Methods in NeuropharmacologyBrian J. Piper, Ph.D., M.S.

Objectives

• Behavior– Motor– Cognition– Emotion– Other

• Neurobiology– Histology– Biochemistry

Institutional Animal Care & Use Committee (IACUC)

• Approves all modern studies involving animals• Criteria (R-R-R)

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

Motor-Function

• Locomotor Activity– Video (manual or automated)– Light-Beams– Running Wheels– Accelerometers

Motor-Function

Rotorod (balance test)

Model of MS (2:00 – 3:30)http://www.youtube.com/watch?v=rw4GbY4xgHA

Learning & Memory

• Standard mazes• Y or T mazes

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

Cincinatti Maze

• Spatial learning & memory• Developed by Charles “Chip” Vorhees• Aversive motivation

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

Object-Recognition Test

• Measure of learning & memory (non-spatial)• Developed in the 1980s• No aversive motivation

Object-Recognition Test

• Measure of learning & memory (spatial)• Developed in the 1980s• No aversive motivation

Emotion

Domain TestAnxiety Open-Field, Elevated-Plus maze, Social-interaction test

Depression Forced Swim Test, Tail Suspension Test

Aggression Resident-intruder

Reinforcement Self-administration

Open-Field

• Total distance can be used to measure locomotor activity in rodents

• Entries into center used to measure “anxiety”

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

Social-Interaction Test

• Developed by Sandra File• Conducted in neutral area• Decreased anxiety therefore more time

interacting

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.

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.

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

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

Hot-Plate Test

• Latency to remove limb from a heated surface is recorded.

• Latency is increased by opiates.

Neurobiological Methods

• Brain surgery• Protein (receptors, transporters) quantification• Hormone measurement• Genetic measurement• Pharmacological Challenges

Types of Uncontrolled Lesions (Human)

• Tumor• Stroke• Traumatic Brain Injury

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

Microtome

• Device to section brain• Brain is frozen• Sliced very thin (e.g. 40 μm or 10-6)

Types of Controlled Lesions (Animal)

• Electrolytic Lesion: destroys cell bodies, axons, dendrites

• Drug lesions (more selective)– Cell bodies: kainic acid– Axons: 5,7-dihyroxytryptamine

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

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

c-fos

• index of neuron activity• Protein expressed 30 min after event• Aggression example

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

Autoradiography

• Histological procedure• 1) tissue is sectioned with microtome• 2) radioactive drug (radioligand) is applied to

the tissue• 3) localization of radioactivity is determined

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

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

Microarray

• Biochemical• Measures large # of mRNA at the same time

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

Genetic Engineering

• Knock-In– Replacing one gene with another – Example: mouse with human apoE

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

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

Adult ecstasy (MDMA) Challenge in Rats Exposed to MDMA as Infants

Piper & Meyer (2007). Neurotoxicology & Teratology, 28, 95-102.

Summary

• Biochemical Procedures– Radioligand Assay

• Histological Procedure– Immunocytochemistry (IHC)– Autoradiography– In Situ Hybridization

Self-test 1

• What test(s) measure spatial learning and memory?

Self-Test 2: What procedure?

Self-test 3

• What neurobiological procedure(s) uses antibodies?