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.

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-Function
Rotorod (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 motivated
1st 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 & Distance
http://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. Emotion
Domain Test
Anxiety Open-Field, Elevated-Plus maze, Social-interaction test
Depression Forced Swim Test, Tail Suspension Test
Aggression Resident-intruder
Reinforcement 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 drugs
http://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=Wq2dyNILb5U
Porsolt RD, Bertin A, Jalfre M. (1977). Behavioral despair in mice: a primary screening test for antidepressants. Archives Internationales de Pharmacodynamie et de Therapie,
229, 327–336 .

18. Tail Suspension Test
• Mouse model of depression
• Latency to stop wriggling is recorded
• Antidepressant drugs increase latency
Steru, L., Chermat, R., Thierry, B., & Simon, P. (1985). The tail suspension test: A new method for
screening 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 area
Goes 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 μm 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

32. Radio-Immuno-Assay
• Radioactivity + Antibody (immuno)
• Quantifies level of hormone (e.g. cortisol)
• Competition assay
– Standard curve with known amounts of antibody
– Unknown quantity of antigen is examined

33. In Situ Hybridization
• Identifies cells making a protein (messenger
Ribo Nucleic Acid mRNA)
• In Situ: Latin for “in position”
• Hybridization: uses cDNA nucleotide base
probes to bind to mRNA
– thymine -> adenine
– guanine -> cytosine

34. Microarray
• Biochemical
• Measures large # of mRNA at the same time

35. Genetic Engineering
• Knock-out
– Altered gene is inserted into fertilized egg
– Heterozygote offspring are bred to produce
homozygotes which will lack protein
– Pro: developmental biology
– Con: compensation

36. Genetic Engineering
• Knock-In
– Replacing one gene with another
– Example: mouse with human apoE

37. Microdialysis
• Procedure to measure neurotransmitter
release from behaving animal
• Steps:
– 1) stereotaxic surgery
– 2) cerebrospinal fluid is removed (and replaced!)
– 3) analytical chemistry on sample

38. Pharmacological Challenge
• Steps:
– Administer drug with known mechanism(s)
– Endpoints measured at regular intervals
– Examples
• Physiological measure: temperature
• Blood sampled for hormone determination (uses RIA too!)
• Behavior: 5-HT syndrome

39. Adult ecstasy (MDMA) Challenge in
Rats Exposed to MDMA as Infants
Piper & Meyer (2007). Neurotoxicology & Teratology, 28, 95-102.

40. Summary
• Biochemical Procedures
– Radioligand Assay
• Histological Procedure
– Immunocytochemistry (IHC)
– Autoradiography
– In Situ Hybridization

41. Self-test 1
• What test(s) measure spatial learning and
memory?

42. Self-Test 2: What procedure?

43. Self-test 3
• What neurobiological procedure(s) uses
antibodies?