Transcript Slide 1

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