Transcript Chapter 5

Chapter 5
Methods and Strategies of
Research
Research methods
Scientific investigation entails a
process of asking questions of nature
The best conclusions about the
physiology of behavior are made not
by any single experiment, but by a
program of research that enables us
to compare the results of studies that
approach the problem with different
methods
Experimental ablation
The removal or destruction of a
portion of the brain of a lab animal;
presumably, the functions that can no
longer be performed are the ones the
region previously controlled
Oldest method in neuroscience
Evaluation the behavioral effects of
brain damage
Lesion study (i.e. experimental ablation)
Must be very careful about interpreting effects of
lesion:
Just because certain functions appear to be missing,
doesn’t mean they were necessary for some type of
behavior
Circuits within the brain perform functions, not
behaviors
No one brain region or neural circuit is solely
responsible for a behavior
Also, all regions of brain are interconnected; i.e. if
you disrupt one neural circuit to affect a certain
behavior, you may end up affecting other
behaviors too
Producing brain lesions
Immediately underneath the skull:
Anesthetize animal, cut scalp, remove part of skull, cut through
dura mater, and use suction device (e.g. pipette) to remove
brain material
For deeper brain tissue
Use electrode to burn away tissue – very general, destroys all
surround tissue, cells
Excitotoxic lesions – e.g. kainic acid – kills neurons by
stimulating them to death; more focused technique
6-hydroxydopamine – chemical selectively taken up by axons
and terminal buttons of NE or DA neurons and acts as poison,
killing them; selective method
However, passing an electrode through or injecting chemical
into brain tissue can destroy other tissue along the way
Control group – sham lesions
Stereotaxic surgery
Brain surgery using a
stereotaxic apparatus
to position an
electrode or cannula in
a specified position of
the brain
In order to predict
location of a brain
area, use stereotaxic
atlas, and use bregma
(the junction of the
sagittal and coronal
sutures of the skull) as
a reference point
Histological methods
After producing lesion and observing effects, must check to make
sure that lesion was in proper area
Do so by fixing, slicing, staining and examining the brain
Fixation and sectioning
Must fix the brain tissue first in order to prevent tissue degradation and
make firmer for slicing; most commonly used is formalin
Before fixing, must perfuse the animal, which is to remove blood and
replace with another fluid (usually saline); this blood cells from being
included in sections while observing under microscope
Once brain has been fixed, it is sliced in to sections using a microtome
Staining
In order to see fine details in sections under a microscope, must stain
the cells
Nissl staining method uses a dye that is taken up by the Nissl
substance in the cytoplasm, making it possible to identify nuclear
masses in the brain
Electron microscopy
Scanning electron microscope – provides less magnification than a
standard EM, but shows objects in 3D
Tracing neural connections
In order to determine where certain neurons send their
axons to (i.e. neural circuits) we must be able to trace
efferent/afferent axons
Tracing efferent axons
Anterograde labeling method – labels axons and terminal
buttons of neurons whose cell bodies are located in a particular
region
Using proteins such as PHA-L
Immunocytchemical methods – uses radioactive antibodies to
indicate presence of proteins
Tracing afferent axons
Retrograde labeling method – labels cell bodes that give rise to
the terminal buttons that form synapses with cells in a particular
region
e.g. fluorogold
Pseudorabies virus – used for transneural tracing, which labels a
series of neurons that are interconnected synaptically
Study of the living human brain
Computerized tomography
(CT scan) – use of a
device that employs a
computer to analyze data
obtained by a scanning
beam of x-rays to produce
a 2D picture of a “slice”
through the body
Magnetic resonance
imaging (MRI) – the interior
of the body can be
accurately imaged;
involves the interaction b/t
radio waves and a strong
magnetic field
Recording of neural activity
Recordings can be made chronically (after
surgery), or acutely (while anesthetized)
Recordings with microelectrodes
Microelectrodes have a fine tip, small enough to record
the electrical activity of individual neurons, called singleunit recording
Recordings with macroelectrodes
Macroelectrodes do not detect the activity of individual
neurons, but of large numbers of neurons; used when
wanting to study a region of the brain
In clinical settings, use electrodes attached to scalp produces an electroencephalogram (EEG) – printout of
electric activity of brain
Recording the brain’s metabolic and
synaptic activity
If neural activity of a particular region of the brain increases,
the metabolic rate of this region increases
Can measure this with radioactive 2-deoxyglucose which enters
the cell
After this is done, the experimenter can remove and slice the
brain to perform autoradiography locates radioactive
substances in a slice of tissue
Another method used involves immediate early genes, which
are turned on when a cell is activated
e.g. Fos – a protein produced in nucleus of neuron; can be stained
for studying
Positron emission tomography (PET) – used for studying
metabolic activity in human brains; reveals the location of a
radioactive tracer (received via injection) in a living brain
Functional MRI (fMRI) – modification of the MRI procedure
that permits the measurement of regional metabolism in the
brain
Measuring the brain’s secretions
In order to determine what NT or
neuromodulators are being released
in areas of brain
Microdialysis – analyzes chemicals
present in the interstitial fluid through
a small piece of tubing made of a
semipermeable membrane that is
implemented in the brain
Stimulating neural activity
Activate neurons by electrical or
chemical stimulation through a
cannula (small metal tube that can be
inserted into the brain of a lab animal
See subsequent effects on behavior
Finding neurons that produce
particular neurochemicals
3 ways:
Localizing chemicals themselves
Localizing enzymes that produce them
Localizing the messenger RNA involved
in their synthesis
In situ hybridization – the production
of DNA complementary to a particular
messenger RNA in order to detect the
presence of the RNA
Localizing particular receptors
2 different procedures:
Autoradiography, using a radioactive ligand that
binds to the receptor
Immunohistochemistry – use protein antibodies
to label the receptors
Through these methods, an experimenter
can not only determine the chemicals that
a neuron prduces, but also what
connections the neuron has with other
parts of the brain; this is called double
labeling
Genetic methods
Twin studies
Compare concordance rate for a trait in pairs of
monozygotic and dizygotic twins
Adoption studies
Compare people adopted early in life with their
biological (to determine genetic effects) and
adoptive (to determine environmental effects)
parents
Targeted mutations
Mutated gene (also called a “knockout gene”)
produced in the lab and inserted into the
chromosomes of mice; fails to produce a
functional protein