Neuropharmacology: Methods

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Lecture from week 5 from a college level neuropharmacology course taught in the spring 2012 semester by Brian J. Piper, Ph.D. (psy391@gmail.com) at Willamette University.

Text of Neuropharmacology: Methods

  • 1. Methods in Neuropharmacology Brian J. Piper, Ph.D., M.S.
  • 2. Objectives Behavior Motor Cognition Emotion Other Neurobiology Histology Biochemistry
  • 3. Institutional Animal Care & Use Committee (IACUC) Approves all modern studies involving animals Criteria (R-R-R)
  • 4. Institutional Animal Care & Use Committee (IACUC) Approves all modern studies involving animals Criteria Alternatives considered (in vitro, computer model) Minimum # of animals Least invasive procedures
  • 5. Motor-Function Locomotor Activity Video (manual or automated) Light-Beams Running Wheels Accelerometers
  • 6. Motor-FunctionRotorod (balance test) Model of MS (2:00 3:30) http://www.youtube.com/watch?v=rw4GbY4xgHA
  • 7. Learning & Memory Standard mazes Y or T mazes
  • 8. Radial Arm Maze # of arms varies: 8, 12, 16+ Can differentiate long-term versus short-term memory Many procedures (e.g. win-shift) Food motivated1st Min: http://www.youtube.com/watch?v=zBNoNoEB1X0
  • 9. Cincinatti Maze Spatial learning & memory Developed by Charles Chip Vorhees Aversive motivation
  • 10. Morris Water Maze Measure of Spatial learning & memory Developed by Richard Morris Measures Time & Distancehttp://www.youtube.com/watch?feature=fvwp&v=kWfNoD_sLww
  • 11. Object-Recognition Test Measure of learning & memory (non-spatial) Developed in the 1980s No aversive motivation
  • 12. Object-Recognition Test Measure of learning & memory (spatial) Developed in the 1980s No aversive motivation
  • 13. EmotionDomain TestAnxiety Open-Field, Elevated-Plus maze, Social-interaction testDepression Forced Swim Test, Tail Suspension TestAggression Resident-intruderReinforcement Self-administration
  • 14. Open-Field Total distance can be used to measure locomotor activity in rodents Entries into center used to measure anxiety
  • 15. Elevated-Plus Maze Developed by Sandra File Rodents chose between open and closed arms Sensitive to anti-anxiety drugshttp://www.youtube.com/watch?v=PLcX2MbpmdY
  • 16. Social-Interaction Test Developed by Sandra File Conducted in neutral area Decreased anxiety therefore more time interacting
  • 17. Porsolt Forced Swim Test Animal model of hopelessness (behavioral despair) Sequence: Day 1 (15 min) Antidepressant Day 2 (5 min) Developed by Porsolt in 1977 http://www.youtube.com/watch?v=Wq2dyNILb5UPorsolt RD, Bertin A, Jalfre M. (1977). Behavioral despair in mice: a primary screening test for antidepressants. Archives Internationales de Pharmacodynamie et de Therapie,229, 327336 .
  • 18. Tail Suspension Test Mouse model of depression Latency to stop wriggling is recorded Antidepressant drugs increase latencySteru, L., Chermat, R., Thierry, B., & Simon, P. (1985). The tail suspension test: A new method forscreening antidepressants in mice. Psychopharmacology, 85, 367-70.
  • 19. Resident-intruder Test Measure of aggression in rodents Conducted in home territory of resident with smaller intruder placed in test areaGoes Fast!http://www.youtube.com/watch?v=YpGRQeY1cx0
  • 20. Drug Self-Administration Animal can press a lever in a Skinner box to receive i.v. administration of a drug Can also be used for drug delivery directly into the brain Measures whether drug is reinforcing (dopamine) Advantage: self-administered Disadvantage: technically demanding
  • 21. Hot-Plate Test Latency to remove limb from a heated surface is recorded. Latency is increased by opiates.
  • 22. Neurobiological Methods Brain surgery Protein (receptors, transporters) quantification Hormone measurement Genetic measurement Pharmacological Challenges
  • 23. Types of Uncontrolled Lesions (Human) Tumor Stroke Traumatic Brain Injury
  • 24. Stereotaxic Surgery Neuroscience procedure to accurately implant device. Animal is anesthetized and skull landmarks are used with stereotaxic atlas. Variations: Cannula: tube for injecting drug Lesion: destroys localized brain tissue
  • 25. Microtome Device to section brain Brain is frozen Sliced very thin (e.g. 40 m or 10-6)
  • 26. Types of Controlled Lesions (Animal) Electrolytic Lesion: destroys cell bodies, axons, dendrites Drug lesions (more selective) Cell bodies: kainic acid Axons: 5,7-dihyroxytryptamine
  • 27. Immunocytochemistry Uses immune system to generate antibodies against a foreign substance (antigen) Antibodies are applied to sectioned tissue Label attached to antibody (fluorescent or color) Location of protein is identified GFAP
  • 28. Immunocytochemistry Uses immune system to generate antibodies against a foreign substance (antigen) Antibodies are applied to sectioned tissue Label attached to antibody (fluorescent or color) Location of protein is identified
  • 29. c-fos index of neuron activity Protein expressed 30 min after event Aggression example
  • 30. Radioligand Binding Biochemical procedure Tissue is ground up Drug with radioactive tag is applied [3H]citalopram Quantity of protein (receptor/transporter) is determined with radioactivity detector
  • 31. Autoradiography Histological procedure 1) tissue is sectioned with microtome 2) radioactive drug (radioligand) is applied to the tissue 3) localization of radioactivity is determined