Midterm NOTES [CH5] - Methods and Strategies of Research.PDF
- 1. Copyright 2001 by Allyn & Bacon Carlson (7e) PowerPoint Lecture Outline Chapter 5: Methods and Strategies of Research This multimedia product and its contents are protected under copyright law. The following are prohibited by law: •any public performance or display, including transmission of any image over a network; •preparation of any derivative work, including extraction, in whole or in part, of any images; •any rental, lease, or lending of the program.
- 2. Copyright 2001 by Allyn & Bacon Experimental Ablation n Ablation involves the destruction of brain tissue followed by an assessment of subsequent changes in behavior n Ablation techniques include l Electrolytic lesions/Radio Frequency lesions l Excitotoxic lesions (kainic acid) l Neurochemical lesions (6-OHDA) l Aspiration l Knife cuts n Distinction between functions and behaviors n Brain lesion studies are complicated by the fact that all regions of the brain are interconnected 5.2
- 3. Copyright 2001 by Allyn & Bacon Stereotaxic Surgery n A stereotaxic instrument holds the head in a fixed position l The instrument has an arm that can move in 3 dimensions l The surgeon can thus position an electrode or other device within a particular sub-cortical structure n A stereotaxic atlas provides a series of drawings of brain structures l Each page is a section of brain relative to a landmark on the skull (such as bregma) 5.3
- 4. Copyright 2001 by Allyn & Bacon Using a Stereotaxic Atlas to Target a Brain Lesion 5.4
- 5. Copyright 2001 by Allyn & Bacon Histological Techniques n Histological techniques are used to verify the placement of a lesion within brain l Perfuse (to remove blood from brain) u Remove brain l Fix brain in formalin to solidify tissue and to prevent autolysis u Slice brain into thin sections (10-80 microns thick) l Use stains to highlight selective neural elements u Myelin (Weil stain) u Cell body (cresyl violet: Nissl substance in cytoplasm) u Membrane (Golgi stain) 5.5
- 6. Copyright 2001 by Allyn & Bacon Defining Neural Connections n Neurons in a given region send axonal outputs (efferents) to other brain regions and receive axonal inputs (afferents) l Tracing efferent connections is done using anterograde labels that are taken up by the cell bodies and transported to axons u “Forward: toward axons from cell bodies” u Inject the lectin PHA-L into a nucleus, wait several days, process brain tissue. u Immunocytochemistry uses a radioactive antibody to PHA-L in order to identify cells containing PHA-L l Tracing afferent connections is done using retrograde labeling u “Backwards: from axons to cell bodies” u e.g. fluorogold is a retrograde tracer 5.6
- 7. Copyright 2001 by Allyn & Bacon Anterograde Tracing 5.7
- 8. Copyright 2001 by Allyn & Bacon Visualizing a Living Human Brain n Computerized tomography (CT) uses an x-ray beam to scan the brain from all angles, these scans are then summarized in an image of the skull and brain (in a horizontal plane) n Magnetic Resonance Imaging (MRI) uses a magnetic field and radio waves to excite hydrogen molecules, the resulting information is combined to form an image of tissue 5.8
- 9. Copyright 2001 by Allyn & Bacon Human MRI (Normal) Images courtesy of Dr. Nancy Andreason 5.9
- 10. Copyright 2001 by Allyn & Bacon Recording Neural Activity n Axons conduct action potentials and neurotransmitters elicit postsynaptic potentials n The electrical events of a discrete region can be recorded using glass microelectrodes (acute recording) or tungsten wire (chronic recording) n Macroelectrodes record the summated electrical activity of large regions of brain l Surface electrodes placed on human scalp are used to record brain activity (electroencephalogram: EEG) 5.10
- 11. Copyright 2001 by Allyn & Bacon Recording Synaptic Activity n Increases in neural activity are associated with increases in metabolic activity in a brain region l The 2-deoxy-glucose (2-DG) method measures relative glucose utilization u 2-DG cannot be metabolized, is trapped in cells and accumulates u Radioactive 2-DG is then quantitated using autoradiography l The c-FOS method measures a nuclear protein (Fos) that is expressed when a neuron is activated u Neuronal activation is associated with activation of genes in the neuron nucleus- can localize Fos within the nucleus, indicates relative degree of activation 5.11
- 12. Copyright 2001 by Allyn & Bacon Human Brain Imaging n The PET scan is a variation of the animal 2-DG technique l Human subject is injected with radioactive 2-DG, which is taken up by brain cells l As the radioactive molecules decay they emit positrons that can be detected by a scanner l A PET scan indicates the relative activity of different brain regions during mental states n Functional MRI (fMRI) scans detect the level of oxygen in brain blood vessels l Current fMRI scanners have a higher resolution than do PET scanners 5.12
- 13. Copyright 2001 by Allyn & Bacon Microdialysis n The secretion of neurotransmitter (NT) within a discrete brain region can be measured using the microdialysis technique l The tip of a microdialysis probe is positioned in a brain region, CSF is flowed inside the membrane, and NT can pass through the semipermeable membrane into the probe l An analytical technique is then used to quantitate the amount of NT in the dialysate 5.13
- 14. Copyright 2001 by Allyn & Bacon Microdialysis Probe Details 5.14
- 15. Copyright 2001 by Allyn & Bacon Artificial Stimulation of Brain n Neurons in a region can be artificially activated to assess the role of that region in behavior l Electrical stimulation involves passing electrical current through a wire inserted into brain l Chemical stimulation can involve infusion of an excitatory amino acid such as glutamate into a region u A cannula implanted into a region can be used to deliver drug solutions into that region u Chemical stimulation can be more specific than electrical stimulation (glutamate activates cell bodies, not axons) 5.15
- 16. Copyright 2001 by Allyn & Bacon Localization of Neurotransmitters n Three approaches to the localization of a neurotransmitter l Peptides are proteins, and proteins can be localized using immunocytochemistry l The enzyme that produces a nonpeptide NT can be assayed using immunocytochemistry u ChAT is the synthesis enzyme for ACh u Neurons that use ACh should contain ChAT l mRNA controls the production of an NT or enzyme u Brain tissue can be exposed to a radioactive solution containing the complement of the mRNA sequence, and autoradiography can be used to localize cells that produce the NT or synthesis enzyme 5.16
- 17. Copyright 2001 by Allyn & Bacon Receptor Localization Techniques n Receptors can be localized in brain tissue using l Autoradiography: u Sections of brain are exposed to solutions containing a radioactive ligand (chemical that binds), washed, and placed on film u The resulting film image shows spots at which radioactivity exposed the film l Immunocytochemistry: u Antibodies are developed for the receptor protein, are tagged with a fluorescent dye u The tissue is exposed to the antibody/dye u The section is then examined under a microscope for the presence of dye in specific regions 5.17
- 18. Copyright 2001 by Allyn & Bacon Genetic Methods n Genetic research methods seek to demonstrate the linkage between genes and behavior n Twin studies examine the impact of varying degrees of genetic similarity on behavioral similarity l Identical twins (MZ) share 100% of their genes while fraternal twins (DZ) share about 50% of their genes l Concordance rate examines the likelihood of whether a twin shares a behavioral trait with the other twin l A higher concordance rate for MZ twins relative to DZ twins suggests a genetic influence for that characteristic 5.18
- 19. Copyright 2001 by Allyn & Bacon Genetic Methods n Adoption studies examine the similarity with regard to a trait for an adopted person compared to their adopted parents and their biological parents n Targeted mutations involve the insertion of defective (knockout) genes into the chromosomes of mice l The target of the mutation is often an enzyme that controls a chemical reaction or a protein that serves as a receptor for a specific neurotransmitter 5.19