Chapter 5 Physiology

Chapter 5

Methods and Strategies

of Research

Neurons in the cortex labeled with a Ruorescent dye.

Chapter Outline

Experimental Ablation

Evaluating the Behavioral Effects of Brain

Damage

Producing Brain Lesions

Stereotaxic Surgery

Histological Methods

Tracing Neural Connections

Studying the Structure of the Living Human

Brain

Recording and Stimulating Neural Activity

Recording Neural Activity

Recording the Brain's Metabolic and Synaptic

Activity

Stimulating Neural Activity

Neurochemical Methods

Finding Neurons That Produce Particular

Neurochemicals

Localizing Particular Receptors

Measuring Chemicals Secreted in the Brain

Genetic Methods

Twin Studies

Adoption Studies

Genomic Studies

Targeted Mutations

Antisense Oligonucleotides

CRJSPR-Cas Methods

105

106 Chapter 5

LO 5.1 Explain what researchers can learn from

lesion studies.

LO 5.2 Compare methods of producing brain

lesions.

LO 5.3 Describe the process of stereotaxic

surgery.

LO 5.4 Summarize the steps of histological

methods.

LO 5.5 Compare techniques for tracing efferent

and afferent axons.

LO 5.6 Contrast methods to study the structure

of the living human brain.

LO 5.7 Compare methods of recording neural

activity.

LO 5.8 Compare methods for assessing

metabolic and synaptic activity.

LO 5.9 Compare methods of neural stimulation.

LO 5.10 Describe methods to identify neurons

that produce a particular neurochemical.

In the summer of 1982, severaJ young people began showing

up at neurology clinics il northern Galifomia displayilg dramatic

-toms (Langston et al., 1983). The most severely affected

patients wem almost co~letety paralyzed. They were unable

to speak clearly, they saivated constantly, and their eyes were

open with a ftxed stare. 0th8'S, less S8'V8(8Iy affected. walked

with a slow, shutting gait and moved with great drfficulty. The

symptoms looked like those of Parkinson's disease. but that

disorder has a Ve<y gradual onset, usually in older adulthood.

These patients wem al in their twenties or earty thirties.

The comnon factor linking these patients was i'ltravenous

drug use. Al of the patients had used a synthetic opiate. The

illicit aug was contartinated with MPTP. a toxic chemical that

damagocl dopaminergic n01Xons and caused the patients' neu•

rological symptoms. Because the symptoms looked li<e those

of Parkinson's disease, the patients were given L•OOPA, the

dopamine p<ecursor drug used to treat this disease. and they

all showed significant improvement in their symptoms. Unfom.J·

nately. the improvement was temporary. and the drug lost its

effectiveness.

Two indMduals affected by the MPTP traveled to SWeden

to receive fetal tissue transplants containing dopamine-secretr,g

neurons. This tissue was transplanted into the caudate and

putamen with the hope that the new neurons from the tissue

LO 5.11 Compare methods to localize particular

receptors.

LO 5.12 Compare methods used to examine

chemicals secreted in the brain.

LO 5.13 Describe how twin concordance rates can

be used to assess genetic contributions to

a behavior.

LO 5.14 Evaluate the role of adoption studies

in assessing genetic contributions to a

behavior.

LO 5.15 Identify genomic techniques used to

study physical and behavioral traits.

LO 5.16 Summarize how targeted mutations can

be used to study genetic contributions to

a behavior.

LO 5.17 Describe how antisense oligonucleotides

function to change behavior.

LO 5.18 Summarize the uses of CRISPR-Cas

methods in neuroscience research.

would survive and begin to produce dopamine, diminishing

the Parkinson's disease•like symptoms that the patients were

experien:ing.

Before the transplant took place, one of the patients

was given an injection of radioactive vOOPA Then. one hall

later, he was given a PET scan. His head was positioned in

the scanner, and f0< the next several minutes the machine

gathered data from SUJaton-»e particles that we<e emitted as

the radioactive L·OOPA in his brain broke down. This data re•

vealed the extent and location of damage to the dopamine

system.

A few weeks lat&<, the patient was admitted to the hospi•

tal for his surgery. Tedmicians removed dopaminergic neurons

from the substantia nigra of several fetal brains and prepared

them for implantation into the patient's brail. The patient was

anesthetized. and the surgeon made cuts in his~ to expose

parts of his skul. The surgeon attached the frame of a st8'90•

taxic apparatus to the patient's skull. made some measure•

ments based on a map of the patient's brain, and then drilled

several holes. He used the stereotaxic apparatus to gt.ide the

injections of the fetal neurons into the patient's caudate nucleus

and put.amen. Onoe the i"fections were completed. the sugeon

removed the st8'80taxic frame and sutured the incisions he had

made in the scalp.

The operation was quite successful , and the patient re•

covered much of n s motor control. A litUe more than a year

later, he was given another injection of radioactive L·OOPA,

and again underwent a PET scan. The results of the sec•

ond scan showed what his recovery implied: The trans•

planted cells had survived and were secreting dopamine.

You can view the results of his PET scans in Figure 5.32 in

this chapter.

This case highlights several importa nt methodological

concepts explored in this chapter. Chemical lesioning, PET

scan imaging,, and stereotaxic surgery are alJ methodologi~

cal tools used by researchers as they try to better under•

stand the structure and function of the nervous system and

develop effective treatments for disease.

Behavioral neuroscience rescard-, involves the efforts

of scientists in many disciplines, including physiology,

neuroanatomy, biochemistry, psychology, endocrinology,

and histology. Pursuing research in behavioral neurosci~

cncc requires skilJ and knowledge in many experimental

techniques. Because different procedures can produce

contradictory results, investigators must be familiar with

the advantages and limitations of lhc methods that are

used. Researchers might receive a puzzling answer, only

to realize later that they were not asking the question

they thought they were. As we will see, the best conclu•

sions about behavioral neuroscience don't come from a

single experiment but from a program of research that en~

ables us to compare the results of studies using different

methods.

An enormous-and potentiaHy confusing-array of

research methods is available to researchers in behavioral

neuroscience. A reader could get lost-or lose interest- if

these methods were presented in a long list. Instead, we

will present some of the most important and commonly

used procedures, arranged by similarities. Our goal is to

make it easier to understand the advantages, disadvan~

tages, and types of information produced by different

types of methods. It will also help explain the strategies

that researchers employ as they follow up the results of one

experiment by designing and conducting another one.

The first module begins with various methods of ex•

perimental ablation. The second looks at how researchers

stimulate and record neural activity. Then the third and

fourth modules examine neurochemical and genetic meth~

ods using in behavioral neuroscience research.

Experimental Ablation

An important research method used to investigate brain

functions involves removing or inactivating part of the

brain and evaluating an animal's subsequent behavior.

This method iscal1ed experimental ablation. Experimental

Methods and Strategies of Research 107

Despite the devastating effects of accidental administration

in this group of patients, MPTP is now considered an impor•

tant tool in Parkinson's disease research. Its neurotoxic effects

make MPTP useftA tor creatilg selective chemical lesions of the

dopamine system and producing a model of the ~toms of

Part<inson's disease. Research8'S oow rety on the MPTP model

in laboratory animals to test the effectiveness of new treatments

f0< the dsease.

ablation can involve removing brain tissue, or damaging

the tissue to disrupt its functioning. Experimental ablation

is one of the oldest methods used in neuroscience.

Evaluating the Behavioral Effects

of Brain Damage

LO 5.1 Explain what researchers can learn from

lesion studies.

A lesion is a wound or injury, and a researcher who destroys

part of U,e brain usually refers to U,e damage as a brain

lesion. Experiments in which part of the brain is damaged

and the individual's behavior is subsequently observed

arc cal1ed lesion studies. Intentional brain lesioning is performed

in animals. In addition, the behavioral results of

naturally occurring lesions, such as those that result from

accide ntal in jurie5, o r 5h'OkC$, can be 5tudied in human rcseard,

participants.

Just what can we learn from lesion studies? The goal is

to discover what functions are performed by different regions

of lhe brain and then to understand how these func~

tions are combined to accomplish particular behaviors. The

distinction behvccn brain function and bel1avior is an important

one. Circuits within the brain perform functions, not

behaviors. No one brain region or neural circuit is solely

responsible for behavior. Each brain region pcrfonns a

function (or set of functions) lhat contributes to the performance

of the behavior.

For example, the act of reading involves functions required

for controlling eye movements, focusing the lens

of lhe eye, perceiving and recognizing words and letters,

comprehending the meaning of the words, and so on. Some

of these functions also participate in other behaviors. For

example, controlling eye movement and focusing arc required

for any task lhat involves looking, and brain mecha~

nisms used for comprehending the meanings of words also

participate in comprehending speech. The researcher's

task is to understand the functions that arc required for

performing a particular behavior and to determine what

circuits of neurons in the brain are responsible for each of

these functions.

Another example that highlights the role of neural

circuits in behavior can be seen in drug•taking behavior.

As you'll read in Chapter I 9, dopamine-secreting cells

108 Chapter 5

located in the ventra) tegmenta1 area have their terminal

buttons located in the nucleus accumbens. This pathway

is involved in the reinforcing effects of many behaviors,

including drug use. Lesioning cells in this pathway docs

red uce drug-taking, but only for certain drugs. Even

lesioning the entire mesolimbic pathway may not com•

pletely eliminate the behavior (Pierce & Kumaresan,

2006). Drug-taking is a complex behavior that involves

multiple circuits in the brain.

Interpreting lesion studies is complicated by the fact

that all regions of the brain are interconnected. Suppose

that we have a good understanding of the functions re•

quired for the performance of a particular behavior. We

find that damage to one specific brain structure impairs

a particular behavior. Can we necessarily conclude that

a function essential to this behavior is performed by cir•

cuits of neurons located in this one specific structure?

Unfortunately, we cannot. The functions we arc inter•

csted in may actually be performed by neural circuits

located elsewhere in the brain. Damage to one structure

may simply interfere with the activity of the neural cir•

cuits in a different structure.

Producing Brain Lesions

LO 5.2 Compare methods of producing brain lesions.

How arc brain lesions produced experimentally? Usually,

a researcher wants to inactivate regions that arc hidden

away in the depths of the brain. Brain lesions of sub•

cortical regions (regions located beneath the cortex) are

usually produced by passing an electrical current through

a stainless steel wire that is covered with an insulating

coating except for the very tip. The wire is then guided

to its destination using exact coordinates to a precise lo,.

cation within the brain. The researcher then activates a

lesion•making device, whid, produces a radio frequency

(RF) current- an alten,ating current of a very high fre•

quency. Passing the RF current through the brain tissue

produces heat that kills cells in the region surrounding

the tip of the electrode.

Lesions produced using this technique destroy

everything in the vicinity of the electrode tip, includ•

ing neural cell bodies and the axons of neurons tha t

pass through the region. A more selective method of

producing brain lesions employs an excitatory amino

acid, such as kniuic ncid, which kills neurons by stimu•

lating them to death. Lesions produced in this way are

referred to as excitotoxic lesions. When an excitatory

amino acid is injected through a cannula (a small metal

tube) into a region of the brain, the chemical destroys

neural cell bodies in the vicinity but spares axons that

belong to different neurons that happen to pass nearby.

(See Figure 5.1.) This selectivity permits the investiga•

tor to determine whether the behavioral effects of de•

st:roying a particular brain structure arc caused by the

death of neurons located there or by the destruction of

axlm!:> that pas:, 11ca rby. Fur t:Xdlllplc, :some n ..- :,t:a rdtcr:;

discovered that RF lesions of a particular region in the

brain stem abolished REM sleep and concluded that this

region was involved in the production of this stage of

sleep. (REM sleep is the stage of sleep during which

Figure 5.1 Two Methods of Producing Brain Lesions

Radio Frequency Lesion

Radio frequency lesions destroy cell

bodies, axons. and t&rminals

in the region or the

e:lectrod&.

ExoitotoxtC Lesion

Ex.citotoxic lesions destroy

ce:11 bOdies in the region

wrie:r& the Chemical is

in;ected.

dreaming occurs. You will learn more about this topic

in Chapter 9.) But later s tudies showed that when kainic

acid was used to destroy the neurons loca tcd there,

the animals' sleep was not affected. Therefore, the RF

lesions must have altered sleep by destroying the axons

that pass through the area.

E\•en more specific methods of targeting and killing

particular types of neurons are available. For example,

molecular biologists have devised ways to conjugnte (attach

together) saporin, a toxic protein, and antibodies that

will bind with particular proteins found only on certain

types of neurons in the brain. The antibodies target these

proteins, and the saporin kills the only cells to which the

proteins are attached.

Note that when subcortical lesions are produced by

passing RF current through an electrode or infusing a

chemical through a cannula, there is always additional

damage caused to the brain. When an electrode or a can•

nula is passed through the brain to get to a targel, it inevitably

causes a small amount of damage even before

turning on the lesion maker or starting the infusion. As a

result, we cannot simply compare the behavior of brain•

lesioned animals with that of unopcrated control animals.

The incidental damage to the brain regions above the

lesion may actually be responsible for some of the behavioral

deficits we see. To control for this, researchers

typkal1y im.-:lm..lic dll dc..kJ itio11a l g roup of an i1m1l:, in <1 lie·

sion study and produce sham lesions. To produce sham

lesions, researchers anesthetize each animal, insert the

electrode or cannula, and lower it lo the proper depth. In

other words, they do everything they would do to pro•

duce the lesion except tum on the lesion maker or start

the infusion. This group of animals serves as a control

group. If the behavior of the animals with brain lesions is

different from that of the sham~perated control animals,

we can conclude that the lesions caused the behavioral

deficits. (A sham lesion sen,es the same purpose as a pla•

cebo does in a pharmacology study.)

Most of the time, investigators produce permanent

brain lesions, but sometimes it is advantageous to disrupt

the activity of a particular region of the brain temporar•

Hy. The easiest way to do so is to inject a local anesthetic

or a drug called nmscimol into the appropriate part of the

brain. The anesthetic blocks action potentials in axons en•

tering or leaving that region, and effectively produces a

temporary lesion (usually called a reversible brain lesion).

Muscimol, a drug that stimulates GABA receptors, in•

activates a region of the brain by inhibiting the neurons

located there. (RecaH that GABA is an important inhibi•

tory neurotransmitter in the brain.) Another technique,

optog-enetics, can also be used to temporarily inhibit, or

in some cases stimulate brain regions. You will read more

about this technique later in the chapter.

Methods and Strategies of Research 109

Stereotaxic Surgery

LO 5.3 Describe the process of stereotaxic surgery.

How do researchers g-et an electrode or cannula to a precise

location in the depths of an animal's brain? The an•

swer is stereotaxic surgery. Stereotaxis refers to the ability

to locate objects in space. A stereotaxic nppnratus contains a

holder that keeps the animal 's head in a standard position

and an arm that moves an electrode or a cannula through

measured distances in all three axes of space. However, to

perform stereotaxic surgery, a researcher first consults a

stereotaxic ntlns.

THE STEREOTAXIC ATLAS A stereotaxic atlas isa book,

website, or sofhvare that contains images that correspond

to frontal sections of the brain taken at various distances

rostral and caudal to bregma. The skull is composed of

several bones that grow together and form sutures (seams).

The heads of babies contain a soft spot at the junction of

the coronal and sagittal sutures called the fo11tmiel/e. Once

this gap closes, the junction is called bregma, from the

Greek word meaning "front of head." No two brains of

animals of a given species are completely identica 1, but

there is enough similarity among individuals to predict the

location of particular brain structures relative to external

features of the head. We can find bregma on a rat's skull,

tnn, a nrl it c;;f'rvM ;ic;;.,. rnnvPniPnt rf'fPrPnrP point Figurp

5.2 is a drawing of a slice of the brain that contains a brain

structure {shown in red) that we are interested in. If we

wanted to place the tip of a wire in this structure (a bundle

of axons called the fomix), we would have to drill a hole

through the skull immedialely above it. Each image of the

stereotaxic atlas is labeled according to the distance of the

section anterior or posterior to bregma. The grid on each

image indicates distances of brain structures ventra l to

the top of the skull and lateral to the midline. To place the

tip of a wire in the fomix, a researcher would drill a hole

above the target and U,en lower the eleclrode through the

hole until the tip was at the correct depth, relative to the

skull height at bregma. By finding a brain structure (which

cannot be seen from the outside of the skull) on one of the

images of a stereotaxic atlas, the researd,er can determine

the structure's location relative to brcgma (which can be

seen from the outside of the skull). Beca use of variations

in different strains and ages of animals, the atlas gives only

an approximate location. Researchers always have to try

out a new set of coordinates, slice and stain the animal's

brain, see the actual location of the lesion, correct lhe num•

be-rs, and try again. (Slicing and staining of brains are described

later.)

THE STERE0TAXIC APPARATUS A stereotaxic

apparatus is a device that i ncludes a head holder, which

mai ntai ns the a nimal?s skull in the proper orientation;

110 Chapter 5

Figure 5.2 Stereotaxic Atlas

This sample page from a stereotaxic atlas of the rat brain shows tlhe target (the fornix) in red.

Labels have been removed for the sake of clarity.

Source: Adapted from Swanson, L. W. (1992). Brain maps: Structure of the rat brain. New York: Elsevier.

Bregma

~~~~~~~~~~~-- ~

In this example, the

target location is

approximately 6 units

inferior of bregma, and

1 unit lateral to bregma

.!!? r--+-_,_-μ,...::;:~=:::::.---r.!!:!~~ ---=~ ~ ::::::...+-+-+---l

§ .__....___ _ ......._ ........ _. _ __._ __ _._ ..........._.~ .............

Units - - - -►

a holder for an electrode or cannula, and a calibrated

mechanism that moves the electrode/cannula holder in

measured distances along the three axes: anterior-posterior,

dorsal-ventral, and lateral- medial. Figure 5.3 illustrates

a stereotaxic apparatus designed for small animals.

The size of the stereotaxic apparatus can be scaled up or

down to be used for different species.

Once a researcher obtains the coordinates from a stereotaxic

atlas, they anesthetize the animal, place it in the apparatus,

and cut the scalp open. The researcher will locate

bregma, dial in the appropriate numbers on the stereotaxic

Figure 5.3 Stereotaxic Apparatus

This apparatus is used for performing brain surgery on rats or mice.

Electrode

in brain

~ Adjusting

knobs

J

-0 '

apparatus, drill a hole through the skull, and lower the device

into the b1rain by the correct amount. Now the tip of the

cannula or electrode is where the researcher wants it to be,

and the researcher is ready to produce the lesion.

Stereotaxic surgery may be used for purposes other than

lesion production. Wires placed in the brain can be used to

stimulate neurons as well as to destroy them, and drugs can

be injected that stimulate neurons or block specific receptors.

A researclher can attach cannulas or wires permanently

by following a procedure that will be described next. In all

cases, once the surgery is complete, the scalp incision is

sewn together,. and the animal is taken out of the stereotaxic

apparatus and allowed to recover from the anesthetic.

Stereotaxic apparatuses are also made for humans.

Sometimes a neurosurgeon produces subcortical lesionsfor

example, to reduce the symptoms of Parkinson's

disease, a trceatrnent you'll encounter in Chapter 16.

Usually, the surgeon uses multiple landmarks and verifies

the location of the wire (or another device) inserted into

the brain by ttaking brain scans or recording the activity

of the neurons in that region before producing a brain

lesion. Deep !brain stimulation is another procedure that

requires using: a stereotaxic apparatus. Deep brain stimulation

is used to treat conditions that include chronic pain,

movement disorders (including Parkinson's disease),

epilepsy, depression, and obsessive-compulsive disorder.

Deep brain stimulation utilizes a stereotaxic apparatus to

implant a permanent electrode into the brain of patients.

(See Figure 5.4.) Rather than produce a lesion, electrical current

passed through the electrode is used to stimulate brain

regions and rieduce symptoms (Holtzheirner & Mayberg,

2011a; Sarem-Aslani & Mullett, 2011). The applications of

Figure 5.4 Stereotaxic Apparatus on a Human Patient

this method continue to grow (for a brief review, see Deeb

et al., 2016; Hariz, 2014).

Histological Methods

LO 4.4 Summarize the steps of histological methods.

After producing a brain lesion and observing its effects

on an animal's behavior, researchers must thinly slice and

stain the brain so that they can observe it under the microscope

and see the location of the lesion. Brain lesioning

can miss the mark, so researchers have to verify the precise

location of the brain damage after testing the animal

behaviorally. To do so, histologists (specialists in these

techniques) must fix, slice, stain, and examine the brain.

Together, these procedures are referred to as histological

methods. (The prefix histo- refers to body tissue.)

Figure 5.5 Microtome and Cryostat

Stage where

frozen brain

is affixed

(a) Microtome

Blade

Methods and Strategies of Research 111

FIXATION AND SECTIONING To study brain tissue, it

must be protected from autolytic enzymes (autolytic means

"self-dissolving"), which will otherwise break down the

tissue,. making it impossible to study. The tissue must also

be preserved to prevent its decomposition by bacteria

or molds. To achieve both of these objectives, neural tissue

is placed in a fixative. Commonly used fixatives are

formalin or paraformaldehyde, aqueous solutions of formaldehydle,

a gas. Fixatives cross-link proteins to strengthen

the very soft and fragile brain tissue and kill any microorganisms

that might destroy it.

Before the brain is fixed (that is, put into a fixative solution),

it is usually perfused. Perfusion of tissue entails

the removal of the blood and its replacement with another

fluid. The animal's brain is perfused because better histological

results are obtained when no blood is present in the

tissue. The animal whose brain is to be studied is humanely

euthanized with an overdose of a general anesthetic. Blood

is removed from the vessels and replaced with a dilute

salt sc,lution. Finally, a dilute fixative solution is pumped

through the tissue, and the brain is removed from the skull

and placed in a container filled with the fixative.

Once the brain has been fixed, it must be sliced into

thin sections and stained for various cellular structures in

order to see anatomical details. Slicing is done with a microtome

or a cryostat. A microtome contains three parts:

a knif,e, a platform on which to mount the tissue, and a

mechamism that advances the knife (or the platform) the

correct distance after each slice so that another section

can be cut. In most cases, the platform includes an attachment

'that freezes the brain to make it hard enough to be

cut inlto thin sections. Figure 5.5 shows a microtome and

-.......--- Temperature

(b) Cryostat

and slicing

controls

Brain is

frozen and

sliced inside

cryostat

112 Chapter 5

a cryostat. A cryostat is similar to a microtome; however,

the entire cutting process occurs within a freezer, allowing

sections to be cut at very cold temperatures. In some cases,

a researcher may need to work quickly with unfixed tissue

and a cryostat may be the tool of choice. After the tissue is

cut, the slices are attached to glass microscope slides. A researcher

can then stain the tissue by putting the entire slide

into various chemical solutions.

STAINING If you looked at an unstained section of

brain tissue under a light microscope, you would be

able to see the outlines of some large cellular masses

and the more prominent fiber bundles. However, no fine

details would be visible. For this reason, the study of

microscopic neuro-anatomy requires special histological

stains. Researchers have developed many different

stains to identify specific subs tances within and outside

of cells. For verifying the location of a brain lesion,

many researchers use one of the simplest stains: a cellbody

stain.

Methylene blue and cresyl violet are two examples of

dyes that stain the cell bodies of brain tissue. The material

that takes up the dye within the cell, known as the Niss[

substance, consists of RNA, DNA, and associated proteins

located in the nucleus and scattered, in the form of granules,

in the cytoplasm. Figure 5.6 shows a frontal section

of a brain stained with cresyl violet. Note that you can observe

fiber bundles by their lighter appearance; they do not

take up the stain. The stain is not selective for neural cell

bodies; all cells are stained, neurons and glia alike. It is up

to the investigator to determine which cell type is whichby

size, shape, and location.

Figure 5.6 Frontal Section of Brain Tissue Stained

with Cresyl Violet

The section is stained with cresyl violet, a cell-body stain. The

arrowheads point to nuclei, or groups of cell bodies.

Source: Histological material courtesy of Mary Carlson.

Other staining techniques frequently used in neuroscience

researc:h include Golgi staining for whole cells, reduced

silver staining for nuclei and cytoskeletal proteins,

and hematoxylin and eosin to distinguish between nuclear

and cytoplasmic inclusions. Finally, the stained sections

are covered with a small amount of a transparent liquid

known as a mounting medium, and a very thin glass coverslip

is placed over the sections. The mounting medium

keeps the coverslip in position.

LIGHT MICROSCOPY Once the tissue has been fixed,

sectioned, stained, and coverslipped, researchers may

use a light microscope to examine tissue mounted on

microscope s:lides. Light microscopes are less expensive

than other types of microscopes and can be used

for a wide v.ariety of research that requires viewing

cells under magnification. (See Figure 5.7.) Light microscopes

typically provide between 40 and 100 times

magnification. This level of magnification allows a researcher

to view whole cells, but other types of microscopy

are requiired to see subcellular structures at higher

magnifica tiom.

ELECTRON MICROSCOPY While many researchers use

light microscopes to examine stained tissue, the light microscope

is limited in its ability to reveal extremely small

details. To seie very small anatomical structures as synaptic

vesicles and details of cell organelles, investigators

Figure 5.7 Light Microscope

A light microscope uses visible light and magnification through

lenses to allow a researcher to view tissue or cell samples.

must use a transmission electron microscope. A beam of

electrons is passed through a thin slice of the tissue to be

examined ( < 100 nm thick). The beam of electrons casts a

shadow of the tissue on a fluorescent screen, which can be

photographed or scanned into a computer. Electron photomicrographs

produced in this way can provide information

about structural details on the order of a few tens of

nanometers. (See Figure 5.8.)

A scanning electron microscope provides less magnification

than a standard transmission electron microscope,

which transmits the electron beam through the tissue.

However, it shows objects in three dimensions. The microscope

scans the tissue with a moving beam of electrons.

The information from the reflection of the beam is received

Figure 5.8 Viewing Neurons Using Different

Microscopes

a) This image shows a stained neuron magnified 1 00x through a

light microscope. b) This image shows a neuron viewed through a

scanning electron microscope. Notice the greater detail and three

dimensional shape visible in this image compared to the image from

a light microscope.

(a)

~ g.,

.g,

·o

~ w z

gi

[ij

~

0

~

w er;

!ii

(b)

Methods and Strategies of Research 113

by a dletector, and a computer produces a remarkably detailed

three-dimensional view. (See Figure 5.8.)

CONFOCAL LASER SCANNING MICROSCOPY Conventional

microscopy or transmission electron microscopy

1require that the tissue be sliced into thin sections.

The rnnfocal laser scanning microscope makes it possible

to see details inside thick sections of tissue (up to

hundreds of micrometers thick) or even in slabs of tissue

maintained in tissue cultures or in the upper layers

of tissue in the exposed living brain. The confocal microscope

requires that the cells or parts of cells of interest

be stained with a fluorescent dye. For example, neurons

that produce a particular peptide can be labeled with a

fluorescent dye. A beam of light of a particular wavelength

is produced by a laser and reflected off of a dichroic

mirro:r- a special mirror that transmits light of certain

wavellengths and reflects light of other wavelengths.

Lenses in the microscope focus the laser light at a particular

depth in the tissue. This light triggers fluorescence

in the tissue, which passes through the lenses and

is transmitted through the dichroic mirror to a pinhole

aperture. This aperture blocks extraneous light. The light

that passes through the aperture is measured by a detector.

Two moving mirrors cause the laser light to scan the

tissue, which provides the computer with the information

it needs to form an image of a slice of tissue located

at a particular depth within the sample. If multiple scans

are m:ade while the location of the aperture is moved, a

stack ,of images of slices through the tissue-remember,

this c.an be living tissue- can be obtained. Figure 5.9

illustrates the use of confocal microscopy to examine two

populations of cells in the hypothalamus of a rat.

Tracing Neural Connections

LO 5.fi Compare techniques for tracing efferent

and afferent axons.

Let's suppose that we were interested in discovering the

neurall mechanisms responsible for reproductive behavior.

To start out, we wanted to study the physiology of

the sexual behavior of female rats. On the basis of some

clues we received by reading reports of experiments by

other researchers published in scientific journals, we performed

stereotaxic surgery on two groups of female rats.

We made a lesion in the ventromedial nucleus of the hypothalamus

(VMH) of the rats in the experimental group and

performed sham surgery on the rats in the control group.

After .a few days' recovery, and on a receptive day of the

estrus cycle, we placed the individual animals with male

rats. The females in the control group engaged in courting

114 Chapter 5

Figure 5.9 Confocal Microscope and Image

(a) A laser scanning confocal microscope is shown in this simplified schematic diagram. (b)i Confocal microscopic image of terminal buttons

stained for the neurotransmitter GABA (red) and cell bodies stained for the neurotransmitter oxytocin (green) in rat hypothalamus.

Source: Courtesy of Dr. William E. Armstrong.

(a)

Inverted

microscope

l

Computer

Monitor

behavior with the males followed by copulation. However,

the females with the VMH lesions rejected the males' attention

and refused to copulate with them. We confirmed with

histology that the VMH was indeed destroyed in the brains

of the experimental animals.

The results of our experiment indicate that neurons

in the VMH appear to play a role in functions required

for copulatory behavior in females. (By the way, it turns

out that these lesions do not affect copulatory behavior

in males.) So where do we go from here? What is the next

step? In fact, there are many questions that we could pursue.

One question concerns the system of brain structures

that participate in female copulatory behavior. Certainly,

the VMH does not function alone. The VMH receives inputs

from other structures and sends outputs to still others.

Copulation requires the integration of visual, tactile,

and olfactory perceptions and organization of patterns of

movements in response to those of the partner. In addition,

the entire network requires activation by the appropriate

sex hormones. What is the precise role of the VMH in this

complicated system?

Before we can hope to answer this question, we must

know more about the connections of the VMH with the rest

(b)

of the brain. \ilvhat structures send their axons to the VMH,

and to what :structures does the VMH, in tum, send its

axons? Once we know what the connections are, we can investigate

the role of these structures and the nature of their

interactions. (See Figure 5.10.)

Figure 5.10 Tracing Neural Circuits

Once we know tlhat a particular brain region is involved in a

particular function, we may ask what structures provide inputs

to the region ancl what structures receive outputs from it.

VMH = ventromedial nucleus of the hypothalamus

~

~~~ ? • "If•-

? ___ .;VMH

How do we investigate the connections of the VMH?

The question cannot be answered by means of histological

procedures that stain all neurons, such as cell-body

stains. If we look closely at a brain that has been prepared

by these means, we will see a large set of all neurons in

the region. But in recent years, researchers have developed

very precise methods that make specific neurons stand out

from all of the others.

TRACING EFFERENT AXONS Eventually, the VMH

must affect behavior by sending axons to parts of the brain

that contain neurons that are responsible for muscular

movements. The pathway is probably not direct. It is likely

that neurons in the VMH affect neurons in other structures,

which influence those in yet other structures until,

eventually, the appropriate motor neurons are stimulated.

To discover this system, we want to be able to identify the

paths followed by axons leaving the VMH. In other words,

we want to trace the efferent axons of this structure. (Look

at Figure 5.10 again.)We can use an anterograde labeling

method to trace these axons. (Anterograde means "moving

forward.") Anterograde labeling methods employ chemicals

that are taken up by dendrites or cell bodies and are

then transported through the axons toward the terminal

buttons.

There are several different methods for tracing the

paths of efferent axons. For example, to discover the destination

of the efferent axons of neurons located within

the VMH, a minute quantity of PHA-L (a protein found

in kidney beans) can be injected into the VMH using a

stereotaxic apparatus. The molecules of PHA-L are taken

up by dendrites and are transported through the soma

to the axon, where they travel by means of fast axoplasmic

transport to the terminal buttons. Eventually, cells

are filled with PHA-L. Figure 5.11 illustrates this process.

After euthanizing the animal, slicing the brain, and

mounting the sections on microscope slides, a special

Methods and Strategies of Research 115

immunocytochemical method is used to make the molecules

of PHA-L visible, and the slides are examined under a microsco,

pe. Figure 5.12 shows how PHA-L injected into the

VMH can be used to identify efferent axons that project to

the pe-riaqueductal gray matter (PAG). The PAG contains

some labeled axons and terminal buttons (gold color),

which proves that some of the efferent axons of the VMH

terminate in the PAG.

Innmunocytochemical methods take advantage of an

immune reaction. The body's immune system has the ability

to jproduce antibodies in response to antigens. Antigens

are proteins (or peptides), such as those found on the

surface of bacteria or viruses. Antibodies, which are also

proteiins, are produced by white blood cells to destroy invading

microorganisms. Antibodies are either secreted by

white blood cells or are located on their surface, in the way

neuroltransmitter receptors are located on the surface of

neuroins. When the antigens present on the surface of an

invading microorganism come into contact with the antibodies

that recognize them, the antibodies trigger an attack

on the invader by the white blood cells.

Molecular biologists have developed methods for

producing antibodies to any peptide or protein. The antibody

molecules are attached to various types of dye

molecules. Some of these dyes react with other chemicals

and stain the tissue a brown color. Others are fluorescent

and glow when they are exposed to light of a particular

wavelength. To determine where the peptide or protein

(the antigen) is located in the brain, the investigator

places. fresh slices of brain tissue in a solution that contains

the antibody/ dye molecules. The antibodies attach

themselves to their antigen. (See Figure 5.13.) When the

investigator examines the slices with a microscope (under

light of a particular wavelength in the case of fluorescent

dyes), they can see which parts of the brain- even which

individual neurons-contain the antigen. For example, a

Figure 5.11 Labeling Efferent Axons

116 Chapter 5

Figure 5.12 Anterograde Tracing Method

PHA-L was injected into the ventromedial nucleus of the

hypothalamus, where it was taken up by dendrites and carried

through the cells' axons to their terminal buttons. The section shows

labeled axons and terminal buttons in the periaqueductal gray matter.

Source: Courtesy of Kirsten Nielsen Ricciardi and Jeffrey Blaustein, University

of Massachusetts Amherst.

researcher might use antibodies for a protein enzyme involved

in the production of GABA to identify GABAergic

cells, or a protein component of the GABA receptor to

identify cells that receive GABAergic messages.

Immunocytochemical methods are an important technique

used to help answer our questions about neural

Figure 5.13 lmmunocytochemical Methods: Dye Binding

circuits in the VMH example. To continue our study of the

role of the Vl\l[H in female sexual behavior, we would find

the structures. that receive information from neurons in

the VMH (such as the PAG) and see what happens when

each of them is lesioned. Let's suppose that damage to

some of these structures also impairs female sexual behavior.

We could then inject these structures with PHA-L

and see where their axons go. Eventually, we will discover

the relevant pathways from the VMH to the motor neurons

whose a,ctivity is necessary for copulatory behavior

(In fact, researchers have done so, and some of their results

are presented in Chapter 10.)

TRACING AFFERENT AXONS Tracing efferent axons

from the VMH will tell us only part of the story about the

neural circuitry involved in female sexual behavior: the part

between the VMH and the motor neurons. What about the

circuits before tthe VMH? Is the VMH somehow involved in

the analysis of sensory information (such as the sight, odor,

or touch of thie male)? Or perhaps the VMH processes the

activating effe:ct of a female's sex hormones on her behavior

through neurons whose axons form synapses there? To

discover the parts of the brain that are involved in the "upstream"

components of the neural circuitry, we need to find

the inputs of the VMH-its afferent connections. To do so,

we will use a retrograde labeling method.

Retrograde means "moving backward." Retrograde

labeling methods employ chemicals that are taken up by

Antibodies bind with proteins or peptides of interest, linking a dye molecule to the site. Uncler a microscope, cells labeled with the dye are

visible. This allows researchers to identify cells based on specific characteristics.

Dye Molecules

(make cell visible under

microscope)

Antibody

----- Specific protein

of interest

(bound to antigen)

Figure 5.14 Retrograde Tracing Method

Fluorogold was injected in the VMH, where it was taken up by

terminal buttons and transported back through the axons to their

cell bodies. The photograph shows these cell bodies, located in the

medial amygdala.

Source: Courtesy of Yvon Delville, University of Massachusetts Medical School.

terminal buttons and carried backward through the axons

toward the cell bodies. The method for identifying the afferent

inputs to a particular region of the brain is similar to

the method used for identifying its efferent outputs. First,

we inject a small quantity of a retrograde labeling chemical

called fluorogold into the VMH. The chemical is taken up

by terminal buttons and is transported backward toward

the cell bodies by means of retrograde axoplasmic transport

to fill the afferent neurons. Similar to the process for investigating

efferent axons, after euthanizing the animal, slicing

the brain, mounting the sections on microscope slides,

and using irnmunocytochemical methods, the brain tissue

is examined under light of the appropriate wavelength. The

molecules of fluorogold fluoresce under this light. Through

this process, we discover that the medial amygdala is one of

the regions that provide input to the VMH. (See Figure 5.14.)

Together, anterograde and retrograde labeling methods

enable us to discover circuits of interconnected neurons

and help to provide us with a "wiring diagram" of the

brain. (See Figure 5.15.)

TRANSNEURONAL TRACING METHODS The anterograde

and retrograde labeling methods identify a single

link in a chain of neurons-neurons whose axons enter or

leave a particular brain region. Transneuronal tracing methods

identify a series of two, three, or more neurons that

form serial synaptic connections with each other. The most

effective transneuronal tracing method uses a pseudorabies

virus-a weakened form of a pig herpes virus that

was originally developed as a vaccine. For anterograde

transneuronal tracing, a variety of the herpes simplex

Methods and Strategies of Research 117

Figure 5.15 Results of Tracing Methods

The figure shows one of the inputs to the VMH and one of the

outputs, as revealed by anterograde and retrograde labeling

methocls.

1a Anterograde tracing:

inject PHA-L in VMH

1 b Then see axons and

terminals in PAG

/ PAG ~

Other~,• Medial ? V/MH

1 • structures? ~~ygdala /

·~ior"'-'. ~~ I

2a Retrograde tracing:

inject fluorogold in VMH

2b Then see cell bodies in

medial amygdala

virus, similar to the one that causes cold sores, is used.

The viirus is injected directly into a brain region, is taken

up by neurons there, and infects them. The virus spreads

throughout the infected neurons and is eventually released

by the terminal buttons, passing the infection to other neurons

that form synaptic connections with them.

After the animal is euthanized and the brain is sliced,

irnmwnocytochemical methods are used to localize a protein

produ,ced by the virus. For example, Daniels and colleagues

(1999) injected pseudorabies virus in the muscles responsible

for female rats' mating posture. After a few days, the rats were

euthanized, and their brains were examined for evidence of

viral infection. The study indicated that the virus found its

way up the motor nerves to the motor neurons in the spinal

co1,d, then to the reticular formation of the medulla, then

to the periaqueductal gray matter, and finally to the VMH.

These results confirm the results of the anterograde and retrograde

labeling methods that were just described.

Studying the Structure of the

Living Human Brain

LO 5.E> Contrast methods to study the structure of the

living human brain.

Although we cannot ethically ask people to submit to lesion

studies for the purposes of research, diseases and

118 Chapter 5

accidents do unfortunately occur that damage the human

brain. If we know where the damage occurs, we can study

the person's behavior and try to make the same sorts of inferences

we make with deliberately produced brain lesions

in laboratory animals. The problem is, where is the lesion?

In the past, a researcher might have studied the behavior

of a person with brain damage and never determined

exactly where the lesion was located. The only

way to be sure was to obtain the patient's brain when

they died and examine slices of it under a microscope.

But it was often impossible to do this. Sometimes the

patient outlived the researcher. Sometimes the patient

moved out of town. Sometimes (often, perhaps) the family

refused permission for an autopsy. Because of these

practical problems, the study of the behavioral effects of

damage to specific parts of the human brain made rather

slow progress.

Advances in X-ray techniques and computers have

led to the development of several noninvasive methods

for studying the anatomy of the living brain. These

advances permit researchers to study the location and

extent of brain damage while the patient is still living.

The first method developed is called computerized

tomography (CT). This procedure, usually referred to as

a CT scan, works as follows: The patient's head is placed

in a large doughnut-shaped ring. The ring contains an

Figure 5.16 Computerized Tomography

X-ray tube and, directly opposite it (on the other side of

the patient's head), an X-ray detector. The X-ray beam

passes through the patient's head, and the detector measures

the arniount of radioactivity that gets through it.

The beam scans the head from all angles, and a computer

translates the information it receives from the

detector into pictures of the skull and its contents. (See

Figure 5.16.)

Figure 5.17 shows a series of CT scans taken through

the head of a patient who sustained a stroke when bleeding

occured in the brain. The scans allow a researcher or

physician to view the location and extent of an injury. In

this case, internal bleeding is indicated by the color blue

added to the scan.

An even more detailed, high-resolution picture of

what is inside a person's head is provided by a process

called magnetic resonance imaging (MRI). The

MRI scanner resembles a CT scanner, but it does not

use X-rays. Instead, it passes an extremely strong magnetic

field through the patient's head. When a person's

head is placed in this strong magnetic field, the nuclei

of spinning hydrogen atoms align themselves with the

magnetic field. When a pulse of a radio frequency wave

is then passed through the brain, these nuclei flip at an

angle to the magnetic field and then flip back to their

original position at the end of the radio pulse. Different

Within a CT scanner, a beam of X-ray is used to image progressive "slices" through tissues of the body, including the brain and skull. Differences

in structure or tissue type, such as tumors or bleeding, can be seen in CT scans.

Source: Based on MedlinePlus, U.S. National Library of Medicine http://www.nlm.nih.gov/medlineplus/ency/imagepages/19237.htm

Methods and Strategies of Research 119

Figure 5.17 CT Brain Scans

Computerized tomography (Cl) brain scans (axial view) were taken through the brain

of a 38-year-old male stroke patient. The stroke occurred four weeks before the scans

were taken. The blue region is an area of internal bleeding, or hemorrhage.

SIMON FRASER/NEWCASTLE HOSPITALS NHS TRUST/Scimce Source

molecules emit energy at different frequencies. The MRI

scanner is tuned to detect the radiation from hydrogen

atoms. Because these atoms are present in different concentrations

in different tissues, the scanner can use the

information to prepare pictures of slices of the brain.

(See Figure 5.18.)

As you can see in Figure 5.18, MRI scans can distinguish

between regions of gray matter and white matter,

so major fiber bundles (such as the corpus callosum)

can be seen. However, small fiber bundles are not visible

on these scans. A special modification of the MRI

scanner permits the visualization of even small bundles

of fibers and the tracing of fiber tracts. Above absolute

zero, all molecules move in random directions because of

thermal agitation: the higher the temperature, the faster

the random movement. Diffusion tensor imaging (DTI)

takes advantage of the fact that the movement of water

molecules in bundles of white matter will not be random

but will tend to be in a direction parallel to the axons that

make up the bundles. The MRI scanner uses information

about the movement of the water molecules to determine

the location and orientation of bundles of axons in

white matter. Figure 5.19 shows a sagittal view of some

of the axons that project from the thalamus to the cerebral

cortex in the human brain, as revealed by DTI. The

computer adds colors to distinguish different bundles of

axons .. The research methods described in this module are

summarized in Table 5.1.

Figure 5.18 MRI Scans of Human Brain

120 Chapter 5

Figure 5.19 Diffusion Tensor Imaging

This image shows a sagittal view of some of the axons that project from the thalamus to the

cerebral cortex in the human brain, as revealed by diffusion tensor imaging.

Source: From Wakana, S., Jiang, H., Nagae-Poetscher, L. M., van Zijl, P. C., and Mori, S. (2004). Fiber tractbased

atlas of human white matter anatomy. Radiology, 230, 77-87. Reprinted with permission.

Thalamus

Table 5.1 Research Methods Related to Ablation

Destroy or inactivate specific brain region Radio frequency lesion

Place electrode or cannula in a specific

region within the brain

Rnd the location of a lesion

Visualize tissue or cell structures

Visualize subcellular structures

Visualize details of cells in thick or living

sections of tissue

Excitotoxic lesion; uses excitatory amino acid

such as kainic acid

Infusion of local anesthetic or drug that

produces local neural inhibition

Infusion of saporin conjugated with an antibody

Stereotaxic surgery

Perfuse brain; fix brain; slice brain; stain sections

Light microscopy

Electron microscopy

Confocal laser scanning microscopy

Identify axons leaving a particular region Anterograde tracing method, such as PHA-L

and the terminal buttons of these axons

Identify location of neurons whose axons Retrograde tracing method, such as ffuorogold

terminate in a particular region

Identify circuits of neurons

Rnd the location of a lesion in living

human brain

TransneuronaJ tracing methods

Computerized tomography (CT scanner)

Magnetic resonance imaging (MRI scanner)

Find the location of fiber bundles in living Diffusion tensor imaging (DTI)

human brain

Dostroys all brain tissue near the tip of the electrode

Dnstroys only cell bodies near the tip of the cannula;

spares axons passing through the region

Temporarily inactivates specific brain region; an animal can

serve as its own control

Dnstroys neurons that contain the antibody; produces

ve•ry precise brain lesions

Consult stereotaxic atlas for coordinates

Often requires histological methods and staining or

immunocytochemical methods to identify cells of interest

Transmission electron microscopes provide structural

information. Scanning electron microscopes provide

th1ree-dimensional information

Can be used to see "slices" of tissue in the living brain;

requires the presence of fluorescent molecules in the tissue

St1ows series of two or more neurons with serial synaptic

connections; often uses herpes simplex (anterograde) or

p~;eudorabies (retrograde) viruses

St1ows "slice" of the brain; uses X-rays

St1ows "slice" of the brain; better detail than CT scan;

us:es a magnetic field and radio waves

St1ows bundles of myelinated axons; uses an MRI scanner

Methods and Strategies of Research 121

Module Review: Experimental Ablation

Evaluating the Behavioral Effects

of Brain Damage

LO 5.1 Explain what researchers can learn from

lesion studies.

By lesioning a part of the nervous system, a researcher

can observe the resulting changes in behavior to determine

the function of that portion of the nervous system.

The goal is to discover what functions are performed by

different regions of the brain and then to understand how

these functions are combined to accomplish particular

behaviors. No one brain region or neural circuit is solely

responsible for a behavior; each region performs a function

(or set of functions) that contributes to the performance

of the behavior.

Producing Brain Lesions

LO 5.2 Compare methods of producing brain lesions.

Brain lesions can be produced by passing an electrical

current through a wire, or by injecting an excitatory amino

acid, selective antibody, or local anesthetic into a specific

brain region. Using an electrical current, an excitatory

amino acid or a selective antibody produces a permanent

lesion. Using a local anesthetic produces a temporary lesion.

Using an electrical current produces a nonselective lesion.

The other methods produce lesions based on the type of

neuron the lesioning agent selectively binds to.

Stereotaxic Surgery

LO 5.3 Describe the process of stereotaxic surgery.

Stereotaxic surgery involves using a stereotaxic atlas to

identify a specific location in the brain. Once the location

has been identified, the researcher places the head in a stereotaxic

apparatus and positions a cannula over the correct

location on the head. The researcher makes an incision

in the scalp of the anesthetized animal (or human), drills

a hole in the skull, and lowers the cannula into place. The

researcher makes the lesion (or in some cases implants an

electrode or transplants tissue), removes the cannula, and

the animal (or human) recovers from the anesthetic.

Histological Methods

LO 5.4 Summarize the steps of histological methods.

Brain tissue is perfused and removed from the skull.

Then, the tissue to be examined is placed in a fixative.

Once the brain is fixed, it is sliced into thin sections on a

cryostat or microtome. The slices are placed on a microscope

slide. Brain tissue must be stained to reveal cellular

and intracellular structures. Cells can be stained for cell

bodies, nuclei, or specific proteins using dyes or specially

labeled antibodies. Depending on the characteristics of

the ce!U or intracellular structures of interest, light microscopes,

transmission electron microscopes, scanning electron

microscopes, or confocal laser scanning microscopes

may be used to study the tissue samples.

Tracing Neural Connections

LO 5.!5 Compare techniques for tracing efferent and

afferent axons.

Tracing efferent axons allows researchers to learn about

the ta1rget locations of sets of neurons. Using an anterograde

labeling method, a chemical is injected into the region

containing cell bodies, taken up with the cells, and

transported to the terminals. Immunocytochemical methods

use antibodies to identify the structures that receive

input from the cell bodies. Tracing afferent axons allows

resear,chers to learn about neurons that provide input to a

regioni of interest. Using a retrograde labeling method, a

chemi,cal is injected into the target region and taken up by

the terminal buttons. The chemical then travels to the cell

body of the neuron. Transneuronal tracing methods can

identilfy a series of two or more neurons with retrograde

(pseudorabies) or anterograde (herpes simplex) viruses.

Studying the Structure of the Living

H111nan Brain

LO 5.16 Contrast methods to study the structure of the

living human brain.

Computerized tomography (CT) uses X-rays to image the

structure of the living human brain. Magnetic resonance

imaging (MRI) uses a magnetic field to image the living

brain and differentiates among different tissue types. Diffusioni

tensor imaging (DTI) uses information about the

movement of water molecules to visualize small fiber

bundl,es not visible in MRI scans.

Thought Question

Henry Molaison (H. M.) became a well-known figure in

psychology and neuroscience after undergoing ablation

of tiss ue in his temporal lobes to reduce seizures. The

surgeiry was performed in 1957. H. M.'s brain and behavior

were documented by physicians and researchers

until his death in 2008. After his death, researchers at the

University of California San Diego carefully preserved,

sectioined, and stained his brain to learn more about it.

Describe the techniques these researchers would need to

use to examine H. M.'s brain after his death.

122 Chapter 5

Recording and Stimulating

Neural Activity

The first module of this chapter dealt with the anatomy of

the brain and the effects of damage to particular regions.

This module considers a different approach: studying

the brain by recording or stimulating the activity of particular

regions. Brain functions involve the activity of circuits

of neurons. Different perceptions and behavioral

responses involve different patterns of activity in the brain.

Researchers have devised methods to record these patterns

of activity or to artificially produce them.

Recording Neural Activity

LO 5. 7 Compare methods of recording neural activity.

Axons produce action potentials, and terminal buttons

elicit postsynaptic potentials in the membrane of the cells

with which they form synapses. These electrical events can

be recorded (as we saw in Chapter 2), and changes in the

electrical activity of a particular region can be used to determine

whether that region plays a role in various behaviors.

For example, recordings can be made during stimulus

presentations, decision making, or motor activities.

Recordings can be made over an extended period of

time after the animal recovers from surgery, for a relatively

short period of time during which the animal is kept anesthetized.

Short-term recordings, made while the animal is

anesthetized, are usually restricted to studies of sensory

pathways. Short-term recordings seldom involve behavioral

observations, since the behavioral capacity of an anesthetized

animal is limited.

RECORDINGS WITH MICROELECTRODES Drugs that

affect serotonergic and noradrenergic neurons also affect

REM sleep. Suppose that, knowing this fact, we wondered

whether the activity of serotonergic and noradrenergic

neurons would vary during different stages of sleep. To

find out, we would record the activity of these neurons

with microelectrodes. Microelectrodes, usually made of

thin wires, have a very fine tip, small enough to record the

electrical activity of individual neurons. This technique is

usually called single-unit recording (a unit refers to an individual

neuron).

Because we want to record the activity of single neurons

over a long period of time in unanesthetized animals,

we want more durable electrodes. Arrays of very fine wires

gathered together in a bundle can simultaneously record

the activity of many different neurons. The wires are insulated

so that only their tips are bare.

Microelectrodes can be implanted in the brains of

animals through stereotaxic surgery and bonded to the

animals' skull. We can then observe both the animal's behavior

during REM sleep and the corresponding activity

of the serotonergic and noradrenergic neurons recorded

by the implanted microelectrodes. (See Figure 5.20.)

The electrical signals detected by microelectrodes are

quite small and must be amplified. Amplifiers used for

this purpose work just like the amplifiers in a stereo system,

converting the weak signals recorded in the brain into

stronger ones. These signals can be displayed and saved on

a computer.

As you wiill learn in Chapter 9, if we record the activity

of these neurons during various stages of sleep, we find

their firing rates fall almost to zero during REM sleep, suggesting

that these neurons have an inhibitory effect on REM

sleep. That is, REM sleep does not occur until these neurons

stop firing.

RECORDINGS WITH MACROELECTRODES Sometimes,

we want to re,cord the activity of a region of the brain as a

whole, not the activity of individual neurons. To do this, we

would use macroelectrodes. Macroelectrodes do not detect

the activity of imdividual neurons; rather, the records that are

obtained with these devices represent the postsynaptic potentials

of mamy thousands-or millions-of cells in the area

of the electrode. These electrodes are sometimes implanted

into the brain or onto the surface of the brain, but many are

temporarily attached to the human scalp with a special paste

that conducts electricity. Recordings taken from the scalp,

especially, represent the activity of an enormous number of

neurons, who:se electrical signals pass through the meninges,

skull, and scalp before reaching the electrodes.

The electrical activity of a human brain recorded

through macroelectrodes is displayed on a polygraph. A

polygraph plots the changes in voltage detected by the electrodes

along a timeline during recording. The polygraph is

displayed on a computer screen. Figure 5.21 illustrates electrical

activity recorded from macroelectrodes attached to

various locations on a person's scalp. Such records are called

Figure 5.20 Implantation of Electrodes

The drawing shows a set of electrodes in a rat brain.

Connecting socket

Electrodes

Dental plastic

Methods and Strategies of Research 123

Figure 5.21 Recording Brain Activity with Macroelectrodes

Macroelectrodes record the summed electrical activity of many neurons. In this example, an electroencephalogram is

created to visually represent the changes in summed postsynaptic poteintials recorded by scalp electrodes.

Left

hemisphere

Right

hemisphere

An electroencepha1logram (EEG)

electroencephalograms (EEGs), or "writings of electricity

from the head." They can be used to diagnose epilepsy or to

study the stages of sleep and wakefulness, which are associated

with characteristic patterns of electrical activity.

In addition to their use in research, clinicians use

macroelectrodes to help treat patients. Occasionally, neurosurgeons

implant macroelectrodes directly into the human

brain. The reason for doing so is to detect the source of abnormal

electrical activity that is giving rise to frequent seizures.

Once the source has been determined, the surgeon can remove

the source of the seizures-usually scar tissue caused by brain

damage that occurred earlier in life. Similarly, another clinical

use of EEG is to monitor the condition of the brain during

procedures that could potentially damage it. One of the authors

of this book (N. C.) observed such a procedure:

Mrs. F. had sustained one mild heart attack, and subsequent

tests indicated a considerable amount of atherosclerosis,

commonly referred to as "hardening of the arteries." Many

of her arteries were narrowed by cholesterol-rich atherosclerotic

plaque. A clot formed in a particularly narrow portion of

one of her coronary arteries, which caused her heart attack. As

the months passed after her heart attack, Mrs. F. had several

transient ischemic attacks, brief episodes of neurological symptoms

that appear to be caused by blood clots forming and then

dissolving in cerebral blood vessels. In her case, they caused

numbness in her right arm and difficulty in talking. Her physician

referred her to a neurologist, who ordered an angiogram (a recording

of heart activity). This procedure revealed that her left carotid

artery was almost totally blocked. The neurologist referred

Mrs. F. to a neurosurgeon, who urged her to have an operation

that would remove the plaque from part of her left carotid artery

and increase the blood flow to the left side of her brain.

The procedure is called a carotid endarterectomy. I was

chatting with Mrs. F. 's neurosurgeon after a conference, and

he happened to mention that he would be performing the operation

later that morning. I asked whether I could watch, and

he a~1reed. When I entered the operating room, scrubbed and

gowned, I found that Mrs. F. was already anesthetized, and the

surgical nurse had prepared the left side of her neck for the incision.

In addition, several EEG electrodes had been attached to

her scalp, and I saw that Dr. L., a neurologist who specializes in

clinical neurophysiology, was seated at his EEG machine.

The surgeon made an incision in Mrs. F. 's neck and exposed

the carotid artery, at the point where the common carotid, coming

from the heart, branched into the external and internal carotid arteries.

Hie placed a plastic band around the common carotid artery and

clamped it shut, stopping the flow of blood. "How does it look?" he

askecl Dr. L. "No good-I see some slowing. You'd better shunt."

The surgeon quickly removed the constricting band and

askecl the nurse for a shunt, a short length of plastic tubing a little

thinnm than the artery. He made two small incisions in the artery

well aibove and well below the region that contained the plaque

and inserted the shunt. Now he could work on the artery without

stopping the flow of blood to the brain. He made a longitudinal

cut in the artery, exposing a yellowish mass that he dissected

away and removed. He sewed up the incision, removed the

shunt, and sutured the small cuts he had made to accommodate

it. "Everything still okay?" he asked Dr. L. "Yes, her EEG is fine ."

Most neurosurgeons prefer to do an endarterectomy by

temporarily clamping the artery shut while they work on it. The

work goes faster, and complications are less likely. Because the

bloodl supply to the two hemispheres of the brain is interconnected

(with special communicating arteries), it is often possible

to shut down one of the carotid arteries for a few minutes without

causiing any damage. However, sometimes the blood flow from

one side of the brain to the other is insufficient to keep the other

side nourished with blood and oxygen. The only way the surgeon

can know is to have the patient's EEG monitored. If the brain is

not mceiving a sufficient blood supply, the EEG will show the

presence of characteristic "slow waves." That is what happened

when Mrs. F. 's artery was clamped shut, and that is why the surgeon

had to use a shunt tube. Without it, the procedure might

have caused a stroke instead of preventing one.

13y the way, Mrs. F. made a good recovery.

124 Chapter 5

MAGNETOENCEPHALOGRAPHY When electrical current

flows through a conductor, it induces a magnetic

field. This means that as action potentials pass down axons

or as postsynaptic potentials pass through dendrites

or sweep across the somatic membrane of a neuron, magnetic

fields are also produced. These fields are very small,

but engineers have developed superconducting detectors

(called superconducting quantum interference devices, or

SQUIDs) that can detect minute magnetic fields.

Magnetoencephalography is performed with neuromagnetometers,

devices that contain an array of several

SQUIDs, oriented so that a computer can examine their

output and calculate the source of particular signals in the

brain. These devices can be used clinically-for example,

to find the sources of seizures so that they can be removed

surgically. They can also be used in experiments to measure

regional brain activity that accompanies the perception of

various stimuli or the performance of various behaviors or

cognitive tasks. (See Figure 5.22.)

An important advantage of magnetoencephalography

is its temporal resolution (ability to quickly show changes in

information). Another technique that you'll read about in this

chapter, functional MRI (fMRI) provides excellent spatial resolution

of regional activity in the brain, but the process is slow

and provides relatively poor temporal resolution. The image

produced by magnetoencephalography is not as detailed as

an fMRI image, but it can be acquired much more rapidly and

can consequently reveal fast-moving events.

Figure 5.22 Magnetoencephalography

An array of SQUIDs in this neuromagnetometer detects regional changes

in magnetic fields produced by the electrical activity of the brain.

Source: Phanie/Science Source

Recording the Brain's Metabolic

and Synaptic Activity

LO 5.8 Compare methods for assessing metabolic

and synaptic activity.

Electrical and magnetic signals are not the only signs of

neural activity. If the neural activity of a particular region

of the brain increases, the metabolic rate of this region

increases, too, largely due to increased operation of ion

transporters in the membrane of the cells, which requires

an increased use of cellular energy. This increased metabolic

rate can be measured. The experimenter injects

radioactive 2-deoxyglucose (2-DG) into the animal's

bloodstream. Because this chemical closely resembles

glucose (the principal food for the brain), it is taken into

cells. This means that the most active cells, which use

glucose at thE! highest rate, will take up the highest concentrations

of radioactive 2-0G. But unlike normal glucose,

2-DG cannot be metabolized, so it stays in the cell.

After administering 2-DG, the experimenter euthanizes

the animal, removes the brain, slices it, and prepares it

for autoradiogi•aphy.

Autoradit0graphy can be translated roughly as "writing

with one's own radiation." Sections of the brain containing

the radioactive 2-DG are mounted on microscope

slides. The slides are then developed, just like photographic

film. The molecules of radioactive 2-DG show

themselves as spots of silver grains in the developed

images. The most active regions of the brain contain the

most radioactivity, showing this radioactivity in the form

of dark spots in the developed images of the tissue.

Figure 5.23 shows the distribution of μ opioid receptors

(described in Chapter 4) in the rat brain. The researchers

modified the autoradiography method described here,

by exposing the tissue to a radioactive ligand (the opioid

receptor antagonist naloxone) before developing the autoradiographic

image. This image shows high concentrations

of opioid receptors in the white areas, including the

olfactory bulb and hippocampus.

Another method of identifying active regions of the

brain capitalizes on the fact that when neurons are activated

(for example, by the terminal buttons that form

synapses with them), particular genes in the nucleus

called immedi,ate early genes are turned on, and particular

proteins are produced. These proteins then bind with

the chromoso,mes in the nucleus. The presence of these

nuclear proteins indicates that the neuron has just been

activated.

One of the nuclear proteins produced during neural

activation is called Fos. You will remember that earlier in

this chapter we began an imaginary research project on the

Figure 5.23 Autoradiography

This horizontal section of a rat brain demonstrates autoradiography.

A radioactive ligand attached toμ opioid receptor. The brain section

was placed on a photographic film. Radiation exposed the film in

the white regions, revealing a high concentration of receptors in the

olfactory bulb, the cerebral cortex, the hippocampus, the striatum,

the thalamus, and the tectum.

Source: National Institute on Drug Abuse, NATIONAL INSTITUTES OF

HEALTH/Miles Herkenham, NIMH/Science Source

neural circuitry involved in the sexual behavior of female

rats. Suppose we want to use the Fos method in this project

to see what neurons are activated during a female rat's sexual

activity. We place female rats with males and permit the

animals to copulate. After euthanizing the animals, we remove

the rats' brains, slice them, and follow a procedure that

stains Fos protein. Figure 5.24 shows the results: Neurons in

the medial amygdala of a female rat that has just mated show

the presence of dark spots, indicating the presence of Fos

protein. Thus, these neurons appear to be activated by copulatory

activity-perhaps by the physical stimulation of the

genitals that occurs then. As you will recall, when we injected

a retrograde tracer (fluorogold) into the VMH, we found that

this region receives input from the medial amygdala.

The metabolic activity of specific brain regions

can be measured noninvasively in a living animal, by

means of functional imaging- a computerized method

of detecting metabolic or chemical changes within the

brain. The first functional imaging method to be developed

was positron emission tomography (PET). First,

a person (or another animal) receives an injection of radioactive

2-DG. (The chemical dose is harmless to humans

and soon breaks down and leaves the cells.) The

person's head is placed in a machine similar to a CT

scanner. When the radioactive molecules of 2-DG decay,

they emit subatomic particles called positrons, which

meet nearby electrons. The particles annihilate each

other and emit two photons, which travel in opposite

paths. Sensors arrayed around the person's head detect

these photons, and the scanner plots the locations from

Methods and Strategies of Research 125

Figure 5.24 Localization of Fos Protein

The photomicrograph shows a frontal section of the brain of a

female rat, taken through the medial amygdala. The dark spots

indicatEl the presence of Fos protein, localized by means of

immunocytochemistry. The synthesis of Fos protein was stimulated

by pernnitting the animal to engage in copulatory behavior.

Source: Courtesy of Marc Tetel, Skidmore College.

which these photons are being emitted. From this information,

a picture is produced of a slice of the brain,

showing the activity level of various regions in that

slice. i(See Figure 5.25).

One of the disadvantages of PET scanners is their operating

cost. For reasons of safety, the radioactive chemicals

that are administered have very short half-lives; that

is, they decay and lose their radioactivity very quickly. For

example, the half-life of radioactive 2-DG is 110 minutes,

and the half-life of radioactive water (also used for PET

scans) is only 2 minutes. Because these chemicals decay so

quickly, they must be produced on-site, in an atomic particle

accelerator called a cyclotron. The cost of PET scanning

also includes the cost of the cyclotron and the salaries of

the personnel who operate it.

Another disadvantage of PET scans is the relatively

poor s.patial resolution (the blurriness) of the images. The

temporal resolution is also relatively poor. The positrons

being emitted from the brain must be sampled for a fairly

long time, which means that rapid, short-lived events

within the brain are likely to be missed. These disadvantages

are not seen in functional MRI, described in the next

parag1raph. However, PET scanners can do something

that functional MRI scanners cannot do: measure the concentration

of particular chemicals in various parts of the

brain. We will describe this procedure later in this chapter.

Cmrently, the brain-imaging method with the best

spatial and temporal resolution is functional MRI (£MRI).

Enginieers have devised modifications to existing MRI

126 Chapter 5

Figure 5.25 PET Scan

Colored positron emission tomography (PEl) scan of the

brain during a speech exercise. The exercise involved reciting

synonyms. The PET scan is superimposed onto a black and

white three-dimensional magnetic resonance imaging (MRI)

scan. The front of the brain is at left. The scan shows the areas

of brain activity (color) associated with speech. These areas are

in the speech cortex of the brain's frontal lobe. The scan shows

increased uptake of radioactive 2-DG in regions of the brain

that are active in the speech task, which indicates an increased

metabolic rate in these areas. Different computer-generated colors

indicate different rates of uptake of 2-DG, with "warmer" color

indicating increased activity.

scanners and their software that permit the devices to acquire

images that indicate regional metabolism. Brain activity

is measured indirectly, by detecting levels of oxygen

in the brain's blood vessels. Increased activity of a brain region

stimulates blood flow to that region, which increases

the local blood oxygen level. The formal name of this type

of imaging is BOLD: blood oxygen level-dependent signal.

Functional MRI scans have a higher resolution than

PET scans do and reveal more detailed information about

the activity of particular brain regions. (See Figure 5.26.)

You will read about many functional imaging studies that

employ fMRI in subsequent chapters of this book.

Stimulating Neural Activity

LO 5.9 Compare methods of neural stimulation.

So fa r, this module has focused on research methods that

measure the activity of specific regions of the brain. But

sometimes we may want to artificially change the activity

of these regions to see what effects these changes have

on behavior. For example, female rats will copulate with

males only if certain female sex hormones are present. If

Figure 5.26 fMRI Scan

This frontal section from a functional magnetic resonance image

(fMRI} shows inc:reased blood flow to brain regions active during a

motor task involving feet and toes. The "warmer" colors represent

higher blood oxygen level-dependent (BOLD) activation in regions

of the motor cortex and cerebellum. SMA = supplementary motor

area

we remove the rats' ovaries, the loss of these hormones

will abolish the rats' sexual behavior. We found in our

earlier studies that VMH lesions disrupt this behavior.

Perhaps if we activate the VMH, we will make up for the

lack of femal,~ sex hormones, and the rats will copulate

again.

ELECTRICAL AND CHEMICAL STIMULATION How

do we activate neurons? We can do so by electrical or

chemical stimulation. Electrical stimulation involves

passing an electrical current through a wire inserted into

the brain usiing stereotaxic surgery. Chemical stimulation

is usually accomplished by injecting a small amount

of excitatory amino acid, such as kainic acid (which in

small doses s:timulates neurons) or glutamic acid, into

the brain. As you learned in Chapter 4, the principal excitatory

neurotransmitter in the brain is glutamic acid

(glutamate), and both of these substances stimulate glutamate

receptors, activating the neurons on which these

receptors are located.

Injections of chemicals into the brain can be done

through an apparatus that is permanently attached to the

skull so that the animal's behavior can be observed several

times. A researcher can place a cannula (a guide cannula)

in an animal's brain using stereotaxic surgery and cement

its top to the skull. A smaller cannula of measured length

can be placed inside the guide cannula and used to inject a

chemical into the brain. Because the animal is free to move

about, it is possible to observe the effects of the injection on

its behavior. (See Figure 5.27.)

The principal disadvantage of chemical stimulation is

that it is slightly more complicated than electrical stimulation

because chemical stimulation requires cannulas,

tubes, special pumps or syringes, and sterile solutions of

excitatory amino acids. However, it has a distinct advantage

over electrical stimulation: It activates cell bodies but

not axons. Because only cell bodies (and their dendrites)

contain glutamate receptors, we can be assured that an

injection of an excitatory amino acid into a particular region

of the brain excites the cells there but not the axons of

other neurons that happen to pass through the region. This

means that the effects of chemical stimulation are more localized

than are the effects of electrical stimulation.

You might have noticed that we just said that kainic

acid, which we described earlier as a neurotoxin, can be

used to stimulate neurons. These two uses are not really

contradictory. Kainic acid produces excitotoxic lesions by

stimulating neurons to death. Whereas large doses of a

concentrated solution kill neurons, small doses of a dilute

solution simply stimulate them.

Figure 5.27 An lntracranial Cannula

a) A guide cannula is permanently attached to the skull. (b) At a later time,

Methods and Strategies of Research 127

When chemicals are injected into the brain through

cannulas, molecules of the chemicals diffuse over a region

that includes many different types of neurons: excitatory

neurons, inhibitory neurons, interneurons that participate

in locatl circuits, projection neurons that communicate with

differe'.nt regions of the brain, and neurons that release or

respond to a wide variety of neurotransmitters and neuromodulators.

Stimulating a particular brain region with

electricity or an excitatory chemical affects all of these neurons,

and the result is unlikely to resemble normal brain

activity, which involves coordinated activation and inhibition

of many different neurons. Ideally, we would like to be

able to stimulate or inhibit selected populations of neurons

in a given brain region.

What about the results of our hypothetical experiment?

In fact (as you will see in Chapter 10), VMH stimulation

does substitute for female sex hormones. Perhaps,

then, the female sex hormones exert their effects in this

nucleus. We will see how to test this hypothesis later in

this chapter.

TRANSCRANIAL MAGNETIC STIMULATION As we

saw earlier in this chapter, neural activity induces magnetic

fields that can be detected by means of magnetoencephalography.

Similarly, magnetic fields can be used

to stimulate neurons by inducing electrical currents in

brain tissue. Transcranial magnetic stimulation (TMS)

uses a coil of wires, usually arranged in the shape of the

numeral 8, to noninvasively stimulate neurons in

the cerebral cortex. The stimulating coil is placed

on top of the skull so that the crossing point in

a thinner cannula can be inserted through the guide cannula into the brain.

Chemicals can be infused into the brain through this device.

the middle of the 8 is located immediately above

the region to be stimulated. Pulses of electricity

send magnetic fields that activate neurons in the

cortex. Figure 5.28 shows an electromagnetic coil

used in transcranial magnetic stimulation and its

placement on a person's head.

Dental

plastic

Guide

cannula

Skull Brain

r-:::::::.----~, rL(

(a) (b)

The effects of TMS are very similar to those of

direct stimulation of the exposed brain. For example,

as you will see in Chapter 6, stimulation of a particular

region of the visual association cortex will disrupt

a person's ability to detect movements in visual

stimuli. In addition, as we will see in Chapters 16

and 17, TMS has been used to treat the symptoms

of neurological and mental disorders. Depending on

the strength and pattern of stimulation, TMS can either

excite the region of the brain over which the coil

is positioned or interfere with its functions.

OPTOG ENETIC METHODS Optogenetic

methods can be used to stimulate or inhibit particular

types of neurons in specific brain regions

128 Chapter 5

Figure 5.28 Transcranial Magnetic Stimulation

Pulses of electricity through the coil produce a magnetic field that

stimulates a region of the cerebral cortex under the crossing point in

the middle of the figure 8.

(Baker, 2011; Boyden et al., 2005; Zhang et al., 2007). Photosensitive

proteins have evolved in many organismseven

single-celled organisms such as algae and bacteria.

Researchers have discovered that one of these proteins,

Channelrhodopsin-2 (ChR2), found in green algae, controls

ion channels that, when open, permit the flow of sodium,

potassium, and calcium ions. When blue light strikes a

ChR2-ion channel, the channel opens, and the rush of

positively charged sodium and ca lei um ions depolarizes

the membrane, causing excitation. A second photosensitive

protein, Natronomonas pharaonis halorhodopsin

(NpHR), is found in a bacterium. This protein controls a

transporter that moves chloride into the cell when activated

by yellow light. This influx of negatively charged

ions hyperpolarizes the membrane, causing inhibition.

The action of both of these photosensitive proteins

begins and ends very rapidly when light of the appropriate

wavelength (blue or yellow) is turned on and off. (See

Figure 5.29).

ChR2 and NpHR can be introduced into neurons by

attaching the genes that code for them into the genetic

material of harmless viruses. The viruses are then injected

into the brain, where they infect neurons and begin

expressing the proteins, which are inserted into the cell

membrane. The genes can be modified so that the proteins

will be expressed only in particular types of neurons.

In this way, researchers can observe the effects of

turning on or off particular types of neurons in a specific

region of the brain.

Because ChR2 and NpHR are activated by light,

researchers must be able to introduce light into the brain.

If the neurons that express these photosensitive proteins

are located in the cerebral cortex, a small hole can be

drilled in the skull, and light-emitting diodes (LEDs) can

be attached diirectly above the hole. To activate photosensitive

proteins in the membranes of neurons deep within

the brain, optical fibers can be implanted by means of

stereotaxic surgery, just like electrodes or cannulas, and

light can be transmitted through these fibers. For example,

Tsai and colleagues (2009) used optogenetic methods

to insert ChR2-ion channels into the membranes of

dopaminergic neurons in the ventral tegmental area of

rats. The inv1:!Stigators found that if these neurons were

stimulated when the rats were in one of two chambers in

Figure 5.29 Optogenetic Methods

Photosensitive proteins can be inserted into neural membranes by

means of genetically modified viruses. Blue light causes ChR2-

ion channels to depolarize the membrane, and yellow light causes

NpHR-ion transporters to hyperpolarize it.

Source: Based on Hausser, M., and Smith, S. L. (2007). Nature, 446, 617--619.

Outside of Cell

ChR2

Blue light 0-ea2+

0 4D 0-Na+

Q () O Ion channel

Q1

NpHR

Yellow light

er---=-<) 00

0

) i (

!

Q O \,on 0 transporter

a testing apparatus, the animals preferred to spend time

in that chamber.

The development of optogenetic procedures has

caused much excitement among neuroscientists because

they suggest ways to study the functions of particular

neural circuits in the brain. Some investigators are also

exploring possible clinical uses of photosensitive proteins.

For example, retinitis pigmentosa is a genetic disease

that causes blindness in humans. People with this

disease are born with normal vision, but they gradually

become blind as the photoreceptor cells in their retinas

degenerate. The retina contains two major categories of

photoreceptors: rods, which are responsible for night vision,

and cones, which are responsible for daytime vision.

The rods of people with retinitis pigmentosa die, but although

the cones lose their sensitivity to light, their cell

Methods and Strategies of Research 129

bodiei; survive. Busskamp and colleagues (2010) used an

optog,enetic method to try to reestablish vision in mice

with a genetic modification that causes them to develop

retinitis pigmentosa. The investigators targeted the animals'

,cones with NpHR. (Because the membranes of photoreceptors

are normally hyperpolarized by light, they

chose to use this protein.) Electrical recording and behavioral

studies found that the treatment at least partially

reestalblished the animals' vision. Furthermore, the same

treatment reestablished light sensitivity in retinal tissue

removed from deceased people who had been diagnosed

with retinitis pigmentosa. These findings provide hope

that forther research may develop a treatment for this

form of blindness.

Table 5.2 summarizes information about the research

methods presented in this module.

Table 5.2 Methods for Recording and Stimulating Neural Activity

Record electrical activity

of single neurons

Record electrical activity

of regions of the brain

Microelectrodes

Macroelectrodes

Microelectrodes can be implanted permanently to record neural

activity as the animal moves

In humans, usually attached to the scalp with a special paste

Record magnetic fields

induced by neural activity

Magnetoencephalography; uses a neuromagnetometer,

which contains an array of SOUIDs

Can determine the location of a group of neurons firing

synchronously

Record metabolic activity

of regions of the brain

2-DG autoradiography

Measurement of Fos protein

2-DG PET scan

Measures local glucose utilization

Identifies neurons that have recently been stimulated

Measures regional metabolic activity of the human brain

Functional magnetic resonance imaging (fMRI) scan Measures regional metabolic activity of the IMng, unanesthetized brain

Measure neurochemicals

in the living human brain

Stimulate neural activity

PET scan

Electrical stimulation

Chemical stimulation with excitatory amino acid

Transcranial magnetic stimulation

Optogenetic methods

Can localize any radioactive substance taken up in the human brain

Stimulates neurons near the tip of the electrode and axons

passing through region

Stimulates only neurons near the tip of the cannula, not axons

passing through region

Stimulates neurons in the human cerebral cortex with an

electromagnet placed on the head

Stimulates (or inhibits) neurons in the brain using light to depolarize

or hyperpolarize the cells

Module Review: Recording and Stirnulating Neural Activity

Recording Neural Activity

LO 5.7 Compare methods of recording neural activity.

Microelectrodes can be used to record the electrical activity

of individual neurons and must be placed into a

single neuron. Macroelectrodes are used to record the

summed electrical activity of many neurons in the vicinity

of !the electrode. Macroelectrodes can be placed in the

brain or on the surface of the scalp. Magnetoencephalography

measures changes in magnetic fields of neurons

and is used to detect groups of synchronously activated

neuroms.

130 Chapter 5

Recording the Brain's Metabolic

and Synaptic Activity

LO 5.8 Compare methods for assessing metabolic and

synaptic activity.

Autoradiography is a postmortem technique that involves

measuring the radioactivity from cells that have taken up

radioactive 2-DG while they are metabolically active prior

to tissue collection. Staining for immediate early genes

reveals the neurons that were recently activated prior to

tissue collection. Positron emission tomography (PET) visualizes

activity of brain regions that are metabolically active

in a living brain (it also uses radioactive 2-DG). Functional

MRI (fMRI) visualizes brain regions that have increased

blood flow and local blood oxygen levels in a living brain.

Stimulating Neural Activity

LO 5.9 Compare methods of neural stimulation.

Electrical and chemical stimulation of neurons is accomplished

by passing an electrical current or injecting a

Neurochemical Methods

Sometimes we are most interested in the location of neurons

that possess particular types of receptors or produce particular

types of neurotransmitters or neuromodulators. We might

also want to measure the amount of these chemicals secreted

by neurons in particular brain regions during particular circumstances.

The following modules describe neurochemical

methods to study the chemicals of the nervous system.

Finding Neurons That Produce

Particular Neurochemicals

LO 5.10 Describe methods to identify neurons that

produce a particular neurochemical.

Suppose we learn that a particular drug affects behavior.

How would we go about discovering the neural circuits

that are responsible for the drug's effects? To answer this

question, let's take a specific example. Physicians discovered

several years ago that farmworkers who had been

exposed to certain types of insecticides (the organophosphates)

had particularly intense and bizarre dreams and

even reported having hallucinations while awake. A plausible

explanation for these symptoms is that the pesticides

acted as a drug that stimulates the neural circuits involved

in the control of REM sleep-the phase of sleep during

which dreaming occurs. (After all, dreams are hallucinations

that we have while sleeping.)

The first question to ask relates to how the organophosphate

insecticides work. Pharmacologists have the

chemical into a specific brain region through a cannula. Optogenetic

methods are used to stimulate or inhibit particular

types of neurons in specific brain regions. In optogenetic

methods, tuming wavelengths of light on and off allows

the researcher to control the activity of the genetically altered

neurons that are light sensitive. Transcranial magnetic

stimulation (TMS) uses a magnetic signal to stimulate or inhibit

neurons beneath the TMS device. Unlike other methods

of stimulalting neural activity, TMS is noninvasive.

Thought Question

Have you heaird about brain-training programs or apps

that claim to activate your brain? Have you wondered

if these claims are accurate or how they could be tested?

Write an email to a friend who is curious about the research

behind measuring brain activation using braintraining

programs. In your message, describe what

technique you predict the researchers used to measure

brain activation in the participants of the study. Explain

why this technique would be appropriate.

answer: These insecticides are acetylcholinesterase (AChE)

inhibitors. As you learned in Chapter 4, acetylcholinesterase

inhibitors are potent acetylcholine agonists. By inhibiting

AChE, the drugs prevent the rapid destruction of ACh

after it is released by terminal buttons and prolong the

postsynaptic potentials at acetylcholinergic synapses.

Now that: we understand the action of the insecticides,

we know that these drugs act at acetylcholinergic

synapses. What neurochemical methods should we use to

discover the sites of action of the drugs in the brain? First,

let's consider methods by which we can localize particular

neurochemicals, such as neurotransmitters and neuromodulators.

(In om case we are interested in acetylcholine.)

There are at least two basic ways of localizing neurochemicals

in the brain: localizing the chemicals themselves or

localizing the enzymes that produce them.

Chemicals that are peptides (or proteins) can be localized

directly by means of irnmunocytochemical methods,

which were described earlier in this chapter. Slices of brain

tissue are exposed to an antibody for the peptide and linked

to a dye (often, a fluorescent dye). The slices are then examined

under a microscope using light of a particular wavelength.

(See Figure 5.30.)

How can a researcher localize chemicals that are not

peptides? In our example, we are interested in studying

acetylcholine, which is not a peptide. Therefore, we cannot

use irnmunocytochemical methods to find this neurotransmitter

directly. However, we can use these methods to

localize the enzyme that produces it. Enzymes are peptides,

and we: can use imrnunocytochernical methods to

localize them. Acetylcholine is synthesized by the enzyme

Figure 5.30 Localization of Peptides

(a) The peptide is revealed by means of immunocytochemistry. The

photomicrograph shows a portion of a frontal section through the rat

forebrain. The gold- and rust-colored fibers are axons and terminal

buttons that contain vasopressin, a peptide neurotransmitter.

(Courtesy of Geert DeVries, University of Massachusetts Amherst.)

(b) An enzyme responsible for the synthesis of a neurotransmitter

is revealed by immunocytochemistry. The photomicrograph shows

a section through the pons. The orange neurons contain choline

acetyltransferase, which implies that they produce (and thus secrete)

acetylcholine. (Courtesy of David A. Morilak and Roland Ciaranello,

Nancy Pritzker Laboratory of Developmental and Molecular

Neurobiology, Department of Psychiatry and Behavioral Sciences,

Stanford University School of Medicine.)

(a)

(b)

choline acetyltransferase (ChAT). Neurons that contain this

enzyme almost certainly secrete ACh. Figure 5.30b shows

acetylcholinergic neurons in the pons that have been identified

by means of immunocytochemistry; the brain tissue

Methods and Strategies of Research 131

was exposed to an antibody to ChAT attached to a fluorescent

dye. In fact, research using many of the methods

descrilbed in this chapter indicates that these neurons play

a role in controlling REM sleep.

Returning to our example of hallucinations following

pesticiide exposure, if we were to conduct an experiment to

confirm the effects of continuous organophosphate exposure

on the ACh system, we might consider comparing the

brain ,changes in a group of rats exposed to organophosphates

with a control group that was not exposed. We could

then examine the brains of animals in these two conditions

to evaluate the effects of organophosphate exposure on the

ACh system. Using immunocytochemical techniques to investigate

ChAT might reveal that organophosphate exposure

resulted in reduced ChAT in the neurons, supporting

our hypothesis that the pesticides had an effect on the ACh

system. To confirm a behavioral effect, we could have also

observed the animals in the REM sleep stage using another

technique in this chapter: EEG.

Localizing Particular Receptors

LO 5.111 Compare methods to localize particular

receptors.

As we saw in Chapter 2, neurotransmitters, neuromodulators,

and hormones convey their messages to their target

cells by binding with receptors on or in these cells. The location

of these receptors can be determined by two different

pwcedures.

The first procedure to determine the location of receptors

uses autoradiography. The basic steps involved

in autioradiography to determine the location of cells that

were metabolically active (by consuming radioactive 2-DG)

were presented earlier in the chapter. In a similar procedure,

autoradiography to determine the locations of receptors

requires us to expose slices of brain tissue to a solution

containing a radioactive ligand for a particular receptor,

instead of radioactive 2-DG that can be used to identify

any metabolically active cell. Next, we rinse the slices so

that the only radioactivity remaining in them is derived

from molecules of the radioactive ligand bound to their receptors.

Finally, the slides are taken into a darkroom and

coated[ with a photographic emulsion and developed to localize

the radioactive ligand and the receptors it is bound

to. (See Figure 5.23.)

Let's apply this method for localizing receptors to the

first lime of investigation we considered in this chapter: the

role of the ventromedial hypothalamus (VMH) in the sexual

behaviior of female rats. So far, we have discovered that lesions

of the VMH abolish this behavior. We also saw that

the behavior does not occur if the rat's ovaries are removed

but that the behavior can be activated by stimulation of the

VMH with electricity or an excitatory amino acid. These results

suggest that the sex hormones produced by the ovaries

132 Chapter 5

act on neurons in the VMH. We could next use autoradiography

to look for the receptors for the sex hormone. We would

expose slices of rat brain to the radioactive hormone, rinse

them, and perform autoradiography. If we did so, we would

indeed find radioactivity in the VMH. (And if we compared

slices from the brains of female and male rats, we would find

evidence of more hormone receptors in the females' brains.)

The second procedure for localizing receptors in the

brain uses another technique you have already encountered

in this chapter: immunocytochernistry. Receptors

are proteins, which means that we can produce antibodies

against them. We expose slices of brain tissue to the appropriate

antibody (often labeled with a fluorescent dye)

and look at the slices with a microscope under light of a

particular wavelength.

We could use this technique to learn more about the

role of sex hormones in the VMH and their involvement in

female sexual behavior. Instead of using autoradiography

to identify the locations of the sex hormone receptors, we

could instead use antibodies for the receptors. This would

allow us to use irnrnunocytochemistry, attach a dye molecule

to the antibodies, and view the labeled receptors

under a microscope. Although this approach will yield the

same results as using autoradiography to localize particular

receptors, the advantages include not needing to obtain

a radioactive ligand and being able to view individually

labeled cells under a microscope.

Measuring Chemicals Secreted

in the Brain

LO 5.12 Compare methods used to examine chemicals

secreted in the brain.

While we can use autoradiography and irnrnunocytochemistry

to measure particular receptors involved in female

sexual behavior in the rat, this information only tells us

about structures located on the postsynaptic cells. Suppose

we are also interested in learning about what chemicals are

secreted by presynaptic cells in the VMH. To measure the

amount of neurotransmitter released in particular regions

of the brain, we use a procedure called microdialysis.

Dialysis is a process in which substances are separated

by means of an artificial membrane that is permeable to

some molecules but not others. A microdialysis probe consists

of a small metal tube that introduces a solution into a

section of dialysis tubing- a piece of artificial membrane

shaped in the form of a cylinder, sealed at the bottom.

Another small metal tube leads the solution away after

it has circulated through the pouch. A drawing of such a

probe is shown in Figure 5.31.

We can use stereotaxic surgery to place a microdialysis

probe in a rat's brain so that the tip of the probe is located

in the region we are interested in (the VMH). We pump a

Figure 5.31. Microdialysis

A dilute salt solution is slowly infused into the microdialysis tube,

where it picks up molecules that diffuse in from the extracellular

fluid. The conten1ts of the fluid are then analyzed.

Dental

plastic""

,r-- Fluid is pumped through inner cannula

n Fluid is collected

r-_::1------:i►and analyzed

Skull Brain

Dialysis tubinq --......._

- I I Substances in extracellular

.__.. A ~ fluid diffuse through the

dialysis tubing

small amount of a solution similar to the extracellular fluid

through one o,f the small metal tubes into the dialysis tubing.

The fluid circulates through the dialysis tubing and

passes throug;h the second metal tube, from which it is

taken for analysis. As the fluid passes through the dialysis

tubing, it collects molecules from the extracellular fluid of

the brain. The molecules are then pushed across the membrane

by the force of diffusion.

We analy:ze the contents of the fluid that has passed

through the dialysis tubing by an extremely sensitive

analytical method. This method is so sensitive that it can

detect neurotransmitters (and their breakdown products)

that hav,e been released by the terminal buttons and

have escaped from the synaptic cleft into the rest of the

extracellular fluid. We find that the cells of the VMH release

a number of different neurotransmitters, including

norepinephrine. Microdialysis reveals that norepinephrine

release is increased when females administered sex

hormones subsequently displayed sexual behavior, but

was not increased when the behavior was absent (Vathy &

Etgen, 1989).

In a few special cases (for example, in monitoring brain

chemicals of people with intracranial hemorrhages or head

trauma) the microdialysis procedure has been applied to

study the human brain, but ethical reasons prevent doing

so for researclh purposes. Fortunately, there is a noninvasive

way to measure neurochemicals in the human brain.

Although PET scanners are expensive machines, they are

also versatile. They can be used to localize any radioactive

substance that emits positrons. Figure 5.32 shows PET

scans of the brain of one of the patients who developed

Parkinson's disease-like symptoms after using a synthetic

Figure 5.32 PET Scans of a Patient with Parkinson's

Disease-like Symptoms

The scans show uptake of radioactive L-DOPA in the basal ganglia

of a patient with Parkinson's disease-like symptoms induced by

a toxic chemical before and after receiving a transplant of fetal

dopaminergic neurons. (a) Preoperative scan. (b) Scan taken 13

months postoperatively. The increased uptake of L•DOPA indicates

that the fetal transplant was secreting dopamine.

Source: Adapted from Widner, H., Tetrud, J., Rehncrona, S., et al. (1992).

Bilateral fetal mesencephalic grafting in two patients with Parkinsonism induced

by 1-methyl-4-phenyl-L,2,3,6-tetrahydropyridine (MPTP). New England Journal

of Medicine, 327, 1556-1563. Scans reprinted with permission.

(a) (b)

Table 5.3 Neurochemical Research Methods

Measure neurotransmitters and

neuromodulators released by neurons Microdialysis

Methods and Strategies of Research 133

opiate in the case that opened this chapter. A stereotaxic

apparatus was used to transplant fetal dopamine-secreting

neurons into his basal ganglia. As you read, PET scans

were taken of his brain before his surgery and a little more

than a year afterward. He was given an injection of radioactive

L-DOPA one hour before each scan was made. As

you learned in Chapter 4, L-DOPA is taken up by the terminals

of dopaminergic neurons, where it is converted to

dopamine; thus, the radioactivity shown in the scans indicates

the presence of dopamine-secreting terminals in the

basal ganglia. The scans show the amount of radioactivity

before (part a) and after (part b) he received the transplant.

As you can see, the basal ganglia contained substantially

more dopamine after the surgery.

Table 5.3 summarizes the research methods presented

in this module.

Identify neurons producing a particular

neurotransmitter or neuromodulator

lmmunocytochemical localization of pepti<Je or protein

lmmunocytochemical localization of enzynne responsible

for synthesis of substance

A wide variety of substances can be analyzed

Requires a specific antibody

Useful if substance is not a peptide or protein

Identify neurons that contain a particular

type of receptor

Autoradiographic localization of radioactiv1a ligand

lmmunocytochemical localization of receptor

Requires a specific antibody

Module Review: Neurochemical Methods

Finding Neurons That Produce Particular

Neurochemicals

LO 5.10 Describe methods to identify neurons that

produce a particular neurochemical.

Using immunocytochemical methods, a researcher could

localize the chemicals themselves or localize the enzymes

that produce the neurochemicals of interest, within specific

neurons.

Localizing Particular Receptors

LO 5."11 Compare methods to localize particular

receptors.

Autoradiography to identify specific receptors involves

imme1:sing brain tissue in a solution containing a radioactive

ligand for the receptor of interest. Slides of the tissue

are th1en developed to expose the locations of the receptors

for the ligand. Immunocytochemistry for specific

receptors involves exposing brain tissue to antibodies

134 Chapter 5

that are selective for a protein on the receptor of interest.

The tissue can then be examined under a microscope.

Measuring Chemicals Secreted in the Brain

LO 5.12 Compare methods used to examine

chemicals secreted in the brain.

First, a microdialysis probe is placed into the brain region

of interest using stereotaxic surgery. A small amount of a

solution similar to extracellular fluid is pumped into the

brain through one of the small metal tubes into the dialysis

tubing. The fluid circulates through the dialysis tubing

and passes through the second metal tube, from which it is

taken for analysis. As the fluid passes through the dialysis

tubing, it collects molecules from the extracellular fluid of

the brain, which are pushed across the membrane by the

force of diffusion. A researcher can then analyze the brain

chemical contents of the fluid that has passed through

the dialysis tubing by an extremely sensitive analytical

method. PET scanning is a noninvasive method, and when

a radioactive ligand is administered, can be used to identify

and quantify chemicals secreted in the living brain.

Genetic Methods

All behavior is determined by interactions between an

individual's brain and his or her environment. Many behavioral

characteristics-such as talents, personality variables,

and mental disorders-seem to run in families. This

suggests that genetic factors may play a role in the development

of physiological differences that are ultimately

responsible for these characteristics. In some cases, the

genetic link is very clear: A defective gene interferes with

brain development, and a neurological abnormality causes

behavioral deficits. In other cases, the links between heredity

and behavior are much more subtle, and special genetic

methods must be used to reveal them.

Twin Studies

LO 5.13 Describe how twin concordance rates can

be used to assess genetic contributions to a

behavior.

A powerful method for estimating the influence of heredity

on a particular trait is to compare the concordance

rate for this trait in pairs of monozygotic and dizygotic

twins. Monozygotic twins (identical twins) have identical

genotypes-that is, their chromosomes, and the genes

they contain, are identical. In contrast, the genetic similarity

between dizygotic twins (fraternal twins) is, on average,

50 percent. Investigators study records to identify pairs of

Thought Question

Throughout this chapter, you have read about a series of

studies intended to understand the neural mechanisms

involved in fomale rat sexual behavior. Many different

methods, including several neurochemical methods,

were used to gather information and improve understanding

of tl11e neural circuits underlying this behavior.

Imagine that you have been asked to submit a grant

proposal for research on the neurochemical basis of an

important behavior. Write a summary of your proposal

identifying:

A. an important behavior (you can select any behavior,

relevant to humans or other animals);

B. a potentiatl neurochemical (for example, this could be a

neurotrans:mitter or hormone);

C. a method! you could use to identify neurons that

produce the chemical; and

D. a method you could use to identify where the receptors

for the chemical are located.

twins in which at least one member has the trait-for example,

a diaginosis of a particular mental disorder. If both

twins have been diagnosed with this disorder, they are said

to be concordant. If only one has received this diagnosis, the

twins are saidl to be discordant. If a disorder has a strong

genetic basis, the percentage of monozygotic twins who are

concordant for the diagnosis will be higher than that for dizygotic

twins. For example, as we will see in Chapter 17,

the concordance rate for schizophrenia in twins is at least

four times higher for monozygotic twins than for dizygotic

twins, a findirng that provides strong evidence for a genetic

component irt the development of schizophrenia. Twin

studjes have found that many individual characteristics,

including personality traits, prevalence of obesity, incidence

of alcohol abuse, and a wide variety of mental disorders,

are inflw~nced by genetic factors.

Adoption Studies

LO 5.14 Evaluate the role of adoption studies in

assessing genetic contributions to a behavior.

Another method for estimating the heritability of a particular

behaviioral trait is to compare people who were

adopted early in life with their biological and adoptive

family members. All behavioral traits are affected to

some degree by hereditary factors, environmental factors,

and an intera<Ction between these factors. Environmental

factors are physical, social, and biological in nature.

For example, the mother's health, nutrition, and drugtaking

behavior during pregnancy are prenatal environmental

factors, and the child's diet, medical care, and

social environment (both inside and outside the home) are

postnatal environmental factors. If a child is adopted soon

after birth, the genetic factors will be associated with the

biological parents, the prenatal environmental factors will

be associated with the biological mother, and most of the

postnatal environmental factors will be associated with

the adoptive family.

Adoption studies require that the investigator knows

the identity of the parents of the people being studied and

is able to measure the behavioral trait in the biological and

adoptive parents. If the people being studied strongly resemble

their biological parents, we conclude that the trait

is probably influenced by genetic factors. If, instead, the

people resemble their adoptive families, we conclude that

the trait is influenced by environmental factors. Remember

that both hereditary and environmental factors are involved

in the expression of a given behavior, and the people

being studied will resemble both their biological and

adoptive families to some degree.

In the laboratory, researchers can model adoption

studies in rodents using cross-fostering methods. In

cross-fostering, newborn pups or litters are exchanged

between mothers, and the behavior and physiology of

the fostered pups can be studied. This approach has been

used to assess the genetic, prenatal, and postnatal environmental

contributions to a wide variety of behaviors.

When the genetic background of the mice is known (as

in many established mouse strains), researchers are able

to obtain more specific information about genetic and

environmental factors involved in complex behaviors.

Cross-fostering can also be used to better understand epigenetic

changes related to behavior (for extensive review,

see McCarty, 2017).

Genomic Studies

LO 5.15 Identify genomic techniques used to study

physical and behavioral traits.

The human genome consists of the DNA that encodes

our genetic information. Because of the accumulation of

mutations over past generations of our species, no two

people, with the exception of monozygotic twins, have

identical genetic information. The particular form of an

individual gene is called an allele. For example, different

alleles of the gene responsible for the production of iris

pigment in the eye produce pigments with different colors.

Genomic studies attempt to determine the location in

the genome of genes responsible for various physical and

behavioral traits.

Linkage studies identify families whose members

vary with respect to a particular trait-for example, the

Methods and Strategies of Research 135

presence or absence of a particular hereditary disease.

A variiety of markers, sequences of DNA whose locations

are ahready known, are compared with the nature of an

individual person's trait. For example, the gene responsible

for Huntington's disease, a neurological disorder discussed,

was found to be located near a known marker on

chromosome 4. Researchers studied people in an extended

family in Venezuela that contained many members with

Huntington's disease and found that the presence or absence

iof the disease correlated with the presence or absence

of the marker.

Similarly, genomewide association studies have been

made possible by the development of methods to obtain

the DNA sequence of the entire human genome. These

studie:s permit researchers to compare all or portions

of the genomes of different individuals to determine

whether differences in the people's genomes correlate

with the presence or absence of diseases (or other traits).

As we: will see in Chapter 17, these studies are beginning

to rev,eal the location of genes that control characteristics

that contribute to the development of various mental

disorders.

Targeted Mutations

LO 5.116 Summarize how targeted mutations can be used

to study genetic contributions to a behavior.

Targetted mutations are mutated genes produced in the

laboraitory and inserted into the chromosomes of animals,

typically mice. In some cases, the genes (also called

knockout genes) are defective: These genes fail to produce

a functional protein. In many cases, the target of the

mutation is an enzyme that controls a particular chemical

reaction. In other cases, the genes (also called knockin

genes) produce a new functional protein to replace a

missing protein, or make increased amounts of a protein.

For example, lack of a particular enzyme interferes with

learniing. (See Chapter 13.) This result suggests that the

enzyme is partly responsible for changes in the structure

of synapses required for learning to occur. In other cases,

the target of the mutation is a protein that itself serves

useful functions in the cell. For example, a particular type

of cannabinoid receptor is involved in the reinforcing and

analgesic effects of opiates. (See Chapter 19.) Researchers

can even produce conditional knockouts that cause the animal

to stop expressing a particular gene when the animal

is given a particular drug. This permits the targeted

gene to express itself normally during the animal's development

and then be knocked out (inactivated) at a

later time. Investigators can also use methods of genetic

engineering to insert new genes into the DNA of mice.

These genes can increase production of proteins normally

found in the host species, or they can produce entirely

new proteins.

136 Chapter 5

Antisense Oligonucleotides

LO 5.17 Describe how antisense oligonucleotides

function to change behavior.

Another genetic method involves molecules that block

the production of proteins encoded by particular genes by

injecting antisense oligonucleotides. The most common

types of antisense oligonucleotides are modified strands

of RNA or DNA that will bind with specific molecules of

messenger RNA and prevent them from producing their

protein. Once the molecules of mRNA are trapped in this

way, they are destroyed by enzymes present in the cell.

(See Figure 5.33.) The term antisense refers to the fact that

the synthetic oligonucleotides contain a sequence of bases

complementary to those contained by a particular gene or

molecule of mRNA.

What role does this method have in helping us to

understand behavior? Destroying proteins in this way

can produce changes in behavior, highlighting the importance

of intracellular proteins in behavior. For example,

injecting antisense oligonucleotides that destroy

receptors for sex hormones in the VMH of female rats inhibited

female sexual behavior (Mani et al., 1994; Pollio

et al., 1993). This method can be used to confirm the importance

of sex hormones in the VMH in contributing to

female sexual behavior in rats. The results obtained using

this technique complement the results of using other

methods highlighted throughout this chapter.

Figure 5.33 Antisense Oligonucleotides

CRISPR-1Cas Methods

LO 5.18 Summarize the uses of CRISPR-Cas methods in

neuroscience research.

All of the proteins used in the nervous system have their

origins in DNA. Proteins contribute to all functions of the

nervous system. To make proteins, cells rely on genetic information

contained in DNA, which is used as a recipe for

synthesizing specific proteins. A set of techniques can be

used to help neuroscientists both understand and modify

sections of DNA that code for specific proteins.

Similar to, using antisense oligonucleotides, CRISPRCas

(or clustered regularly interspaced short palindromic repeat)

methods alter the production of proteins; however,

this technique involves changes to the DNA instead of the

mRNA. To ere.ate changes in the DNA of a cell, CRISPR uses

a Cas protein to identify target sites in the double strand of

DNA and break both strands at that site. Once the strands

are broken, cells use one of two different pathways to repair

the DNA damage: non-homologous end joining (NHEJ) or

homology-directed recombination (HOR). If the cell uses

NHEJ pathways, the result is to mutate or delete the targeted

genetic sequence. This inactivates the gene, creating a

gene knockou1t. If the cell uses HOR, a new genetic sequence

can be inserted into the cut strands of DNA. Researchers can

develop these replacement sequences and introduce them

into the cell, precisely controlling the change in the genetic

sequence. (See Figure 5.34.)

Antisense oligonucleotides block the production of proteins encoded by particular genes.

Antisense DNA

/ ~ Ribosome Amino acids Protein

Figure 5.34 CRISPR Methods

1. A guide RNA and a CAS9 are joined.

The RNA-CAS9 complex

recognises the DNA fragments

to be modified

/

CAS9

3. The "scissors" cut the exact

sequence in the DNA chain

I ii 111 Iii II lillllll

111111111 1111111111

RNA

Methods and Strategies of Research 137

2. The guide RNA targets

a specific gene sequence and

aligns it to CAS molecular

scissors

Act;ve sites of CAS9

(metlecular scissors)

ii I ii Iii II ii I

11 11111111111

4a. Now the DNA can be modified. For example,

a disease-causing gene can be silenced to

prevent disease

=====:::;:, ,m====;:-,, .WU

4b .... or a fective gene can be fixed

b addin orrective DNA sequence

CRISPR-Cas methods have been used to study the

role of genes in behavior in a number of different species

used in neuroscience research, ranging from invertebrates

to primates. The technique has been used to model neurodegenerative

diseases that are caused (at least in part) by

genetic mutations, including Parkinson's, Huntington's,

and Alzheimer's diseases. Some research has begun to

explore the possibility of using this technique to generate

Table 5.4 Genetic Research Methods

personalized interventions for brain diseases with genetic

bases {Heidenreich & Zhang, 2016). Future applications of

this technique could include reprogramming undifferentiated

cells to be used to treat neurodegenerative disease or

model the kinds of environment-dependent gene expression

seen in epigenetics (Savell & Day, 2017).

Table 5.4 summarizes information about the research

metho,ds presented in this module.

Twin studies

Adoption studies

Comparison of concordance rates of monozygotic and dizygotic twins estimates heritability of trait

Similarity of offspring and adoptive and biological parents estimates heritability of trait

Targeted mutations

Antisense oligonucleotides

CRISPR-Cas methods

Inactivation, insertion, or increased expression of a gene

Bind with messenger RNA; prevent synthesis of protein

Create breaks in DNA, insert new genetic sequence to inactivate or alter protein production.

138 Chapter 5

Module Review: Genetic Methods

Twin Studies

LO 5.13 Describe how twin concordance rates can be

used to assess genetic contributions to a behavior.

Concordance rates help researchers understand the

contributions of genetic differences to variations in

behavior. For example, if a disorder has a strong genetic

basis, the percentage of monozygotic twins who are

concordant for the diagnosis will be higher than that for

dizygotic twins.

Adoption Studies

LO 5.14 Evaluate the role of adoption studies in

assessing genetic contributions to a behavior.

Adoption studies help researchers understand the

genetic and environmental contributions to a behavior.

Cross-fostering can be used to assess the genetic and environmental

contributions to behavior and physiology in

lab animals.

Genomic Studies

LO 5.15 Identify genomic techniques used to study

physical and behavioral traits.

Linkage studies identify families whose members vary

with respect to a particular tr ait- for example, the

presence or absence of a particular hereditary disease.

Genomewide association studies permit researchers to

compare all or portions of the genomes of different individuals

to determine whether differences in the people's

genomes correlate with the presence or absence of

diseases (or other traits).

Chapter Review Questions

1. Discuss the research method of experimental ablation:

the rationale, the evaluation of behavioral effects

resulting from brain damage, and the production of

brain lesions.

2. Describe stereotaxic surgery.

3. Describe research methods for preserving, sectioning,

and staining the brain and for studying its parts and

interconnections.

4. Describe research methods for tracing efferent and

afferent axons.

Targeted Mutations

LO 5.16 Summarize how targeted mutations can be used

to study genetic contributions to a behavior.

Targeted mutations are used to change the production of

a specific proltein. The resulting change in behavior can

be associated with the mutated protein.

Antisense Oligonucleotides

LO 5.17 Describe how antisense oligonucleotides

func:tion to change behavior.

Antisense olig;onucleotides can be used to block the production

of a specific protein, revealing its role in a behavior.

CRISPR-Ca.s Methods

LO 5.18 Summarize the uses of CRISPR-Cas methods

in neuroscience research.

CRISPR-Cas methods can be used to change sequences

of DNA, revealing the roles DNA play in behavior.

Thought Question

Humans have a variety of behavioral responses to tasting

the chemical Jphenylthiocarbamide (PTC). The majority

(about 75 percent) of people perceive this harmless chemical

as bitter-tasting and respond to it with avoidance and

negative facial reactions. A minority of people perceive

PTC as tasteless and show no behavioral response. The

gene for the PTC taste receptor was identified in 2003.

Using one or more of the methods in this section, describe

a study to investigate the genetic contribution to

PTC tasting and behavior.

5. Describe research methods for studying the living

human brain.

6. Describe how the neural activity of the brain is measured

andl recorded, both electrically and chemically.

7. Describe how neural activity in the brain is stimulated,

both chemically and electrically, and the behavioral

effects of brain stimulation.

8. Discuss research techniques to identify genetic factors

that may iinfluence behavior.