Neuropharmacology: Methods
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Transcript of Neuropharmacology: Methods
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?