1. Methods and Strategies of Research
Chapter 5
Methods and Strategies of Research

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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 single-unit 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. Genetic methods Twin studies Adoption studies Targeted mutations
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