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Neurological Basis of Behavior (PSY - 610)

Lesson 13

RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION

Objectives:

To familiarize the students with:

· The various techniques used to study the brain and its function and structures. Invasive vs.

Non- Invasive methods, Stereotaxic surgery.

· Stereotaxic Atlas used for brain surgery and manipulation.

· Histological and cytological methods for Cell staining, Radio labeling, Fluorescence,

autradiography, Lesioning and electrical stimulation, single cell recordings, push-pull cannulae.

· The advanced techniques such as MRI, fMRI, CAT, PET, CT, EEG, EOG, EMG, X ray etc.

Research techniques in the Neurosciences:

Measuring, researching, and studying brain which is an extremely complex systems with 1011 (100

billion) neurons, with multiple synapses and 1015 (1000 trillion) connections. This is as complex a

system as the Milky Way. On the average 1 cubic millimeter of the brain has about 109 (billion)

synapses. We need to understand the physical structures (from visible to naked eye to barely visible with

e-microscope), functional organization (from a single neuron to an anatomical area working together as

an orchestra), and the ongoing activity of a few milliseconds to watching a brain grow across time. The

techniques and methods have grown enormously since study of the brain began, with more

sophistication and finer measurements possible now than ever before.

There is good news for those who wish to study the brain:

The brain similar in structures, neuroanatomical organization, function, and development to

animals on the phylogenetic scale, therefore it is possible to study the primate brain or the rat

brain and see commonalities and extrapolate to the human brain.

The brain structures are grouped together simplifying the study of the brain therefore it is easy

to locate, to verify, and to study the areas which are connected. For example the thalamic nuclei

are connected with the central cortical areas and if we damage cortical areas this leads to

degeneration of the corresponding thalamic nuclei.

The human brain cells require similar nutrients and materials as other living cells, therefore the

neurons, and other structures as well as the biochemical processes can be studied in other

animals, in vivo ( within) or in vitro (outside the living system).

Development of Techniques clarify the role of different areas, several techniques can be utilized

simultaneously.

There is a consistency of patterns of responses within cells and neuroanatomical areas, (

electrophysiological, neurochemicals, other chemicals, ionic changes etc)

Then there is the bad news:

· The brain is extremely complex with intricate networks; it is not easy to study one area

independently.

· Identifying one area or process is not enough as areas/ processes are interconnected and there are

several levels of functioning and controls (chemical, physical, electrical, ionic etc).

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Neurological Basis of Behavior (PSY - 610)

· The brain functions at molecular and molar levels (the invisible behavior of molecules and neurons

to the more visible motor and behavioural actions), where the movement/action of molecules are

translated into gross behaviors.

· Measurements of these behaviours can be made at each of these levels, and tiny miniscule

molecular communication and movement of ions and amino acids across gaps which are in

Angstroms result in major behavioural changes. What role does each play?

· Many interconnections and many neurochemicals work in one setting in one synapse. Isolating and

studying one leads to an incomplete picture.

Inspite of all these difficulties scientists have been able to locate and identify functions using the

techniques which we would discuss in the lesson today.

Techniques

There are as many as researchers working in the brain areas, they vary in the kind of area the scientists

are working in or the area of their interest.

Techniques vary depending on:

1. Area, on which the researchers are working with, and whether they are working with simpler or

complex behaviors, whether the brain areas are at the lower levels (motor basal ganglia) or

higher levels (thalamus) even higher (motor cortex).

2. Techniques available. Although there is a wide range of techniques available but as more

advanced techniques become available with advancement of technology these open up more

areas that the scientists can work on.

3. The research goal whether it is exploration, understanding, or manipulation, to develop

theoretical frameworks or to describe functions of brain areas.

Basically all methods aim to understand, control, simplify and objectify behaviour at microscopic a

macroscopic/gross levels. The simplest methods and techniques would

measure neurons, their size and their interconnections (structural /histological procedures) or,

measure the electrical activity of cells or measure and manipulate their metabolic processes

(glucose, oxygen, blood flow)

The more complex methods would be using

Radiolabelling, neurophysiological or neuro psychological tests, behavioural paradigms.

Far more complex methods for measuring ongoing functioning would be advanced technologies such as

MRI's, PET scans etc).

There is a variation in the subjects of brain sciences investigation. A wide range of animal species are

used as subjects from the lowest organism with the simplest nervous system such as the Aplysia (much

work has been done on learning and memory using this organism), to highest order primates such as the

Chimpanzees.

Animal's studies: these can be studied using in vivo techniques, microelectrodes, invasive techniques

which allow manipulation of the brain measuring electrical potentials of various areas creating electrical

stimulation or lesioning studies creating animal models.

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Neurological Basis of Behavior (PSY - 610)

Human studies post mortem As we cannot manipulate the human brain to bring about changes ( except

the mad scientists in films !) therefore post mortem studies are used which have provided a great insight

into brain damage and through that the brain functioning. For example we know more about Alzheimer's

after studying brain of patients and the areas of damage identified. Also studies following brain trauma

can help identify areas linked with behaviours as in the cases of Phineas Gage, and HM (ref: Bridgeman

58-63, Pinel 104-131).

Neuroanatomical studies: These are to identify brain areas and their structures and function: Therefore

this involves techniques which are mainly histological and invasive.

Histology: This is the study of brain cells and their projections after the removal of the brain

which requires special training.

Staining techniques: where a certain dye or chemical is used to identify the structure form or

connections of cells and their locations.

Invasive: where we actually invade the intact brain to create a lesion, or introduce radioactive

substances, or stimulate or use in combination. Changes in behaviors are noted if we are

working in a living animal and later brain studied histologically.

Histology:

We need to carry out histology for verification of location of lesion or the stimulation sites (cell, fibers,

connections) after our experiments are over. Histology is primarily used for location, structure, functional

organization of the CNS. Various techniques and histological procedures are available which are carried

out on the intact brain. This can be done only after the experiments are over and the animals sacrificed,

brain taken out of the cranial bone and sectioned. These are steps which are followed as follows:

First step, Perfusion of the circulatory system of the live but anesthetized animal through heart using

saline to wash out the blood from the entire system so that it does not coagulate.

Second step After the saline comes through clear the fixative is perfused into the heart using the same

needle. The fixative, formalin (formaldehyde) is injected to prevent degeneration of tissue, fixes

protein bonds at the point of death.

Third step: Hardening the tissue by a) freezing using the freezing microtome (a machine which cuts

thin slices of tissues). We have to be very careful, because if it freezes hard the brain tissue would

become brittle and break or it remains too soft, it cannot be cut.

Fourth Step: Embedding: one of the procedures is to embed the brain before cutting. It can be

Nitrocellulose which would allow thin sections of the brain to be cut.

Fifth Step: Staining the cells. Many different stains are available and each with its special

characteristics. The stains bring out different structures of the cell or their projections.

Types of Stains more frequently used:

Nissyl blue: this is a special stain in which only the cell bodies are stained in a typical blue. This

was developed by Nissyl, one of the pioneers of histology.

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Neurological Basis of Behavior (PSY - 610)

Weil Stain: Myelin stained brown: stains only cell projections/ fibers with myelin .

Golgi-Cox Silver: heavy metals such as silver absorbed by tissue- only 2-3% neurons stained,

but completely-can see soma, dendrites, and axons to the branches.

Histoflourescence: This method utilizes a special technique in which the cells are exposed to

dry Formaldehyde gas. Cells fluoresce in different colors. This can be sued to identify

neurotransmitters the areas of heavy concentration. This technique was used for noradrenaline

and Dopamine neurons, the NA fluoresce bright yellow. This can be used to identify single cell

or a group of cells.

Tracers: These are methods which are a special procedures involving injecting the animal while the

animal is alive. This can be used to where the tracer chemical end up- can be used in learning and

memory and the animal injected while it is performing. Or the animals can be injected with tracers during

early brain development and then the tracers located in later period to see how far neurons have traveled

during the early phase.

Neuroanatomical tracing methods are therefore used to follow up projections of neurons their

connections. There is wide range and types of tracers which can be used depending upon the area of

investigation and the interest of the researcher.

Procedure is to Inject a tracer into the nucleus of a neuron or other areas such as the synapse (where two

neurons connect), and to follow it through.

Anterograde tracer: The direction of transport is toward the periphery i.e. when the tracer is

carried away from cell soma to projections, (dendrites, and their branches).

Retrograde tracer: Where the direction of transport is towards the cell i.e. when the tracers

move towards the cell soma. This kind of tracer enters from the synapse. This shows connections

and interactions of a neuron.

The three major techniques are as follows:

Radio labeling, autoradiography (writing with own radiation (cell's)),Horse Radish Peroxidase: In

all these techniques a chemical or other Substance normally used in the brain (and is of interest) is tagged

with a radioactive tracer for follow up, and injected in the brain.

Radiolabelled (radioactive) Glucose: radiolabelling glucose to check out brain glucose cues. A

substance, 2-DG (2-Deoxy glucose) is injected and it enters the cell like a virus. It uses the cells

own metabolic mechanism (all cells use glucose during activity). This travels wherever the

glucose would have gone and it stays there. It emits radioactivity when exposed on to a

photographic plat.

Similarly radio-labeling other substances such as Amino acids for locating and identifying

amine activity, endorphins for brain opioid sites etc. This is done using the receptor binding

autoradiography for the neurotransmitter or drug which binds to a receptor, it is thus radio

labelled. Neural tissue is then exposed to a labelled ligand (molecule that binds to a target) and

these areas show up as radioactive on the photographic plate or under the microscope.

HRP: Horse Radish Peroxidase- this is an enzyme which travels through the retrograde

axoplasmic transport system moving through axons to the cytoplasm and traveling further to the

cells, their projections i.e. the dendrites. This is capable of breaking down certain peroxide

molecules turn into soluble salts, which are then are taken up by the terminal boutons. This

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Neurological Basis of Behavior (PSY - 610)

technique works well with a single cell as well. The stains glow or flouresce under specific

wavelengths light. This is also one of the histoflourescence techniques.

Histoflourescence: a technique developed by Falck and Hillarp in Sweden in the 1960's where

the monoamines exposed to formalin fixative, glow when exposed to flourescent light. This was

used to identify the locations and projections of Neurotransmitters.

Immunocytochemistry: introducing an antigen, to create antibodies (monoclonal antibodies).

Can use specific proteins for specific Neurotransmitters. The regions of radioactive accumulation

or dyes show up under microscope as location of the neuroprotein (the antigen). Thus we can use

labelled antibodies for target sites called labeled ligands.

References

1. Kalat J.W (1998) Biological Psychology Brooks/ Cole Publishing

2. Carlson N.R.(2005) Foundations of Physiological Psychology ( 5th Edition) Allyn and Bacon, Boston

3. Pinel, John P.J. (2003) Biopsychology (5th edition) Allyn and Bacon Singapore

4 Bloom F., Nelson and Lazerson (2001) Behavioural Neuroscience: Brain Mind and Behaviour (3rd

edition) Worth Publishers

5. Bridgeman, B (1988) The Biology of Behaviour and Mind. John Wiley and Sons New York

6. Brown, T.S. And Wallace.(1980) P.M Physiological Psychology

Academic Press New York

7. Seigel, G.J. (Ed. in chief) Agranoff, B.W, Albers W.R. and Molinoff, P.B. (Eds) (1989) Basic

Neurochemistry: Molecular, Cellular and Medical Aspects

8. Kolb, B and Whishaw, I.Q (1985) Fundamentals of Human Neuropsychology. (2nd edition) Freeman

and Company, New York