Methods and Strategies of Research 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 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

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 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