Methods of study and research techniques

Class By: Monojit (Assistant Professor, SoS JU)

 !! Syllabus

Objectives

• To introduce the concept of brain-behaviour relationships, behaviour genetics

• To acquaint the students with the basic biological processes affecting psychological processes.

Syllabus

• Unit 1: Introduction to biological basis of behavior (10 Hours)

Nature and scope of physiological psychology; Methods of study and research techniques: Neuroanatomical,
Neuroelectric, Neurochemical, and imaging techniques.
• Unit 2: Behavior genetics (8 Hours)
Nature and scope, Methods of study and research techniques: Family, twin, adoption methods; Genetic principles and
mechanism of animal and human behavior, chromosomal functions.
• Unit 3: Nervous System and Neuronal Functions (4.5 Hours)
Major divisions and functions of nervous system: central and autonomic nervous systems.

9/26/2023 Mono’s Class 2

 1 Techniques

• Visualizing the Human Brain

• Computerized Tomography
• Magnetic Resonance Imaging
• Positron Emission Tomography
• Microscopic Approaches to Brain Anatomy
• Recording Brain Electrical Activity
• The Electroencephalogram
• Magnetic Recording
• Microelectrode Recording
• Patch Clamp
• Brain Stimulation
• Magnetic Stimulation
• Neurochemical Approaches
• Chemical Stimulation
• Microiontophoresis
• Microdialysis
• Brain Lesion Analysis

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 2 Brain Anatomy: Microscopic Approaches
The first compound microscopes date to 1590, but it was the Dutch, Antony Van Leeuwenhoek in the mid-seventeenth century.

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 2 Brain Anatomy: Microscopic Approaches

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 2 Brain Anatomy: Microscopic Approaches
• The optical microscope, also referred to as a light
microscope, is a type of microscope that commonly uses
visible light and a system of lenses to generate magnified
images of small objects.
• Transparent objects can be lit from below and solid objects
can be lit with light coming through (bright field) or around
(dark field) the objective lens.

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 2 Brain Anatomy: Microscopic Approaches
• Phase-contrast imaging is a method of imaging that has a
range of different applications. It exploits differences in the
refractive index of different materials to differentiate
between structures under analysis.
• Phase contrast takes advantage of the fact that different
structures have different refractive indices, and either bend,
refract or delay the light passage through the sample by
different amounts. The changes in the light passage result in
waves being 'out of phase' with others. This effect can be
transformed by phase contrast microscopes into amplitude
differences that are observable in the eyepieces and are
depicted effectively as darker or brighter areas of the
resultant image.

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 2 Brain Anatomy: Microscopic Approaches
• Dark-field microscopy (also
called dark-ground
microscopy) describes
microscopy methods, in both
light and electron
microscopy, which exclude
the unscattered beam from
the image. As a result, the
field around the specimen
(i.e., where there is no
specimen to scatter the beam)
is generally dark.

• Dark-field microscopy is well suited for uses involving live and unstained biological
samples, such as a smear from a tissue culture or individual, water-borne, single-celled
organisms

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 2 Brain Anatomy: Microscopic Approaches

Reference: https://en.wikipedia.org/wiki/File:Dark_field_and_phase_contrast_microscopies.ogv
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 2 Brain Anatomy: Microscopic Approaches
• Differential interference contrast (DIC) microscopy, also known as Nomarski
interference contrast (NIC) or Nomarski microscopy, is an optical microscopy
technique used to enhance the contrast in unstained, transparent samples. DIC
works on the principle of interferometry to gain information about the optical
path length of the sample, to see otherwise invisible features.

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 2 Brain Anatomy: Microscopic Approaches
• An inverted microscope is a microscope with its light source
and condenser on the top, above the stage pointing down,
while the objectives and turret are below the stage pointing
up. It was invented in 1850 by J. Lawrence Smith, a faculty
member of Tulane University.
• These microscopes may also be fitted with accessories for
fitting still and video cameras, fluorescence illumination,
confocal scanning and many other applications.
• Inverted microscopes are useful for observing living cells or
organisms at the bottom of a large container (e.g., a tissue
culture flask) under more natural conditions than on a glass
slide, as is the case with a conventional microscope.

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 2 Brain Anatomy: Microscopic Approaches
• A fluorescence microscope is an optical microscope that uses
fluorescence instead of, or in addition to, scattering,
reflection, and attenuation or absorption, to study the
properties of organic or inorganic substances.

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 2 Brain Anatomy: Microscopic Approaches

• Confocal microscopy, most frequently confocal laser

scanning microscopy or laser confocal scanning
microscopy, is an optical imaging technique for increasing
optical resolution and contrast of a micrograph by means
of using a spatial pinhole to block out-of-focus light in
image formation.

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 2 Brain Anatomy: Microscopic Approaches

Reference: https://en.wikipedia.org/wiki/File:Dark_field_and_phase_contrast_microscopies.ogv
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 2 Brain Anatomy: Microscopic Approaches

Reference: https://www.youtube.com/watch?v=a0G7iyz4McM
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 2 Brain Anatomy: Microscopic Approaches

Reference: https://www.youtube.com/watch?v=s6KqJS1GZNE
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 3 Brain Anatomy: Microscopic Approaches/ Histology
The study of tissues and cells under a microscope is called histology.

0. Grossing
1. Tissue fixation
• This is a crucial step in tissue preparation, and its purpose is to
prevent tissue autolysis and putrefaction.
2. Specimen Transfer to Cassettes
3. Tissue Processing
• Processing tissues into thin microscopic sections is usually done
using a paraffin block, as follows:
• Dehydration, which involves immersing your specimen in
increasing concentrations of alcohol to remove the water and
formalin from the tissue.
• Clearing, in which an organic solvent such as xylene is used to
remove the alcohol and allow infiltration with paraffin wax.
• Embedding, where specimens are infiltrated with the
embedding agent – usually paraffin wax. The tissue becomes
surrounded by a large block of molten paraffin wax, creating
what is now referred to as the “block”. Once the block
solidifies, it provides a support matrix that allows very thin
sectioning.

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 3 Brain Anatomy: Microscopic Approaches/ Histology
4. Sectioning
• Your tissue specimen is now ready to be cut into sections that can be placed
on a slide.
• Blocks are chilled on a refrigerated plate or ice tray for 10 minutes before
sectioning.
• A microtome is used to slice extremely thin tissue sections off the block in
the form of a ribbon.
• Once cut, the tissue ribbons are carefully transferred to a warm water bath.
Here they are allowed to float on the surface and can then be scooped up
onto a slide placed under the water level. Charged slides work best for this
process – they improve tissue adhesion to the glass and help to reduce the
chance of sections washing off the slide during staining.
• Slides should be clearly labeled, and then allowed to dry upright at 37oC
for a few hours to gently melt the excess paraffin wax, leaving the tissue
section intact.
5. Staining
• Most cells are transparent and appear almost colorless when unstained.
Histochemical stains (typically hematoxylin and eosin) are therefore used
to provide contrast to tissue sections, making tissue structures more visible
and easier to evaluate. Following staining, a coverslip is mounted over the
tissue specimen on the slide, using optical grade glue, to help protect the
specimen.

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 3 Brain Anatomy: Microscopic Approaches/ Histology

Reference: https://www.youtube.com/watch?v=7-LIbAWPc-g
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 3 Brain Anatomy: Neuro anatomy specific staining
• Staining is a procedure to selectively darken or color particular features of the
sectioned tissue.
• By choosing an appropriate stain, different features of the tissue are
highlighted.
Specialized staining:
Golgi Stain
• The Golgi silver impregnation technique is a simple histological procedure
that reveals complete three-dimensional neuron morphology. This method is
based in the formation of opaque intracellular deposits of silver chromate
obtained by the reaction between potassium dichromate and silver nitrate Camillo Golgi
(black reaction).
• The Golgi method is used for for visualizing single nerve cells.
Nissl’s Stain
• The Nissl method refers to staining of the cell body, and in particular Golgi Stain
endoplasmic reticulum. This is done by using various basic dyes (e.g. aniline,
thionine, or cresyl violet) to stain the negatively charged RNA blue and is used
to highlight important structural features of neurons. The Nissl substance
(rough endoplasmic reticulum) appears dark blue due to the staining of
ribosomal RNA, giving the cytoplasm a mottled appearance. Individual
granules of extranuclear RNA are named Nissl granules (ribosomes). DNA
present in the nucleus stains a similar color.
• Nissl staining is useful for visualizing the distribution of cell bodies in the
specimen.
Franz Alexander
Nissl

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 3 Brain Anatomy: Neuro anatomy specific staining
Luxol fast blue stain
• Luxol fast blue stain, abbreviated LFB stain or simply LFB, is a
commonly used stain to observe myelin under light microscopy, created
by Heinrich Klüver and Elizabeth Barrera in 1953.
• LFB is commonly used to detect demyelination in the central nervous
system (CNS).
• Myelin can be lost in a variety of conditions or disease states. For
example, multiple sclerosis (MS) is a common neurological condition
resulting in a dysfunctional immune system-mediated loss of myelin, Mouse Brain Luxol fast + Hematoxylin
which causes symptoms such as muscle weakness, paresthesias, and
fatigue.
• Traumatic brain injury, such as a concussion, can also lead to
demyelination.
• Luxol fast blue is a copper phthalocyanine dye that is soluble in alcohol
and is attracted to bases found in the lipoproteins of the myelin sheath.
• Under the stain, myelin fibers appear blue, neuropil appears pink, and
nerve cells appear purple. Tissues sections are treated over an extended
period of time (usually overnight) and then differentiated with a lithium
carbonate solution.

Micrograph of the pons using a hematoxylin & eosin-luxol fast blue stain.
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 3 Brain Anatomy: Neuro anatomy specific staining
Weil stain
• The Weil stain is a basic stain used for the revelation of myelinated axons.
Myelinated area appear darker.
• Weil’s stain is a modification for paraffin sections of the Weigert-Pal-
Kulschitsky technique. The underlying principle of these methods
involves the reduction of chrome salt to chromium dioxide by myelin. The
chromium subsequently acts as a mordant for the haematoxylin,
intensifying the stain.

Bielschowsky's silver stain Weil-Myelin Stain on a 40 micron Rhesus

• Bielschowsky's silver stain is a very useful tool to detect nerve fibers. It Monkey section.
can be used to stain axons, neurofibrils and senile plaques in the central
nervous system. This method is easy to perform and is routinely used in
the study of Alzheimer's disease together with antibody staining.
• The Bielschowsky technique is a silver staining method used in
histochemistry for the visualization of nerve fibers, including multipolar
interneurons in the cerebellum.
• Based on modifications of Cajal’s method.

Bielschowsky silver stain showing the processes of basket cells in the cerebellum.

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 3 Brain Anatomy: Neuro anatomy specific staining
Cajal's Gold Sublimate
• Used for astrocyte detection.
• A method for demonstrating astrocytes by impregnation in a solution containing
gold chloride and mercuric chloride.

Ｋlüver-Barrera（KB）staining
• Double staining with Nissl staining using cresyl violet and LFB staining of myelin
sheaths.
• The Kluver-Barrera staining method (K. B. method) presented by Kluver and
Barrera in 1953 who got a hint from Kosaki's study, has an advantage thatnervous
cells and myeline sheathes can be observed on the same slice. Proliferation of reactive astrocytes. Cajal Gold sublimate.

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 4 Brain Anatomy: Immuno Histochemistry
Glial fibrillary acidic protein (GFAP) is a protein specifically found in the cytoskeleton of
astrocytes.

Glial cells. A, B, C and D: Astrocytes in the telencephalon. B: Neuron and glia are
different in size. A, B and C: Protoplasmic astrocytes. D: Fibrous astrocytes. E:
Bergmann glia in the cerebellum.

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 5 Brain Anatomy: Immunofluorescence

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 6 Brain Anatomy: Tracing
Neuronal tracing, or neuron reconstruction is a technique used in neuroscience to determine the pathway of the neurites or
neuronal processes, the axons and dendrites, of a neuron.
• From a sample preparation point of view, it can be:
• Anterograde tracing, for labeling from the cell body to synapse.
• Retrograde tracing, for labeling from the synapse to cell body. E.g., PLA-L
• Viral neuronal tracing, for a technique which can be used to label in either direction.
• Manual tracing of neuronal imagery.

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 6 Brain Anatomy: Tracing
• Traditional tracing compounds either produce a colorimetric product or are fluorescent.
• While dyes usually spread unidirectionally, there are some reports of bi-directionality.
• For predominately anterograde tracing, biotinylated dextran amines or fluorescent
molecules like rhodamine-isothiocyanate are used.
• For predominantly retrograde tracing, albumin protein labelled with horseradish
peroxidase, plant lectins (wheat germ agglutinin) or fluorescent molecules such as
Fluoro-Gold were used.
• To facilitate cellular uptake of these dyes in vivo, microinjection, pressure injection or
iontophoresis (applying small electric currents via inserted electrodes) may be applied,
but these methods can have a negative impact on cellular health.
• Another limitation of traditional tracing compounds is that their diffusion across the
synaptic cleft is usually severely impaired, and only few reports exist about dye
molecules that are transported from one neuron to another at synapses resulting in faint
signals.

Immunohistochemistry with anterograde tracing

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 6 Brain Anatomy: Tracing
• Viral vector technology, have been developed to study neuronal
connectivity.
• These methods are less neurotoxic and better compatible with
other neuroscience methods such as electrophysiological
recordings.
• The origins of these methods are naturally occurring viruses that
infect, persist, and migrate within neurons, and that are also
capable of spreading across synaptic connections.
• Most famous is the Rabies Virus (RABV) which moves
retrogradely from a peripheral site of infection to the central
nervous system where it replicates, spreads, causes neurotoxicity,
and is lethal if left untreated.
• Herpes Simplex Virus (HSV) also migrates through neurons
where it can replicate and by doing so spread across several
synaptic connections.
• When using these viruses to mark neuronal connectivity a large
majority of neuronal cells are labeled overtime due to the
continued replication of the virus.
• While this can be useful, if too many neurons are labeled, it can
be difficult to accurately map network connections

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 6 Brain Anatomy: Tracing

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 6 Brain Anatomy: Tracing

Neuroanatomical tract-tracing techniques that did go viral

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 7 Neurochemical: Neurotransmitters

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 7 Neurochemical: Microdialysis

• The technique of microdialysis enables

sampling and collecting of small-molecular-
weight substances from the interstitial
space. It is a widely used method in
neuroscience and is one of the few
techniques available that permits
quantification of neurotransmitters,
peptides, and hormones in the behaving
animal.
• Stereotaxic Surgery is needed to place the
probes in place.
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 7 Neurochemical: Microdialysis

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 7 Neurochemical: Microdialysis

Reference: https://www.youtube.com/watch?v=kak5UAntnwQ
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 7 Neurochemical: Microdialysis

Reference: https://www.youtube.com/watch?v=_WcHlf_crSU
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 8 Neurochemical: In-situ Hybridization
• In-situ hybridization is a technique that allows for precise localization of a specific segment of nucleic acid within a histologic
section. It can be done using enzyme conjugated or fluorescent (FISH) attached single strand RNA probes.

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 8 Neurochemical: In-situ Hybridization

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 8 Neurochemical: In-situ Hybridization

Reference: https://www.youtube.com/watch?v=DEIL3KeXL9w
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 9 Neuroablation: Brain Damage
Experimental neuroablation is a safe procedure in which a portion of nerve tissue is destroyed or removed to cause an
interruption neuronal signals in that area. Nerve ablation can be done in different ways. For example, it can be done using heat,
cold, or chemicals, it may be called radiofrequency ablation, cryoablation, neurotomy, or rhizotomy.
• Lesion studies directly relate brain dysfunction — in the form of a lesion — to behavioral deficits.
• Focal area of brain damage is correlated with the development of a defect in some aspect of cognition or behavior, and then
an inference is made that the damaged brain region is part of the neural substrate for the impaired function.
• The study of patients with brain lesions has made major historical contributions to cognitive neuroscience.
• Lesions give us insight into the causally necessary function of brain structures, whereas electrophysiology and fMRI reflect
mere correlations with psychological processes.
• Lesions show us dissociations in cognition we could never have hypothesized, and thus can radically change our model of the
architecture of the mind.
• And careful characterization of the deficits following lesions and their change over time provides clinically valuable
information not only about the constellation of impairments produced, but also about their compensation and possible
resolution over time.
• Major disadvantage is-
• Very less lesion studies are done
• Every patient is different in idiosyncratic ways, the lesions are far too coarse since they cannot be experimentally
produced, and fMRI has already made the lesion method obsolete
• Ethics, family and societal pressure.

https://doi.org/10.1007/978-1-4757-2055-6_3
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 9 Neuroablation: Brain Damage

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 9 Neuroablation: Brain Damage

Rats administered septal lesions after

parturition became hyperresponsive,
ceased all maternal behaviors, and
cannibalized their pups.

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 9 Neuroablation: Brain Damage

Radiofrequency ablation

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 9 Neuroablation: Brain Damage

Thermal ablation (MRI guided)

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 9 Neuroablation: Brain Damage

Neurotomy

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 9 Neuroablation: Brain Damage

Stereotaxic Surgery

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 10 Neuroelectrochemical: Patch clamp
Nerve cells regulate their electrical activity by controlling small pores or channels in
their outer membrane.
• A patch clamp is an adaption of the glass micropipette method in which a small
amount of suction is applied to the fluid-filled recording electrode.
• If the tip of the electrode is placed on the outer surface of the cell membrane, a
tight mechanical and electrical seal results. The result is that the electrode
measures electrical current only from the portion of the membrane that is
clamped to the electrode. In this way, the activity of individual membrane
channels can be measured.

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 10 Neuroelectrochemical: Patch clamp

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 10 Neuroelectrochemical: Patch clamp

Reference: https://www.youtube.com/watch?v=mVbkSD5FHOw
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 11 Neurostimulation: Recordings
• Electroencephalography is a method to record an electrogram of the spontaneous electrical activity of the brain.
• The biosignals detected by EEG have been shown to represent the postsynaptic potentials of pyramidal neurons in the
neocortex and allocortex.
• Electroencephalography is a method to record an electrogram of the
spontaneous electrical activity of the brain.
• The biosignals detected by EEG have been shown to represent the
postsynaptic potentials of pyramidal neurons in the neocortex and
allocortex.
• It is typically non-invasive, with the EEG electrodes placed along the scalp
(commonly called "scalp EEG") using the International 10-20 system, or
variations of it.

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 11 Neurostimulation: Recordings
• Voltage fluctuations measured by the EEG bioamplifier and
electrodes allow the evaluation of normal brain activity
including the posterior dominant rhythm (PDR), first
described by Hans Berger.
• EEG can detect abnormal electrical discharges such as sharp
waves, spikes or spike-and-wave complexes that are seen
in people with epilepsy, thus it is often used to inform the
medical diagnosis.
• EEG can detect the onset and spatio-temporal evolution of
seizures and the presence of status epilepticus.
• It is also used to help diagnose sleep disorders, depth of
anesthesia, coma, encephalopathies, cerebral hypoxia
after cardiac arrest, and brain death.
• EEG used to be a first-line method of diagnosis for tumors,
stroke and other focal brain disorders, but this use has
decreased with the advent of high-resolution anatomical
imaging techniques such as magnetic resonance imaging
(MRI) and computed tomography (CT).

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 11 Neurostimulation: Recordings

Reference: https://www.youtube.com/watch?v=tZcKT4l_JZk&t=40s
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 11 Neurostimulation: Recordings

Reference: https://www.youtube.com/watch?v=i2St7BeRz6A&t=349s
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 11 Neurostimulation: Recordings

Reference: https://www.youtube.com/watch?v=XMizSSOejg0
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 11 Neurostimulation: Recordings

Reference: https://www.youtube.com/watch?v=kFghQLm2ND8&t=14s
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 11 Neurostimulation: Recordings
• Electrocorticography, involving surgical placement of
electrodes, is sometimes called "intracranial EEG".
• Cortical mapping for epilepsy surgery.
• ECoG has recently emerged as a promising recording
technique for use in brain-computer interfaces (BCI).
• BCIs are direct neural interfaces that provide control of
prosthetic, electronic, or communication devices via direct
use of the individual's brain signals.

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 11 Neurostimulation: Recordings
• Magnetoencephalography, is the measurement of the
magnetic field generated by the electrical activity of
neurons. It is usually combined with a magnetic resonance
imaging to get what is called magnetic source imaging.
• It is a functional neuroimaging technique for mapping brain
activity by recording magnetic fields produced by electrical
currents occurring naturally in the brain, using very
sensitive magnetometers. Arrays of SQUIDs
(superconducting quantum interference devices) are
currently the most common magnetometer, while the SERF
(spin exchange relaxation-free) magnetometer is being
investigated for future machines.

• Use:
• Brain connectivity and neural oscillations
• Focal epilepsy
• Experimental fetal imaging
• Traumatic brain injury

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 12 Neurostimulation: Stimulations

• Electrical stimulation of the brain (ESB) is an effective

means of demonstrating functional neural connections
between two brain regions.
• If an electrical stimulation of one area evokes an electrical
response in another, there must be some functional pathway
linking the two regions of the brain.
• Using specialized techniques, stimulation may be confined to
a single nerve cell.
• Usually, however, a population of cells is activated in the
region of the electrode.
• It is generally believed that using electrodes with 1 mm2
exposed tip and passing about 1 milliampere (mA) of current
stimulates about 1 cubic millimeter of brain tissue, although
a larger region may be affected under many circumstances.
• Usually, the effects of ESB are apparent immediately
following stimulation.
• Electrical stimulation of the lateral hypothalamus - an area
deep within the brain related to feeding - may have no
immediate effect.
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 12 Neurostimulation: Stimulations
• The problem for a neurosurgeon attempting to remove diseased tissue in regions of the brain that support higher mental
functions - such as language - is that those functions are not always carried out by exactly the same brain areas, particularly
in individuals with a long history of brain disease.
• ESB provides the "gold standard" by which the functional properties of a region of brain may be determined before the
tissue is surgically removed.
• For this reason, ESB is often carried out with the surface of the brain exposed during neurosurgery. The patient is
conscious, since it is necessary to determine whether stimulation of a particular cortical region affects speech perception of
production. Such an operation is possible because the brain itself does not have pain receptors; only a topical anesthetic is
required to deaden the nerves of the scalp and skull.
• As different regions of the brain are stimulated, speech perception and production are tested.
• Electrical stimulation of the human brain is also carried out - although much less frequently - by using electrodes that have
been surgically implanted within the brain of patients undergoing prolonged (e.g. several weeks) of monitoring often for
the localization of epileptic disorders. Studies of such patients have also contributed to the understanding of certain higher
mental functions.

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 12 Neurostimulation: Stimulations

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 12 Neurostimulation: Stimulations
Magnetic Stimulation:
• Recently, a new - completely noninvasive - procedure for stimulating the neurons has been developed, using focused magnetic
fields rather that electrical current. By using a small (10-15cm) coil placed against the surface of the scalp, a 1- to 2-tesla
focused magnetic field may be generated. This field is capable of locally exciting the regions of the underlying brain and
inducing electrical discharges from that tissue. In this way, functional activity of the brain can be determined in normal
individuals who are not undergoing neurosurgery.

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 12 Neurostimulation: Stimulations
Chemical Stimulation:
• Similar analysis can be carried out on a much more localized basis, by specifically introducing the chemical to a particular
brain region. Using a small injection tube, called a cannula, any pharmacological agent can be placed in a restricted brain
region.
• In animal research, a sturdy, large-bore guiding cannula is surgically implanted before testing is to take place. After the animal
has recovered, a small injection cannula can be inserted through the guide to deliver the agent to the target structure.

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 12 Neurostimulation: Stimulations
Microiontophoresis:
• Injecting chemical into the brain using a cannula affects - of necessity - a large number of cells, since a cannula is
comparatively large with respect to the size of these cells.
• Microiontophoresis provides a more precise means of stimulating single nerve cells with chemical agents. In this method, a
cluster of micropipettes is employed.
• One is used as a microelectrode to record the electrical activity of the target cell. The other pipettes are filled with specially
prepared solutions of the chemicals to be tested. These solutions are ionized, or electrically charged. BY passing a small
electrical current through a pipette containing an ionized solution, molecules of the substance can be released from the pipette
onto the target cell. Iontophoresis means literally "carried by ions." Microiontophoresis is the most precise form of chemical
stimulation of the brain possible today.

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 Q&A

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 Food for Thought
▪ How can you use a transgenic expression system in neuroimaging?
▪ What is HPLC and how it is used to detect neurochemicals?
▪ What is difference between frequency, amplitude, and wavelength?
▪ Design a hypothetical experiment and explain hoe patch clamp will be
used in that experiment?
▪ Differentiate between MRI, PET, and CT.
▪ What actually goes on in the brain that produces different waves in
EEG?

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 Chill

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