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RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION

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Neurological Basis of Behavior (PSY - 610)
VU
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)
VU
· 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)
VU
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)
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·
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)
VU
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
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Table of Contents:
  1. INTRODUCTION:Descriptive, Experimental and/ or Natural Studies
  2. BRIEF HISTORICAL REVIEW:Roots of Behavioural Neurosciences
  3. SUB-SPECIALIZATIONS WITHIN THE BEHAVIORAL NEUROSCIENCES
  4. RESEARCH IN BEHAVIOURAL NEUROSCIENCES:Animal Subjects, Experimental Method
  5. EVOLUTIONARY AND GENETIC BASIS OF BEHAVIOUR:Species specific
  6. EVOLUTIONARY AND GENETIC BASIS OF BEHAVIOUR:Decent With Modification
  7. EVOLUTIONARY AND GENETIC BASIS OF BEHAVIOUR:Stereoscopic vision
  8. GENES AND EXPERIENCE:Fixed Pattern, Proteins, Genotype, Phenotypic
  9. GENES AND EXPERIENCE:Mendelian Genetics, DNA, Sex Influenced Traits
  10. GENES AND EXPERIENCE:Genetic Basis of behavior, In breeding
  11. GENES AND EXPERIENCE:Hybrid vigor, Chromosomal Abnormalities
  12. GENES AND EXPERIENCE:Behavioral Characteristics, Alcoholism
  13. RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION
  14. RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION:Activating brain
  15. RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION:Macro electrodes
  16. RESEARCH METHODS AND TECHNIQUES OF ASSESSMENT OF BRAIN FUNCTION:Water Mazes.
  17. DEVELOPMENT OF THE NERVOUS SYSTEM:Operation Head Start
  18. DEVELOPMENT OF THE NERVOUS SYSTEM:Teratology studies, Aristotle
  19. DEVELOPMENT OF THE NERVOUS SYSTEM:Stages of development, Neurulation
  20. DEVELOPMENT OF THE NERVOUS SYSTEM:Cell competition, Synaptic Rearrangement
  21. DEVELOPMENT OF THE NERVOUS SYSTEM:The issues still remain
  22. DEVELOPMENT OF THE NERVOUS SYSTEM:Post natal
  23. DEVELOPMENT OF THE NERVOUS SYSTEM:Oxygen level
  24. Basic Neuroanatomy:Brain and spinal cord, Glial cells, Oligodendrocytes
  25. Basic Neuroanatomy:Neuron Structure, Cell Soma, Cytoplasm, Nucleolus
  26. Basic Neuroanatomy:Control of molecules, Electrical charges, Proximal-distal
  27. Basic Neuroanatomy:Telencephalon, Mesencephalon. Myelencephalon
  28. Basic Neuroanatomy:Tegmentum, Substantia Nigra, MID BRAIN areas
  29. Basic Neuroanatomy:Diencephalon, Hypothalmus, Telencephalon, Frontal Lobe
  30. Basic Neurochemistry:Neurochemicals, Neuromodulator, Synaptic cleft
  31. Basic Neurochemistry:Changes in ionic gates, The direct method, Methods of Locating NT
  32. Basic Neurochemistry:Major Neurotransmitters, Mesolimbic, Metabolic degradation
  33. Basic Neurochemistry:Norepinephrine/ Noradrenaline, NA synthesis, Noadrenergic Pathways
  34. Basic Neurochemistry:NA and Feeding, NE and self stimulation: ICS
  35. Basic Neurochemistry:5HT and Behaviors, Serotonin and sleep, Other behaviours
  36. Basic Neurochemistry:ACH and Behaviors, Arousal, Drinking, Sham rage and attack
  37. Brain and Motivational States:Homeostasis, Temperature Regulation, Ectotherms
  38. Brain and Motivational States:Biological Rhythms, Circadian rhythms, Hunger/Feeding
  39. Brain and Motivational States:Gastric factors, Lipostatic theory, Neural Control of feeding
  40. Brain and Motivational States:Resting metabolic state, Individual differences
  41. Brain and Motivational States:Sleep and Dreams, Characteristics of sleep
  42. Higher Order Brain functions:Brain correlates, Language, Speech Comprehension
  43. Higher Order Brain functions:Aphasia and Dyslexia, Aphasias related to speech
  44. Higher Order Brain Functions:Principle of Mass Action, Long-term memory
  45. Higher Order Brain Functions:Brain correlates, Handedness, Frontal lobe