Zebrafish: An emerging model in drug abuse research

Kevin S. Jones, Ph.D.Assistant. Professor PharmacologyThe College of Osteopathic Medicine of the PacificWestern University of Health Sciences

Charles Drew University 4th Annual Drug Abuse Research Symposium September 26,2008

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

~30 hpf (hours post fertilization)

1 mm

Embryonic

> 3 months

Adult

5 dpf

1 mm

Larval

Zebrafish as a model organism

Advantages Disadvantages1. Small size

2. High fecundity

3. Sequenced genome

4. Optical transparency

5. External Development

6. Genetic manipulation

7. Automated analysis

Evolutionarily distant from humans

Limited behavioral repertoire

Larval zebrafish (10dpf)

96 well plateU.S. Penny

How can zebrafish be used to study drug abuse?

Recreational “Illy” (PCP) Smoker

pcp

PCP (phencyclidine) blocks NMDA receptors

PCP

PCP

kd < 5 nM

MK-801

MK-801 is a potent analogue of PCP

Psychostimulants cause movement disorders

Actions of psychostimulantsDrug-treated

HUMANDrug-treated

RodentsHallucinations YES ???

Delusions YES ???

Thought Disorder YES YES

Movement Disorders YES YES

Zebrafish have NMDA receptors

• zNMDAR is ~90 % IDENTICAL to human NMDAR

•Expressed as early as 24 hpf

Developmental Dynamics 2005 234: 756-766

Objective:

Characterize the behavioral effects of NMDA receptor blockade in zebrafish

• 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

Data Acquisition

Methods

MethodsData Analysis

• Video recordings analyzed off-line with particle tracking software

Vehicle-treated MK-801-treated

Prototypical Data

Dose Response of MK-801-induced locomotion

EC50 ~ 10.3 uM

Locomotor kinetics

t (h)

Spe

ed

(mm

/sec

)

Developmental Sensitivity

The antipsychotic, ziprasidone,attenuate the locomotor effects of MK-801

Ziprasidone targets several receptors

Target Receptor D2 D3 5HT2A 5HT2C 5HT1A 5HT1D α1

Ki 4.8 7.2 0.4 1.3 3.4 2 10

Dopamine antagonists do not disrupt MK-801-indcued locomotion

MPTP pre-treatment diminishes locomotor effects of MK-801

Δ S

PE

ED

(mm

/sec

)

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 importantmolecular 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