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Transcript
Charles Drew University 4th Annual Drug Abuse Research Symposium September 26,2008 Zebrafish: An emerging model in drug abuse research Kevin S. Jones, Ph.D. Assistant. Professor Pharmacology The College of Osteopathic Medicine of the Pacific Western University of Health Sciences What is a “zebrafish”? The zebrafish, Danio rerio, is: • Tropical, fresh water fish native of India. •Member of the teleost class (bony fish). •Well established model for vertebrate development. •Readily accessible to transgenesis. Zebrafish have 3 developmental stages Adult Embryonic 1 mm ~30 hpf (hours post fertilization) > 3 months Larval 1 mm 5 dpf Zebrafish as a model organism Advantages Disadvantages 1. 2. 3. 4. 5. 6. 7. Evolutionarily distant from humans Limited behavioral repertoire Small size High fecundity Sequenced genome Optical transparency External Development Genetic manipulation Automated analysis Larval zebrafish 96 well plate (10dpf) U.S. Penny How can zebrafish be used to study drug abuse? Recreational “Illy” (PCP) Smoker PCP (phencyclidine) blocks NMDA receptors PCP PCP pcp MK-801 is a potent analogue of PCP MK-801 kd < 5 nM Psychostimulants cause movement disorders Drug-treated HUMAN Drug-treated Rodents Hallucinations YES ??? Delusions YES ??? Thought Disorder YES YES Movement Disorders YES YES Actions of psychostimulants Zebrafish have NMDA receptors Developmental Dynamics 2005 234: 756-766 • zNMDAR is ~90 % IDENTICAL to human NMDAR •Expressed as early as 24 hpf Objective: Characterize the behavioral effects of NMDA receptor blockade in zebrafish Methods Data Acquisition • Individual zebrafish larvae are placed into single wells of a multi well plate • Multi-plate illuminated from below with a 15w lightbox • Larvae given 5 minutes to acclimate • Subsequent activity recorded for 5 minutes on digital camera Methods Data Analysis • Video recordings analyzed off-line with particle tracking software Prototypical Data Vehicle-treated MK-801-treated Dose Response of MK-801-induced locomotion EC50 ~ 10.3 uM (mm/sec) Speed Locomotor kinetics t (h) Developmental Sensitivity The antipsychotic, ziprasidone, attenuate the locomotor effects of MK-801 Ziprasidone targets several receptors Target Receptor D2 D3 Ki 4.8 7.2 5HT2A 5HT2C 5HT1A 5HT1D 0.4 1.3 3.4 2 α1 10 Dopamine antagonists do not disrupt MK-801-indcued locomotion (mm/sec) Δ SPEED MPTP pre-treatment diminishes locomotor effects of MK-801 VEH 500 uM MPTP 1 mM MPTP Summary • Zebrafish retain the most reliable symptom of NMDA blockade… increased locomotion. • Antipsychotic meds block behavioral effects in zebrafish. • Zebrafish may prove useful for elucidating important molecular pathways of drugs of abuse Future Directions • • • • Elucidate molecular mechanism(s). Examine developmental sensitivity. Probe for behavioral sensitization. Molecular neurotoxicology. Acknowledgments Mr. John Chen (Pomona College) Ms. Roshni Patel (Pomona College) Biology Dept Pomona College Pomona College—Dean’s office