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NeurologicalBasis of Behavior (PSY - 610)

Lesson14

RESEARCHMETHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION

Objectives:

To familiarize the studentswith

· Thevarious techniques used to study the brain and its function andstructures.

· Invasivevs. Non- Invasive methods,Stereotaxic surgery.

· StereotaxicAtlas used for brain surgery and manipulation,

· Hislotogical and cytological methods forCell staining, Radiolabelling, Flourescence,

autoradiography,Lesioning and electricalstimulation, single cell recordings, push-pull

cannulae.

· Theadvanced techniques such as MRI,fMRI, CAT, PET, CT, EEG,EOG, EMG, X Rayetc

Histological,electrical chemical and pharmacologicalprocesses and techniques why so many?

Advancedtechnologies and methodologies

In order to identify brain areas specific sites and relate behaviors and functions with these sites,

traditional neuro anatomy uses techniques which are invasive. These techniques actually involve

entering the brain to record, to lesion or to bring about a change in the normal brain areas/systems.

There are many techniques which are used to do such as stimulation of brain areas using electrical

current to those areas, damaging (lesioning) areas of interest, drawing chemicals from a site through

push-pull cannulae technique and many others. Again, these can be used in combination with each other

or with other techniques (usually histology follows such manipulations). Keeping in view the

complexity of the brain, each of these techniques requires skills and training in order to the surgery-

brain surgery of animals. This requires being well trained in using the Stereotaxic instrument and the

Stereotaxic atlas

Stereotaxic instrument and surgery

Stereotaxicinstrument allows the restriction of the animal's body and head so that it is immovable,and

sensitive insertion of fine electrodescan be made. Using the Stereotaxic instrument the scientist can

place electrodes or conduct other surgicalprocedures using precisepositioning. These are in 3

dimensions, anterior-posterior (A-P: front-back), dorsal-ventral (D-Vtop-down), Medial-Lateral(M-L:

centersides). The Stereotaxicinstrument is made of steelwith several mainparts.

a) It has a head holder (for making sure that there are no head movements),

b) The ear bars which support the head in a position by locking onto the ear bones,

c) The electrode holder which can move in three dimensions described above (sideways, front and

back, up and down using a system which allows movement of electrode holder to follow exact

measurements.

Howdoes one use the Stereotaxic instrument? We follow the specifications given in the Stereotaxic

atlas (check out figure: one pageout of the rat brain atlas). These are prepared afterstudying dissections

of several hundred brains, here arespecific atlases for eachspecies (monkeys, mice, cats,dogs, etc)

Stereotaxicatlas: This is like a geographical atlas where dimensions are given to arrive at particular

location.These locations areidentified using two majorsutures (lines where bones of the skull join) and

the point they meet. In the anterior we use Bregma, Lambda is the posterior line. Using these as the

reference points we can move in anydirection in accordance with the atlas, and drill a hole in the skull

to lower the electrode in place.

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NeurologicalBasis of Behavior (PSY - 610)

Activatingbrain: Thiscan be done using electrodes(to pass the electricalcurrent) or through chemical

stimulation

Electrical stimulation. Electrodes (extremely thin for microelectrode, and insect pins for

macrolectrode) are prepared using insulated needles, leaving .5mm uninsulated tip to allow

current to pass through in the e brain are stereotaxically implanted in the brain. Insertions are

carried out using directions from the stereotaxic atlas placing the animal in the Stereotaxic

apparatus. The electrodes are implanted using dental cement to fix electrode for permanent use.

Once the electrode is set then weak electrical current can be passed through the electrodes to

the neurons to see the effect on behavior. Bipolar Electrodes are used to stimulate and measure

electrical charge and behavior at the same time. Behavioral and electrical response is dependent

upon the site in the brain, the amount of current/ charge the intensity and duration of

stimulation.

Animal'sstudies usingelectrical stimulation have providedresearchers a lot of information

about the brain. For example, one of the major behavior is selfstimulation, and the discovery of

selfstimulation areas thoseareas in which animals sendthemselves electrical current to the

pointthat they forego food and water to get this current in their brains ii) unilateralelectrical

stimulation of the substantia nigra( rich in Dopamine) and circling behavioriii) aggression.

In Humans, extremelyinteresting studies were carriedout by Roger Penfield and colleagues

who have identified areas by carryingout electrical stimulationprior to surgery to seewhich

areasand behaviors are going to affected, this led to the development and preparation of the

Motor homunculus ( dictionary of motor movements)

Microiontophoresis: This is an interesting method in which the chemical response of the

postsynaptic neuron is measured using a extremely thin doublebarreled glass pipette. Thetip of

innerpipette (which contains saline) is inserted into the membrane of the connecting cells. The

cellsoma is then stimulated with a weak current; this is thenpassed to stimulate the neuronal

endingleading to an electrical chemical discharge. This discharge is then is pulledout for

analysis, this requires extreme precision andskill.

Chemicalstimulation: pushpull cannulae. This is a method similar to the one abovebut

instead of the electrodes we can usethin glass cannulae to insertchemicals and immediately

drawout metabolites for analysis of activity and NT. This we canuse to measure effects of

stimulation or inhibition by using appropriate chemical solutions.

Recording on going activity: we cancheck for Excitatory or inhibitory electricalaftereffects

on an instrument known as the oscilloscope.

Inactivatingthe brain; theseare the procedures in which we can inactivate the parts of the brain by

using specialized procedure.

Ablation:This is a procedure in which we remove a majorpart of the brain to studywhat

behaviors and functions would be affected.There are various techniques, these are discussed

belowthis is irreversible, as once we make the changes, lesions, we cannot reverse the process.

Suction or aspiration: In thistechnique brain tissue is sucked out through a glasspipette. This

requires extreme precision. Also it is difficult to go through thicker white matter on the surface

to get to the inner deeper areas.Ablation or suction is not a very commonly used procedure

because we may be damaging largerarea without knowing or intending to do so.

Radio frequency lesions: in this procedure we use the alternating highfrequency currentfrom

tip of electrode which is placed in a preciselocation in the brain. Theheat from the current

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NeurologicalBasis of Behavior (PSY - 610)

destroys the tissue and the sizeregion of damage is the same as that of the tip of the electrode.

Theintensity and duration of current, the area in which we have lesionedwould also be

important in the effects. It is a fairly safe procedure as the electrode passing through toptissue

leaves the overlying areas unharmed. This is also irreversibledamage.

Knife cuts: verythin knifes are used to section out /cut or to damage connections between

brainareas. This helps us isolateare using precision knifecuts. What about

remaining/adjoiningareas? How has the damageaffected their functioning?This is also

irreversibledamage

Cryogenic freeze: (reversible). In this technique we freeze the brain area with cryoprobe (a

thinpipe with some mechanism to cool/ or artificial ice).The coolant is inserted and as soon as

the brain area freezes, it stopsfunctioning. We can assessbehaviors which do notoccur. This

brainareas returns functioning when the it becomes warm For otherreversible techniques, local

anestheticsare also used effectively ( the sodium amytal test for language functioning and

assessingbrain hemispheric differences)

Lesioningirreversible This is an irreversible technique, meaningthat the damage is permanent.

Lesioningcan be electrical, (or radiolesioning as discussedearlier).

Electrolytic:Thisinvolves passing a highfrequency direct electricalcurrent through the

stereotaxically placed electrodes and preciselocation/area is damaged throughelectrolysis.

There is minimal damaged and veryfocused in the area.

Chemical:Thisinvolves injecting selective neurotoxins (poisons) and other degenerative

substanceswhich can kill offliving neurons. There aresubstances such as 6-OHDAwhich are

selective toxins for dopamine. This is retrograde and travels fromthe synapse to the cellbody.

Bilaterallesions: Theseare lesions carried out on the areas on both sides (left and the right

hemisphere), therefore the effect is more intense.

Unilaterallesions involveonly one side of the brain:This is good experimental design as one

sideacts as the control for the other, but the changes may be small and may not be easy to

detect.

Shortcomings of this technique: Lesions require histological verificationsafter behavioral

testsare over. Further, the areas adjacent to the lesions are also affectedand the behaviors

observed and tested may be confounded by these. There aredifficulties in interpretation as

there is the irritation of neighboring neurons. Furthermore there are issues of adaptability of the

brainareas (to the damage),plasticity of the brain (recoveryand learning).

Points to ponder: Do thesechanges measure actual changes?

Electrophysiological methods: these are methods which measure changes of the electrical potential

and charge in the brain

Single microelectrode recording: a single thin neuron about 5-10 UM (micromolar), or a

microelectrode (1-3 UM) glass tubing or steel pens are used to record electrical potential. This

is how the all-or none axonal activity was measured and identified. This is also how responses

to a single stimulus single resulting in neuronal firing were first measured by Hubel and Weisel

using the visual systems of the kittens (intracellular/ extracellular recordings are possible with

this technique.

Macro electrodes: these involve inserting large electrodes in large neurons. The large tips help

measure evoked potentials (EP's) of these areas, (response to a stimulus or stimulation. There

are many neurons firing in an area, and these are then magnified to a point one can hear the loud

firing of the neurons in a typical EP average evoked potential.

Surface/Scalp electrode in a region: Electroencephalography recordings are done for the human

brain and recording can show variation (during sleep) and other behaviours.

NeurologicalBasis of Behavior (PSY - 610)

References:

1. Kalat J.W (1998) BiologicalPsychology Brooks/ ColePublishing

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

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

4 Bloom F, Nelson and Lazerson (2001),Behavioral Neuroscience: Brain, Mind and Behaviors (3rd

edition)Worth Publishers NewYork

5. Bridgeman,B (1988)The Biology of Behaviour and Mind. John Wileyand Sons NewYork

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

Neurochemistry:Molecular, Cellular and Medical Aspects.

Note:References#2, 3, and 4 are followedmost closely, as they have been used in teaching as well;

furtherindividual references/pages arealso given on the powerpoints of each lesson

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