of a substance in the
collected dialysate from the probed brain region compared to concentration in the
perfusion solution.
Replacement  One of the guidelines for animal welfare stating that scientists must do as
much research without using animals as possible, including using computational methods
and cell culture methods. Also applies to choice of a less-sentient animal species, such
as choosing to study invertebrates over vertebrates or mice rather than monkeys.
Reporter gene/protein  A visible, nonendogenous gene or protein that is controlled
under the same promoter as a gene of interest to report the expression pattern of
that gene. Alternatively, a reporter gene is fused to a gene of interest to report the
subcellular localization of a reporter fusion protein.
Rescue experiment  An experiment in which an aspect of the nervous system that is
lost is deliberately reintroduced to determine if it restores a normal phenotype.
Resident intruder assay  A social behavioral assay in rodents that measures territorial
behavior in males by adding an intruder animal into the cage of a resident
animal and measuring specific aggressive behaviors.
Resistance (R)  An electrical property that restricts the movement of charge, for
example, across a membrane.
Resolution  In microscopy, the minimum distance by which two points can be
separated and still distinguished as two separate points.
Resolving power  See Resolution.
Resting potential  The voltage across the cell membrane of a cell at rest, generated
by differential buildup of ionic charges on the intracellular vs. extracellular sides
of the membrane. Neurons typically have a resting potential of about −70 mV.
Restriction digest  The process of using restriction enzymes to cut DNA into
fragments.
Restriction endonuclease  See Restriction enzyme.
370 Glossary
Restriction enzyme  An enzyme that recognizes and cuts specific sequences of
DNA.
Restriction site  An approximately four to eight unique base pair sequence that a
particular restriction enzyme recognizes and cuts.
Retrograde tracer  A chemical probe that labels an axon path by being transported
from the synaptic terminal back up to the cell body, revealing what areas of the
brain project to the location where the tracer was deposited.
Retrovirus  A type of virus that uses RNA rather than DNA as its genetic material.
Reversal potential  The membrane voltage at which there is no overall flow of ions
across the membrane. Determining this potential can be used to identify the ion
species that passes through a particular channel.
Reverse genetics  The directed mutagenesis of a specific gene so that a scientist can
determine if it is necessary for a certain phenotype to occur. Contrast with forward
genetics.
Reverse microdialysis  The use of a microdialysis probe to introduce chemical
substances into a brain region.
Reverse transcriptase  The enzyme that uses RNA as a template for creating strands
of DNA, called complementary DNA (cDNA).
Reverse transcription  The process of creating complementary DNA (cDNA) from
RNA templates using reverse transcriptase.
Reverse transcription PCR (RT-PCR)  A PCR reaction that amplifies a cDNA
fragment generated from reverse transcription of an RNA fragment.
Ribosome  Macromolecular complexes of RNA and protein that translate mRNA into
a protein by assembling amino acids into polypeptide chains.
RNA-induced silencing complex (RISC)  A complex of proteins that uses siRNA as
a template for finding a complementary mRNA to degrade.
RNA interference (RNAi)  The process of silencing gene expression through
endogenous cellular mechanisms that degrade mRNA.
RNA polymerase  The enzyme that binds to and transcribes RNA molecules from
DNA templates.
RNA splicing  The removal of introns and joining of exons by a spliceosome of a
newly transcribed mRNA before it leaves the nucleus for translation.
Rotarod  A rodent behavioral assay of muscle coordination and balance. Consists of
a rotating rod on which rodents must walk and maintain their balance. This device
can also be used to examine motor learning, as animals will improve after multiple
trials of walking on the rotarod.
RT-PCR  See Reverse transcription PCR.
Run  In a brain imaging experiment, one complete scan of the brain, during which a
volume of information is collected.
Sagittal plane  The plane dividing the brain into left and right portions such that the
brain is complete in the superior to inferior and anterior to posterior planes.
Glossary  371
Sanger dideoxy chain termination method  A technique for determining the
sequence of a DNA molecule.
Scanning electron microscope (SEM)  A form of microscopy that uses electron
interactions at the surface of a specimen to generate an image of the specimen’s
surface that appears three dimensional.
Screen  A method used to determine which nuclei, neurons, genes, or proteins may be
important for a particular biological process.
SDS-PAGE  See Sodium dodecylsulfate polyacrylamide gel electrophoresis.
Secondary neurosphere assay  An assay that measures whether cells taken from a
primary neurosphere are able to form new neurospheres, indicating the cell is still
proliferative.
Secondary antibody  An antibody that binds to a primary antibody.
Sectioning  The process of physically or optically creating thin sections of a specimen
to gain greater access and/or visibility of internal structures.
Selection cassette  A sequence within a genetic targeting construct that encodes genes
that can be used to identify cells that have properly incorporated the targeting construct.
Typically, this cassette includes a positive selection gene, such as neomycin
resistance gene, that can be used to kill cells that have not incorporated the construct.
It also typically includes a negative selection gene, such as thymidine kinase, that can
be used to kill cells that have improperly incorporated the targeting construct.
Session  In a brain imaging experiment, the scheduled time when an actual experiment
is conducted.
Short hairpin RNA (shRNA)  A strand of RNA containing complementary sequences
at either end that cause it to fold onto itself, producing a hairpin shape.
ShRNA  See Short hairpin RNA.
Signal transduction  The conversion of an extracellular cue, such as a ligand binding
to a receptor, into a signal that begins an intracellular signaling cascade.
Single-proton emission computerized tomography (SPECT)  A functional brain
imaging technique in which radioactive probes injected into the blood generate
high-energy photons that can be detected using a gamma camera placed around a
subject’s head.
SiRNA  See Small interfering RNA.
Slice  In brain imaging experiments, two-dimensional representations of a plane of the
brain that is one voxel thick.
Slice culture  A primary tissue culture technique in which sections of brain tissue are
sliced using a vibratome and then placed in a culture medium. These slices can be
used acutely for short-term experiments, or organotypically, for multiple days.
Small interfering RNA (siRNA)  Short (19–23 bp) double-stranded RNA fragments
used as a template in the RNAi pathway to target mRNA molecules for degradation.
Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)  A
technique used to separate proteins based on their size and folding properties. Often
used while performing a western blot.
372 Glossary
Somatic cell  A differentiated cell that is not part of the germline (eggs or sperm),
such as skin or blood cells.
Southern blot  A nucleic acid hybridization technique that can identify the presence
of a specific sequence of DNA.
Spatial resolution  The minimum size that can be resolved to distinguish individual
components of an organ or tissue. Compare to temporal resolution.
Spike  Another name for an action potential.
Spike sorting  Differentiating action potentials recorded in an extracellular
electrophysiology experiment and assigning them to different neurons based on
their pattern, shape of spiking activity, and signal size.
Spliceosome  The macromolecular complex that catalyzes RNA splicing.
Splitting  See Passaging.
Startle response assay  A behavioral assay that measures the ability of an animal to
exhibit a startle response, such as an eye blink or sudden muscle contraction, in
response to the presentation of an unexpected sensory stimulus. This assay can be
used to indicate whether sensory processing is intact.
Stem cell  A pluripotent cell that has an unlimited ability to renew itself.
Stereomicroscope  A microscope that magnifies small objects and provides a threedimensional
perspective for examining the surface of brains or large neural structures.
Often used for dissections, surgeries, or examining electrodes and implants.
Stereotaxic instrument  A piece of equipment that stabilizes an animal’s head and
allows precise positioning for injections or implants.
Stereotaxic surgery  A survivable procedure in which a scientist physically
penetrates the brain of an animal under anesthesia for the injection of substances
or implantation of devices.
Sterile  Aseptic and free from microorganisms. Important condition for performing
surgeries on live animals or culturing cells and tissue outside of an organism.
Sterile field  A dedicated aseptic surface where sterile instruments can be placed
when not in use.
Sterilizer  A small device filled with heated glass beads used to sterilize tools and fine
instruments when autoclave sterilization is inconvenient, such as between surgeries.
Sticky end  Single-stranded DNA overhang generated by some restriction enzyme
digests. Sticky ends can be matched to a complementary sticky end produced by
the same restriction enzyme on a different sample of DNA. Compare to blunt end.
Structural brain imaging  Techniques that are used to resolve brain anatomy in a
living subject without physically penetrating the skull. Examples include cerebral
angiography, CT scans, and magnetic resonance imaging (MRI).
Subcloning  The process of copying or moving a fragment of DNA from one recombinant
DNA construct into a new vector to create a new recombinant DNA construct.
Sucrose preference test  A behavioral measure of anhedonia in rodents. A scientist
places a bottle of plain tap water and a bottle containing sweet water in a rodent’s
cage. Normal rodents prefer sweet water, but rodent models of depression do not.
Glossary  373
Sulci  The indentations created by the folds in the brain (plural for “sulcus”).
Suture  Either (1) a seam between different parts of the skull or (2) a thread used after
a surgery to close a wound.
Synapto-pHlorin  A pH-sensitive fluorescent protein variant of GFP that is used to
examine synaptic vesicle recycling and release. It does not fluoresce when present
inside synaptic vesicles, but does fluoresce when the vesicle fuses during exocytosis.
Systems neuroscience  The branch of neuroscience that examines the coordinated
activity of neural ensembles or the firing properties of individual neurons that
generate behavior and cognition.
T1  In MRI and fMRI, the time (in milliseconds) required for a certain percentage of
protons that had been excited by radiofrequency pulses to realign in the longitudinal
direction.
T1-weighted image  An MRI image formed using T1 signal intensity.
T2  In MRI and fMRI, the time (in milliseconds) required for a certain percentage of
protons that had been excited by radiofrequency pulses to relax in the transversal
direction.
T2-weighted image  An MRI image formed using T2 signal intensity.
T-maze  A behavioral apparatus with two arms oriented in opposite directions, giving
it a T shape. A Drosophila T-maze contains an additional loading arm to move flies
into the choice point between the T. This maze can be used to test sensory
preferences as well as learning and memory.
Tail flick assay  A rodent assay for nociception in which painful heat or cold is
applied to the tail, causing the rodent to rapidly flick its tail to the side.
Tail suspension assay  An assay for depression in which a rodent is held by the
tail and the amount of time the animal struggles is measured. Rodent models of
depression stop struggling sooner than normal rodents but struggle for a normal
amount of time if given antidepressant medication.
Talairach space  A coordinate system based on the stereotaxic measurements of
a single postmortem brain used to normalize MRI data so that anatomical
comparisons can be made among different brains.
Tamoxifen  A molecule that is able to bind to the estrogen receptor, causing its translocation
from the cytoplasm to the nucleus. Useful to exert temporal control over
the Cre/lox system when Cre recombinase is fused to the estrogen receptor.
Task paradigm  A strategy for presenting stimuli to subjects during an experiment.
In human brain imaging experiments, these paradigms are typically classified as a
blocked design, event-related design, or mixed design.
Temporal delay  In functional brain imaging techniques, the time delay between the
presentation of a stimulus and measurement of changes in neural activity.
Temporal resolution  The minimum distance in time necessary to distinguish between
discrete neural events.
Tesla  Units of measurement for magnetic field strength. Conventional MRI scanners
create external magnetic fields at 1.5–3 T.
374 Glossary
Tet-off/Tet-on system  An inducible promoter system that allows transgene expression
to be either turned off (Tet-off) or turned on (Tet-on) through the use of an antibiotic
called doxycycline.
Tetrode  A bundle of four microelectrodes used to perform extracellular
electrophysiological recordings, often in vivo recordings in awake, behaving
rodents.
Tetrodotoxin (TTX)  A toxin produced by specific species of fish, such as the
pufferfish, that binds pores of voltage-gated sodium channels to block action
potentials. The purified chemical is often used as a pharmacological agent to
block action potentials or sodium channel current.
Thermocycler  A machine that controls the heating and cooling of samples, most
often used for PCR.
Time of flight (TOF) detector  In mass spectrometry, an instrument that measures
the mass to charge ratio of molecules in the gas phase.
Total internal reflection fluorescence (TIRF) microscopy  A form of microscopy
used to image molecules and/or events that occur at the cell surface. A TIRF microscope
uses a rapidly decaying evanescent wave of fluorescence excitation to restrict
imaging to a thin (∼100 nm) region of contact between a specimen and the surface
it is on (usually a glass coverslip).
Tracer  A chemical that can be injected and transported along neural processes to
highlight axonal paths and connections.
Transcranial magnetic stimulation (TMS)  A noninvasive method used to manipulate
neural activity in the brain. A coil is positioned adjacent to a subject’s head.
This coil generates magnetic field pulses that induce electrical activity in superficial
brain structures. Depending on the specific sequence and strength of pulses, TMS
can reversibly activate or inactivate brain regions.
Transcription  The process of synthesizing RNA from a DNA template, performed
by the enzyme RNA polymerase.
Transcription activator-like effector nucleases (TALENs)  Artificially engineered
restriction enzymes composed of a DNA-binding domain and a DNA-cleavage domain.
Transcriptional start site  The location within a gene where RNA polymerase begins
transcription.
Transcription factor  A DNA-binding protein that recognizes a specific genetic regulatory
element and regulates transcription by recruiting or blocking RNA polymerase.
Transduction  The process of using a nonreplicating viral vector to deliver foreign
DNA into a cell.
Transfection  The process of delivering DNA to cells using nonviral methods.
Transformation  The process of introducing a DNA vector into competent cells.
Transgene  A foreign gene expressed in an organism that does not normally express the gene.
Transgenic organism  An organism that carries foreign DNA that it does not
normally express.
Translation  The process by which a ribosome synthesizes a chain of amino acids
based on an mRNA template.
Glossary  375
Transmission electron microscopy (TEM)  A form of microscopy that transmits an
electron beam through a thin specimen to generate an image based on differences
in electron density of the material. Produces extremely high-resolution images of
cellular ultrastructure.
Transposable element  See Transposon.
Transposase  An enzyme that acts on specific DNA sequences at the end of a transposon
to disconnect the sequence from flanking DNA and insert it into a new target DNA site.
Transposition  The natural process of a transposon moving to different positions
within the genome.
Transposon  A genetic sequence capable of translocating to different positions within
the genome.
Transsynaptic tracer  A tracer capable of passing through synapses useful for
labeling multisynaptic connections.
Trophoblast  Cells in a blastocyst that form the outer spherical shell and eventually
develop into the placenta.
Two-photon microscopy  A specialized form of fluorescent microscopy that sends
two relatively low energy pulses of laser light that can add up to excite fluorophores
in a restricted section of a specimen. Regions beyond the restricted section do not
receive enough energy for excitation, greatly reducing out-of-focus fluorescence.
UAS  See Upstream activation sequence.
Ultrasonic neuromodulation  A noninvasive neuromodulation technique in which
focused sound waves slightly heat an area of the brain, causing transient effects.
Unconditioned stimulus (US)  In classical conditioning, the salient stimulus that
eventually becomes associated with a previously neutral stimulus.
Unipotent  A cell that is capable of dividing but only producing a single cell type.
Upright microscope  A light microscope in which the objective lens is above the
specimen and the light source and condenser are beneath the specimen.
Upstream activation sequence (UAS)  A strong promoter derived from yeast that is
activated by the Gal4 transcription factor.
Validity  A subjective measure of how relevant an animal model is toward
understanding humans.
Vector  See DNA vector.
Vertical pole test  A behavioral paradigm used to assay motor coordination and
balance in rodents.
Vibratome  A device that uses a vibrating blade to section a fresh, unfrozen specimen
into tissue slices that can be kept alive.
Viral gene delivery  The delivery of DNA constructs into cells in vivo or in vitro
using one of many available viral vectors.
Visual cliff assay  An assay used to examine normal visual function in rodents.
Voltage (V)  An electromotive force that exists across a membrane.
376 Glossary
Voltage clamp  An electrophysiology technique that allows a scientist to hold the
membrane potential at a constant voltage to measure currents generated by ions
moving across the membrane.
Voltage-sensitive dye imaging (VSDI)  A cell or tissue visualization technique that uses
dyes that shift their absorption or emission fluorescence based on the membrane
potential, thus allowing scientists to visualize changes in membrane potential over time.
Voltammetry  A method used to measure the presence and relative amounts of
neurochemicals in living tissue.
Volume  In brain imaging experiments, a complete scan of the brain composed of
multiple, individual slices.
Von Frey assay  A behavioral test used to assay noxious mechanical and pinch
stimuli in rodents.
Voxel  A three-dimensional unit of space, often used to describe a unit of brain space
during whole-brain imaging experiments.
Voxelwise analysis  A strategy for analyzing data in a brain imaging experiment, in
which each voxel of data is examined for significant changes in signal intensity.
Western blot  A method used to detect the expression of a protein in a biological
sample.
Whole-cell recording  A patch clamp recording technique in which a scientist places a
glass pipette adjacent to a cell membrane, forms a tight gigaseal with the membrane,
and provides enough suction so that the membrane patch is removed, allowing the
interior of the pipette to become continuous with the cytoplasm of the cell.
Whole-mount preparations  A histological preparation in which a chunk of tissue or
an entire small animal brain is mounted onto a slide for analysis.
Wide-field fluorescent microscopy  See Epifluorescent microscopy.
Within-subjects study  A study that compares the effects of two different stimuli
within the same subject pool.
Worm tracker  Computer software that can automatically track locomotor behavior
in Caenorhabditis elegans.
X-ray  A structural visualization technique used to image parts of the body with
natural contrast, such as a bone in tissue. This technique is inadequate by itself to
visualize individual structures within the brain, but serves as the basis of cerebral
angiogram and computerized tomography technology.
Xylazine  A chemical sedative and analgesic, often used in combination with
ketamine to anesthetize small rodents.
Yeast artificial chromosome (YAC)  A large (100–2000 kb) DNA vector capable of
replicating in yeast. Useful for manipulating large sequences of DNA, such as in
the generation of transgenic or knockout constructs.
Yeast two-hybrid assay  A method that takes advantage of yeast transcriptional
machinery to determine whether two proteins directly interact with each other.
Zinc finger nucleases  Artificially engineered restriction enzymes consisting of a zinc
finger DNA-binding domain fused to a DNA-cleavage domain.
377
Index
Note: Page numbers followed by “f”, “t”, and “b” indicate figures, tables and boxes, respectively.
A
Ablation, 187–188
Absolute recovery, 83
Action potential, 91
ionic basis of, 92f
Activity sensors, 172t
Acute cultures, 302
Adeno-associated virus, 250
Adenovirus, 248
Adobe Photoshop®, 136
Aequorin, 175
Affinity chromatography, 319
Agonist, 189–190, 190f
Allatostatin receptor (Alstr), 253, 257
Amino acids, 208–209
Amperometry, 84–85
Amplifier, 92–93, 94b–96b
Anatomical landmarks, 74–75, 74f
Animal behavior, 39–40
behavioral assay. See Behavioral assay
Animal use, ethical consideration, 42b
Anisotropic diffusion, 13
Antagonist, 189–190, 190f, 198t–199t
Anterograde tracers, 162–164, 162f, 163t
Antibodies
interference, 308
making and using, 314–318
protein-specific antibodies, in technique, 318t
Antigens, 314
Anxiety, 58–60
defensive marble burying, 59
elevated plus maze, 59, 59f
Geller-Seifter conflict test, 59–60
open field test, 58, 58f
Aptamers, 315–318
Archaerhodopsin (Arch), 256–257
Archaerhodopsin-3 (Arch), 194
ArcLight, 254
Array tomography, 159–160
Assaying cellular function, 169–170
cell proliferation with thymidine analogs,
169–170, 170f
protein trafficking with pulse-chase labeling,
170
Ataxia, 46
Ataxin, 191, 257
Attention and impulsivity, 56
five choice serial reaction time task, 56
go/no-go task, 56
Autofluorescence, 128–129
Autoradiography, 154b–156b
B
Bacterial artificial chromosome (BAC)
constructs, 259
Bacteriophage, 227–228
Base pair, 206
Basic fibroblast growth factor (bFGF), 304
Basic Local Alignment Search Tool
(BLAST), 214
Basophilic stain, 150
Behavioral assay
behavioral paradigm selection, 41–42
model organism selection, 40–41
using animal behavior as a model for, 43–44
Between subjects, 26–27
Bimolecular fluorescence complementation
(BiFC), 180
Binary expression systems, 260f–261f, 261
Binary transgenic systems, 260f–261f,
261–265
Cre/Lox system, 263–264
Flp/Frt system, 264
Gal4/UAS system, 262–263
promoters in, 262t
Tet-off/Tet-on system, 264–265
378 Index
Biochemical assays. See individual entries
Biolistics, 243
Biological ballistics. See Biolistics
Blastocyst, 277–278
inserting into female mouse, 278
Bleed-through, 139
Blocked designs, 27–28
Blood oxygen level-dependent (BOLD)
effect, 14–17, 15f
Blunt ends, 220–221
Brain atlases, 74–75, 74f
Brain
composition, 3f
X-ray technology, 4f
Bregma, 74–75
Brightfield microscopy, 126–127, 140t–142t
Brodmann’s areas, 32–33
C
Caenorhabditis elegans (C. elegans),
211b, 267–268, 289
advantages and disadvantages, 212t
behavioral paradigms, 65–67
locomotor behavior, 66
sensory behavior, 66–67
Calcium indicator dyes, 174
genetically encoded, 173–175, 176f
Canine adenovirus (CAV), 248
Cannulae, 80–81
Capsaicin receptor (TrpV1), 257
Carbon fiber microelectrode, 85
cDNA microarray screen, 215–216, 215f
Cell body stains, 150–152
Cell
Cell-attached mode, 105
culture. See Cell culture
flow of information in, 205f
fractionation, 323, 324f
Cell culture, 295
equipment, 296–297
immortalized cell lines, 298–301
manipulating cells in, 307–308
media, 298
primary culture. See Primary cell/tissue culture
stem cell cultures. See Stem cell cultures
techniques, 295–310
Central dogma of molecular biology, 204
DNA, 204–206
transcription, 206–208
translation, 208–209
Cerebral angiogram, 3
Cerebral angiography, 3, 5f
Cerebrospinal fluid (CSF), 2
Channelrhodopsin-2 (ChR2), 194, 253,
256–257
Chemical gene delivery, 244–247
calcium phosphate transfection,
244–246, 245f
lipid transfection, 246–247
Chemical mutagenesis, 210
Chemosensory jump assay, 64
Chimeras, 278
Chromatin immunoprecipitation (ChIP),
338–341, 340f
Chromatography, 319, 319f
Chromogenic labels, 154b–156b
Classical conditioning, 55b
Clear lipid-exchanged acrylamide-hybridized
rigid imaging/immunostaining/in
situ hybridization-compatible tissue
hydrogel (CLARITY), 149–150
Clone, 227b
Cloning DNA, 227–231
ligation, 229–230
purifying DNA from host cells, 231
transformation, 231
vector. See Vectors
Cloning vectors, 228
Clozapine-N-oxide (CNO), 257
Co-culture systems, 307–308
Codon, 208–209
Cognitive neuroscience, 24
Cohesive ends, 220–221
Co-immunoprecipitation (Co-IP), 327, 328f
Colorimetric labels, 154b–156b
Competent cells, 231
Complementary DNA (cDNA), 215–216
library, 223b–224b
Complementary strands, 206
Complementation test, 213
Compound microscope, 121–125, 123f–124f
Computer, 92–93, 94b–96b
Computerized tomography (CT), 4–6, 5f
Condenser, 122
Conditional knockout mice, 282–284
with the Cre/lox system, 281, 283f
Conditioned stimulus (CS), 55b
Confluence, 297
Confocal microscopy, 129–132, 131f
advantages and disadvantages, 140t–142t
Construct validity, 43–44
Contextual fear conditioning, 55b
Coomassie blue, 321
Coronal plane, 149
Countercurrent apparatus, 63, 63f
Index  379
Cranial windows, 81
Cre recombinase, 263, 282–283
and lox recombination system, 281
CRISPR (clustered regularly interspaced short
palindromic repeat), 285–286
Cas9 nuclease system, 274, 285–287, 286f
Cross talk. See Bleed-through
Cross-linking fixatives, 146
Cryostat, 148, 148t
Cued fear conditioning, 55b
Current (I), 90
clamp mode, 96–99
I/V curves, 98b–99b
Cyclic voltammogram, 84, 84b
Cytokines, 313
D
Darkfield microscopy, 127f, 140t–142t
Data analysis
current over time, 100b
I/V curves, 98b–99b
spikes, 101b–103b
Defensive marble burying, 59
Dehydrating fixatives, 146
Dependent variable, 26
Depolarization, 90–91
Depression, 60–61
forced swim test, 60, 61f
sucrose preference test, 60–61
tail suspension assay, 60
Designer receptors exclusively activated by
designer drugs (DREADDs),
192–193, 257
Dicer, 287–288
Differential interference contrast (DIC)
microscopy, 127, 140t–142t
Diffuse optical imaging (DOI), 22
Diffusion, 13
Diffusion MRI, 12–13
Diffusion tensor imaging (DTI), 13, 13f
Digoxigenin, 154b–156b
Diphtheria toxin receptor, 191, 257
Dipstick assay. See Olfactory avoidance assay
Direct IHC, 158–159
Dissecting microscope, 125
Dissociated cell cultures, 303–304
DNA, 204–206
cloning. See Cloning DNA
identifying. See Identifying DNA
ladder, 226
libraries, 223b–224b
microinjection, 240–241
molecular structure of, 205f
purifying from host cells, 231
sequencing, 233–234, 234f
synthesis, 224
vector, 227–229
Dominant negatives, 290–291, 291f
Double helix, 206, 207f
Double-inverse orientation (DIO), 263–264
Doxycycline, 264
Drosophila melanogastor, 211b
advantages and disadvantages, 212t
behavioral paradigms, 61–65, 289
flight, 62–63
learning and memory, 65. See also Learning
and memory
locomotor behavior, 62
sensory function, 63–65. See also
Sensory function
social behaviors, 65
E
Electroencephalography (EEG), 20, 21f
Electrolytic lesion, 189
Electron microscope tomography, 135, 135f
Electron microscopy, 133–135, 134f
Electron tomography (ET), 135, 140t–142t
Electrophoretic mobility shift assay
(EMSA), 338–339, 338f
Electrophysiology tissue preparations,
108–113
in vitro recordings, 108–112. See also In vitro
recordings, in electrophysiology
in vivo recordings, 112–113. See also In vivo
recordings, in electrophysiology
Electrophysiology, 81–82
Electroporation, 241–242, 242f
in utero, 243f
Electrospray ionization (ESI), 329b–330b
Elevated plus maze, 59, 59f
Embryonic lethal, 278–280
Embryonic stem (ES) cells, 304–305
inserting into blastocyst, 277–278
selecting, 276–277, 277f
Emission filter, 128
Endogenous genes
genetically perturbing, 274f
manipulating, 273–294
Ensembl, 214
Enzymatic histochemistry, 160, 161f
Enzyme-linked immunosorbent assay
(ELISA), 323–324, 325f
Epidermal growth factor (EGF), 298, 304
380 Index
Epifluorescent microscopy, 129,
131f, 140t–142t
Epitope, 314
Equipment, for cell culture, 296–297
biosafety hood, 296
cell incubator, 296
microscope, 297
refrigerator, 297
specially prepared cell culture
flasks/plates, 297
water bath, 297
Estrogen receptor (ER), 283–284
and tamoxifen system, 283–284
Ethnology, 40
Event-related designs, 27–28
Event-related potentials (ERPs), 20
Excitation filter, 128
Excitatory postsynaptic potential
(EPSP), 90–91
Exon, 208
Explant cultures, 303
Expression vector, 228–229
Extracellular recording, 89–90,
99–104, 101f
F
Face validity, 43–44
Faraday cage, 94b–96b
Fast-scan cyclic voltammetry, 84
Fiber stains, 152
Fibroblast growth factor (FGF), 298
Flat skull position, 78
Flex construct. See Double-inverse orientation
(DIO)
Flight simulators, 62–63, 62f
Flippase recognition targets (Frts), 264
Flippase recombinase (Flp), 264
Frt recombination system, 282–283
Floxed sequence, 263. See also Lox sites
Fluorescence microscopy, 128–133
Fluorescence photoactivation localization
microscopy (FPALM), 122b
Fluorescence recovery, after photobleaching, 180
Fluorescence resonance energy transfer
(FRET), 177–180, 179f
Fluorescent activity indicators, 82
Fluorescent labels, 154b–156b
Fluorodeoxyglucose (FDG), 17
Fluorophores, 128, 154b–156b
Footprint pattern assay, 46, 48f
Forced swim test, 60, 61f
Formalin assay, 51
Forward genetic screen, 209–213
Founders, 266–267
Fractional intensity change, 171–173
Freezing microtome, 147–148
Functional brain imaging
electroencephalography, 20, 21f
experimental design and analysis, 24–35.
See also Functional brain imaging
experiments
figure preparation, 33–35, 34f
fMRI. See Functional magnetic resonance
imaging (fMRI)
magnetoencephalography, 20–22, 21f
optical imaging, 22–24, 23f
positron emission tomography, 17–19, 19f
single-proton emission computerized
tomography, 19–20, 21f
techniques, comparison of, 23t
Functional brain imaging experiments,
35b–36b
conducting, 29–31
design and analysis, 24–35
human experiment. See Human experiments
pilot experiments, 28
planning, 25–29
practical consideration, 25
structure of, 26–27
task paradigm, 27–28
Functional magnetic resonance imaging
(fMRI), 14–17
animal experiment, 18b
BOLD effect, 15f
example, experiment, 16f
G
Gain-of-function experiment, 186–187
Gal4, 262
UAS system, 262–263, 263f
Gal80, 263
Gaussian distribution curve, 101b–103b
GCaMPs, 254
Gel electrophoresis, 229f
isolation and characterization of DNA
fragments using, 226–227
Gel shift assay. See Electrophoretic mobility
shift assay (EMSA)
Gel-filtration chromatography, 319
Geller-Seifter conflict test, 59–60
Gene delivery strategies, 239–252
chemical gene delivery. See Chemical gene
delivery
choosing, 251t
Index  381
physical gene delivery. See Physical gene
delivery
viral gene delivery. See Viral gene
delivery
Gene gun, 243, 244f
Gene products, disrupting, 287–291
dominant negatives, 290–291, 291f
morpholinos, 289–290, 290f
RNA interference, 287–289
Gene targeting, 273, 279f
analyzing genotype after, 281f
classical methods, 274–284
nuclease-based methods,
284–287, 284f
Genes, 203–204
review of, 204–209
screening. See Genetic screens; In silico
screens; Molecular screens
Genetic model organisms, 211b
relative advantages and disadvantages of
different organisms, 212t
Genetic screens, 209–213
forward genetic screen, 210–213
reverse genetic screen, 213
Genetically encoded calcium indicators
(GECIs), 173–175, 176f, 254
imaging synaptic transmission, 176–177
Genetically encoded voltage indicators
(GEVIs), 171, 173, 254
calcium indicator dyes, 174. See also
Calcium indicator dyes
imaging calcium dynamics,
173–175, 175f
Genomic DNA library, 223b–224b
Genotype, 203
Germ cells, 278
Gold labels, 154b–156b
Golgi stain, 152, 153f, 153b
Graded potential, 90–91
Gray matter, 2
Green fluorescent protein (GFP), 253
Ground electrode, 94b–96b
Ground-state depletion (GSD), 122b
Growth factors, 313
Guide RNA (gRNA), 285–286
Gyri, 33
H
Halorhodopsin (NpHR), 194, 256–257
Hanging wire assay, 46–47
Hargreaves assay, 50
Headstage, 94b–96b
Health Insurance Portability and
Accountability Act’s (HIPAA)
Privacy Rule, 28–29
Hemoglobin, 14
Herpes simplex virus (HSV), 249–250
Heterologous expression systems, 109
Histochemical stains, 151t. See also
individual entries
hM3Dq, 257
hM4Di, 257
Hodgkin, Alan, 105
Holding potential, 96–99
Homologous recombination, 275, 275f
Homology arms, 275–276
Homology-directed repair (HDR), 284–285
Hooke, Robert, 117
Horizontal plane, 149
Hot plate assay, 50–51, 51f
Human experiments
data acquisition, 30–31
figure preparation, 33–35, 34f
manipulating neural activity, 31–32
planning, 28–29
postexperimental data analysis, 32–35
working with humans, 29–30
Huxley, Andrew, 105
Hybridization, 234
Hyperpolarization, 90–91
Hypothesis generating, 209–210
Hypothesis testing, 209–210
I
I/V curves. See under Current (I)
Ibotenic acid, 190
Identifying DNA, 231–236
northern blot, 236
nucleic acid hybridization techniques,
234–236
sequencing, 233–234, 234f
Southern blot, 235–236
Imaging voltage, 171–173
voltage-sensitive dyes, 171–173, 173f
Immediate early genes (IEGs), 168
Immortalized cell lines, 298–301,
299t–300t
versus primary cell culture, 302t
Immunoblot. See Western blot (WB)
Immunoelectron microscopy (IEM), 326–327
Immunofluorescence (IF), 158
Immunohistochemistry (IHC), 158–160, 159f,
280, 325–326
Immunopanning, 303–304
382 Index
Immunoprecipitation, 319–320, 320f
Implants, for long-term access, 80–85
amperometry, 84–85
cannulae, 80–81
cranial windows, 81
electrophysiology, 81–82
fluorescent activity indicators, 82
microdialysis, 82–84
neural activity in vivo, 81, 84–85
neural chemistry in vivo, 82
sealable chambers, 80
voltammetry, 84–85
In silico screens, 214
BLAST, 214
Ensembl, 214
In situ hybridization (ISH), 156–158, 157f, 280
In vitro recordings, in electrophysiology,
108–112
experiment, 110b–111b
heterologous expression systems, 109
primary cultures, 109
slice cultures, 111–112
In vivo recordings, in electrophysiology,
112–113
acute versus chronic, 112
anesthetized versus awake animals, 112–113
In vivo techniques
manipulating neural activity, 86
measuring neural activity, 81
measuring neural chemistry, 82
microdialysis, 77–78, 82–84
Independent variable, 26
Index of refraction, 118–120
Indirect IHC, 158–159
Induced pluripotent stem (iPS) cells, 304,
306–307
Infection, 239, 307
Inhibitory postsynaptic potential (IPSP), 90–91
Insertional mutagenesis, 210
Inside-out recording, 107
Institutional Animal Care and Use Committee
(IACUC), 42b, 75
Institutional Review Board (IRB), 28–29
Intracellular recording, 89–90, 101f, 105
Intracellular signaling, 312–313, 312f
experiment, 333b–334b
Intracerebroventricular (i.c.v.) injection, 190
Intraperitoneal (i.p.) injection, 190
Introns, 208, 259
Inverted microscope, 124–125, 125f
Ion-exchange chromatography, 319
Irradiation mutagenesis, 210
Isoflurane, 77
Isolating DNA fragments, 220–227
isolation and characterization of, 226–227
polymerase chain reaction, 221–226. See
also Polymerase chain reaction (PCR)
restriction enzymes, 220–221, 221f
Isotropic diffusion, 13
J
JAK-STAT pathway, 313, 314f
Janus Kinases (JAKs), 313
K
Ketamine, 77
Kinase assay, 336, 336f
Knock down gene expression, 287
Knockin animal, 273
Knockin mouse, 282
Knockout animal, 273
Knockout mouse, 275–282
breeding offspring mice, 278–280
conditional, 282–284
creating targeting construct, 275–276, 276f
injecting and selecting ES cells, 276–277
inserting blastocyst into female mouse, 278
inserting ES cells into blastocyst, 277–278
using in experiment, 280–282
validating, 280
L
Label specific neural cell types, 158t
lacZ, 253
Lambda, 74–75
Learning and memory, 65
nonspatial, 54–56, 55b
spatial, 52–54
Lectins, 164
Leica (microscope manufacturer), 139
Lentivirus, 250
Lesion. See Ablation
Light emitting diodes (LEDs), 195
Light microscopy, 126–127, 127f
Linkage analysis, 213
Lipid transfection, 246–247
Lipofection, 246, 246f. See also Lipid
transfection
Liposomes, 246
Local field potentials, 101–103
Localized potential, 90–91
Locomotor activity, 44–45
exploratory, 46f
home cage activity, 44–45, 45f
Index  383
open field locomotion test, 45
running wheel, 44
Loss-of-function experiment, 186–187
Loudspeaker system, 92–93, 94b–96b
Lox sites, 282–283
Luciferase, 253
assay, 341–342, 341f
M
Magnetic resonance imaging (MRI), 6–12
diffusion. See Diffusion MRI
electromagnetic basis of, 6–7, 8f
features, 11
functional. See Functional magnetic
resonance imaging (fMRI)
image generation, 8–11
slice selection, 11–12
subject in, 7f
Magnetoencephalography (MEG),
20–22, 21f
Magnification, 118
Magnifying glass, 123f
Making transgenic organisms, 265–269
other organisms, 269
transgenic flies, 267, 268f
transgenic mice, 265–267, 266f
transgenic worms, 267–268, 269f
Manipulating neural activity, 185–186
electrical manipulation, 189
experimental logic of, 186–187
genetic manipulation, 191–192
methods of, 186f, 198t
optogenetic manipulation, 193–196. See
also Optogenetic manipulation; neural
activity
pharmacogenetic manipulation, 192–193
pharmacological manipulation, 189–191
physical manipulation, 187–189. See
also Physical manipulation; neural
activity
Mass spectrometry, 327, 329b–330b
Matrix-assisted laser desorption ionization
(MALDI), 329b–330b
Meganucleases, 285
Microarray, 215
Microdialysis, 82–84, 82f
Microdrive, 94b–96b
Microelectrode, 92–93, 94b–96b
Microinjection, 240–241
DNA, 241f
Microiontophoresis, 190–191
Micromanipulator, 94b–96b
Microscope, 94b–96b
Microscopy, 117–118. See also
individual entries
comparison of different forms of,
130f, 140t–142t
data preparation. See Microscopy data
preparation
fundamental parameters in, 118–121
principles, 118–126
resolving power, 118, 119f
Microscopy data preparation, 136–139
image interpretation, 137–139, 138f
image processing, 136–137
merging images, 137f
Microstimulation, 189
Microtome, 147–148, 148t
Midsagittal plane, 149
Mixed designs, 27–28
MNI (Montreal Neurological Institute)
template, 32–33
Molecular cloning, 219–220, 227
definition, 219
isolating DNA fragments, 220–227
Molecular screens, 214–216
cDNA microarray screen, 215–216, 215f
RNAi screen, 216
Monoclonal antibodies, 314–315
versus polyclonal antibodies, 315f
producing, 316f
Morpholinos, 289–290, 290f
Motor coordination and balance, 45–48
footprint pattern assay, 46
hanging wire assay, 46–47
rotarod, 46, 47f
vertical pole test, 47–48
Multielectrode array (MEA), 81, 103, 104f
Multipotent, 304
Muscimol, 190
Myelin, 152
N
Near-infrared spectroscopy (NIRS), 22
Neher, Erwin, 105
Neomycin resistance gene (neoR), 276–277
Nervous system visualizing, tissue
preparation, 146–150
embedding, 147
fixation, 146–147
intracellular labeling, 153, 154b–156b
juxtacellular labeling, 153, 154b–156b
neuroanatomical planes, 149f
sectioning, 147–150, 148f
384 Index
Neural activity in vivo, 81
Neural chemistry in vivo, 82
Neural progenitors, 305
Neural stem cells (NSCs), 304–306
Neuroethnology, 41b
Neuromodulation techniques, used in
humans, 197
Neuronal differentiation, 301f
Neurons, electrical properties of, 90–92
Neurospheres, 306, 306f
NIH ImageJ®, 136
Nikon (microscope manufacturer), 139
Nissl stains, 150
Nomarski microscopy, 127, 140t–142t.
See also Differential interference
contrast (DIC) microscopy
Nonhomologous end-joining (NHEJ),
284–285
Nonhuman primate behavioral
paradigms, 67–69
Nonratiometric dyes, 174
Nonspatial learning and memory, 54–56
delayed/nonmatch to sample, 55–56
novel object recognition, 54–55
Northern (RNA) blot, 225–226, 235f, 236
Nuclease, 284–285
Nuclease-based gene targeting methods,
284–287, 284f
Nucleic acid hybridization techniques,
225–226
Numerical aperture (NA), 118–120, 120f
O
Objective lens, 122
Ocular lens, 122
Ohm’s law, 90
Olfaction, 50
nociception, 50–51
Olfactory avoidance assay, 64
Olfactory jump response. See Chemosensory
jump assay
Open field locomotion test, 45
Open field test, 58, 58f
Operant conditioning, 55b
Optical imaging, 22–24, 23f
Optogenetic manipulation, neural
activity, 193–196
genetically encoded actuators, 193f
methods of delivering light, to nervous
system, 195, 195f
optogenetic transgenes. See Optogenetic
transgenes
Optogenetic transgenes, 193–194
optogenetic inhibition, 194
optogenetic stimulation, 194
Optogenetics, 193
stimulation of projections using, 196f
strategies for studying neural circuits
using, 196
Organotypic slice culture, 302
Oscilloscope, 92–93, 94b–96b
Outside-out recording, 107
P
P elements, 267, 268f
Packaging cells, 247–248
Particle-mediated gene transfer, 243
Passaging, 298
Patch clamp techniques, 89–90, 101f, 105–108
relative advantages and disadvantages
of, 107t
Pavlovian conditioning. See Classical
conditioning
Perforated patch, 107–108
Perfusion, 146–147
Peri-stimulus time histogram (PSTH),
101b–103b
Phage. See Bacteriophage
Pharmacogenetics, 192
Pharmacology, 308
Phase-contrast microscopy, 127, 140t–142t
Phenotype, 203
Phosphorylation, 335f
Photoactivated localization microscopy
(PALM), 122b
Photoactivation, 180
Photobleaching, 128–129, 180, 181f
Photoconversion, 154b–156b, 180
Phototaxis, 63
Phototoxicity, 128–129
Physical gene delivery, 239–243
biolistics, 243
categories, 240t
electroporation, 241–242, 242f
microinjection, 240–241
Physical manipulation, neural activity,
187–189
ablation, 187–188
thermal cooling, 188–189
Plasmids, 227–228
Pluripotent, 304
Polyclonal antibodies, 314–315
versus monoclonal antibodies, 315f
producing, 317f
Index  385
Polymerase chain reaction (PCR),
221–226, 221f, 280
modifying DNA sequences using, 225
PCR cycle, 222–224
quantitative real-time, 225–226
reverse transcription, 225
standard PCR, 221–224
Porsolt test. See Forced swim test
Position effects, 259–261
Positron emission tomography (PET), 17–19, 19f
Positron-emitting isotope, 17
Poststimulus time histogram, 101b–103b
Posttranslational modifications (PTMs), 208–209
investigating, 331–337, 335t
specific antibodies, 337, 337f
specific assays, 336–337
Prepulse inhibition (PPI), 50b
Primary antibody, 158–159
Primary cell/tissue culture, 301–304, 301f
dissociated cell cultures, 303–304
explant cultures, 303
versus immortalized cell lines, 302t
slice culture, 302
Primate behavioral task, 68f
Primers, 221
Proboscis extension response (PER), 64–65
Progenitor cells, 304
Promoters, 206, 258
Protein expression, investigating, 320–327
ELISA. See Enzyme-linked immunosorbent
assay (ELISA)
immunohistochemistry, 325–326
methods of measuring, 321t
radioimmunoassay, 324–325
western blot. See Western blot (WB)
Protein kinases, 313
Protein–DNA interactions, 337–342
chromatin immunoprecipitation,
339–341, 340f
electrophoretic mobility shift assay,
338–339, 338f
luciferase assay, 341–342, 341f
Protein–protein interaction, 327–331
co-immunoprecipitation, 327, 328f
protein affinity chromatography, 328–330
yeast two-hybrid assay, 330–331, 331f–332f
Proteins, 203–204, 311
definition, 203
and DNA interaction. See Protein–DNA
interactions
expression. See Protein expression,
investigating
fundamental tools used, 313–320
and protein interaction. See Protein–protein
interaction
purifying, 318–320
review of, 204–209
tagged, 317b
Proteomics, 329b–330b
Protons
alignment in magnetic field, 6, 8f
process around axis, 7, 9f
Protospacer adjacent motif (PAM),
286–287
Pseudopregnant, 265
Pulse-chase labeling, 170
Q
Quantitative real-time PCR (qRT-PCR),
225–226, 225f
Quantum dots, 154b–156b
R
Radioactive amino acids, 164
Radioactive labels, 154b–156b
Radiofrequency (RF) pulses, 8–9, 9f
Radioimmunoassay (RIA),
324–325, 326f
Raster plot, 101b–103b
Ratiometric dyes, 174
Recognition sites, 220
Recombinant DNA plasmid, 230f
Recombinant DNA technology, 219–238
Recombineering, 229–230, 231b
Refractive index, 118–120
Region-of-interest (ROI) analysis, 33
Relative recovery, 83
Reporter genes, 160, 161f, 177, 254
Reporter proteins, 327
Resistance (R), 90
Resolution, 118
diffraction limit of, 122b
Resolving power, 118, 119f. See also
Resolution
Resting potential, 90
ionic basis of, 91f
Restriction digest, 220–221
Restriction endonucleases. See
Restriction enzymes
Restriction enzymes, 220–221, 221f
Restriction sites, 220
Retrograde tracers, 162–164, 162f, 163t
Reverse genetic screen, 209–210, 213
Reverse genetics, 273–274
Reverse microdialysis, 83
386 Index
Reverse transcription PCR (RT-PCR), 225
Reward-related behaviors, 56–57
conditioned place preference, 57
self-administration, 56–57
Ribosomes, 208–209
RNA
RNA-induced silencing complex (RISC),
287–288
polymerase, 206
splicing, 208f
RNA interference (RNAi) screen, 216,
287–289, 288f
Rodent behavioral paradigms, 44–61
anxiety, 58–60. See also Anxiety
attention and impulsivity, 56
depression, 60–61. See also Depression
locomotor activity, 44–45. See also
Locomotor activity
motor coordination and balance, 45–48. See
also Motor coordination and balance
nonspatial learning and memory. See
Nonspatial learning and memory
reward-related behaviors, 56–57
sensory function, 48–50. See also
Sensory function
social behaviors, 57–58
spatial learning and memory. See Spatial
learning and memory
S
Sagittal plane, 149
Sakmann, Bert, 105
Sanger dideoxy chain-termination method,
233–234
Saturated structured-illumination microscopy
(SSIM), 122b
Scanning electron microscopy (SEM),
133–135, 140t–142t
Sealable chambers, 80
Secondary antibody, 158–159
Sensory behavior, 66–67
chemosensation, 67
mechanosensation, 66–67
thermosensation, 67
Sensory function, 48–50, 63–65
formalin assay, 51
Hargreaves assay, 50
hot plate assay, 50–51, 51f
olfaction, 64. See also Olfaction
prepulse inhibition (PPI), 50b
startle response assay, 49
tail flick assay, 50
taste, 49, 64–65
T-maze, 64f
vision, 63
visual cliff assay, 48, 49f
Von Frey assay, 51
Short hairpin RNA (shRNA), 288–289, 289f
Signal Transducers and Activators of
Transcription (STATs), 313
Signal transduction, 313
Single electrodes, 81
Single-proton emission computerized
tomography (SPECT), 19–20, 21f
Slice culture, 302
Slices, 12f, 28–29
definition, 11
selection, 11–12
Small interfering RNA (siRNA), 287–288
Social behaviors, 57–58, 65
aggression, 65
courtship, 65
resident-intruder assay, 57
social approach/avoidance, 58
Sodium dodecyl sulfate (SDS) polyacrylamide
gel electrophoresis (PAGE), 320–321
Southern (DNA) blot, 220–2270, 235f, 280
Spatial learning and memory, 52–54
Barnes maze, 53, 53f
Morris water maze, 52–53, 52f
radial arm maze, 53–54, 54f
Spatial resolution, 11
Spike, 91. See also Action potential
Spike sorting, 103–104
Spliceosome, 208
Splitting, 298
Startle response assay, 49
Static markers, nervous system’s
function, 168–170
assaying cellular function. See Assaying
cellular function
assaying neural activity, 168–169, 169f
Stem cell cultures, 304–307
embryonic stem cells, 304–305
induced pluripotent stem cells, 306–307
neural stem cells, 305–306
Stereomicroscope, 125–126, 126f
Stereotaxic instrument, 74–75, 75f
Stereotaxic surgeries
and in vivo techniques, 73–88
in nonhuman primates, 79–80
in rodents. See Stereotaxic surgeries; in rodents
Stereotaxic surgeries, in rodents, 73–80
accessing brain, 78
anesthesia, 77–78
Index  387
finishing surgery, 79
penetrating brain, 78–79
positioning in instrument, 78
sterile environment, 76
Sterile environment, 76
equipment, 77f
Sticky ends, 220–221
Stimulated emission depletion (STED), 122b
Stochastic optical reconstruction microscopy
(STORM), 122b
Structural brain imaging, 2–13
cerebral angiography, 3, 5f
computerized tomography, 4–6, 5f
magnetic resonance imaging. See Magnetic
resonance imaging (MRI)
Subcloning, 227, 232b–233b
Sulci, 33, 34f
Synaptic transmission, 176–177
dynamics, 177f
FM dyes, 176
pH-sensitive fluorescent proteins, 176–177
T
T1-weighted image, 10–11, 10f
T2-weighted image, 10–11, 10f
Tagged proteins, 317b
Tail flick assay, 50
Talairach space, 32–33
TaqMan assay, 225f
Task paradigms, 27–28
relative advantages and disadvantages of
different paradigms, 28t
Temporal delay, 15–17
Temporal resolution, 15–17
Tetracycline transactivator (tTA) protein, 264
Tetraethylammonium (TEA), 189–190
Tetrodes, 103, 104f
Tetrodotoxin (TTX), 189–190
The Principles of Humane Experimental
Research (Russell and Burch), 42b
Thermal cooling, 188–189
Thermocyclers, 224
Thymidine analogs, 169–170
Time of flight (TOF) detector, 329b–330b
Total internal reflection fluorescence (TIRF)
microscopy, 132–133, 133f, 140t–142t
Transcranial magnetic stimulation (TMS),
31–32, 31f, 197
Transcription, 206–208
of DNA into RNA, 207f
Transcription activator-like effector nucleases
(TALENs), 285
Transcription factors, 206, 259
Transcriptional start site, 259
Transfection, 239, 307
calcium phosphate transfection,
244–246, 245f
lipid transfection, 246–247
Transgenes, 253–258
to ablate neurons, 257
commonly used, 255t–256t
to disrupt endogenous gene function,
257–258
to manipulate neural activity, 256–257
to measure neural activity, 254
overexpression of endogenous genes, 258
reporter genes, 254
Transgenic organisms, 253–272
binary systems. See Binary transgenic
systems
definition, 253–254
making. See Making transgenic organisms
transgenes. See Transgenes
transgenic construct, 258–261, 258f
Translation, 208–209
of mRNA to protein, 209f
Transmission electron microscopy (TEM),
133–134, 140t–142t
Transposable elements, 267
Transposase, 267
Transposition, 267
Transposons, 210, 267
Transsynaptic labeling, 164–165
plant lectins, 164
radioactive amino acids, 164
viruses, 165
Trophoblast, 277–278
Two-photon laser scanning microscopy
(TPLSM), 132
Two-photon microscopy, 132, 140t–142t
U
Ultrasonic neuromodulation (USNM), 32, 197
Unconditioned stimulus (US), 55b
Unipotent, 304
Untranslated regions (UTRs), 208
Upright microscope, 124–125, 125f
Upstream activation sequence (UAS), 262
V
Vectors, 227–229
features, 228
types of, 228–229
viral, in neuroscience, 249t
388 Index
Vertical pole test, 47–48
Vibration isolation system, 94b–96b
Vibratome, 148, 148t
Viral gene delivery, 247–250, 247f
adeno-associated virus, 250
adenovirus, 248
canine adenovirus, 248
herpes simplex virus, 249–250
lentivirus, 250
in neuroscience, 249t
Visible spectrum of light, 121f
Visual cliff assay, 48, 49f
Visualizing circuitry, 162–165
anterograde tracers, 162–164, 162f, 163t
retrograde tracers, 162–164, 162f, 163t
transsynaptic labeling. See
Transsynaptic labeling
Visualizing gene and protein expression,
156–160
enzymatic histochemistry, 160, 161f
immunohistochemistry, 158–160, 159f
in situ hybridization, 156–158, 157f
reporter genes, 160, 161f
Visualizing nervous system, function, 167–168
neural activity. See Visualizing neural activity
static markers of activity. See Static markers,
nervous system’s function
Visualizing nervous system, structures,
145–146
morphology, 150–153
tissue preparation. See Nervous system
visualizing, tissue preparation
Visualizing neural activity, 171–177
imaging voltage, 171–173. See also Imaging
voltage
Visualizing protein function, 177–180
bimolecular fluorescence
complementation, 180
comparison of techniques, 178t
fluorescence recovery after
photobleaching, 180
fluorescence resonance energy transfer,
177–180
photoactivation, 180
photoconversion, 180
time-lapse imaging with reporter
genes, 177, 179f
Voltage clamp, 109
Voltage-sensitive dye imaging (VSDI), 171
Voltammetry, 84–85
analyzing and depicting data, 85f
Von Frey assay, 51
von Leeuwenhoek, Anton, 117
Voxel, 11, 28–29
Voxelwise analysis, 33
W
Western blot (WB), 280, 320–323, 322f–323f
White matter, 2
Whole brain imaging, 1–2
functional brain imaging, 14–24. See also
Electroencephalography
(EEG); Functional magnetic
resonance imaging (fMRI);
Magnetoencephalography (MEG);
Optical imaging; Positron emission
tomography (PET); Single-proton
emission computerized tomography
(SPECT)
structural brain imaging, 2–13. See also
Cerebral angiography; Computerized
tomography (CT); Magnetic resonance
imaging (MRI)
Whole-cell recording, 106–107
Whole-mount preparations, 149
Wide-field fluorescent microscopy, 129
Within subjects, 26–27
Worm Tracker (software), 66, 66f
www.brain-map.org (online resource), 157
X
Xanthomonas bacteria, 285
Xenopus laevis oocytes, 263f, 289–290
X-gal, 333b–334b
X-ray technology, 2–3, 4f
Xylazine, 77
Y
Yeast protein. See Gal80
Yeast two-hybrid assay, 330–331, 331f–332f
Z
Zebra fish (Danio rerio), 129, 211b, 212t,
263f, 289–290
Zeiss (microscope manufacturer), 139
Zinc finger nucleases (ZFNs), 285