1. Alterations in Expression Levels of Synapsin IIa After Haloperidol Treatment in Zebrafish Embryos (Danio Rerio) Rachel Klein, Department of Biological Sciences, York College of Pennsylvania Results 1500bp 200bp 0µM Synapsin IIa4.5µM Synapsin IIa 9µM Synapsin IIa 0µM GAPDH 4.5µM GAPDH 9µM GAPDH 0µM ACTB4.5µM ACTB 9µM ACTB 500bp ladder100bp ladder Figure 1. Semi-quantitative PCR analysis of syanpsin IIa (lanes 2-4), GAPDH (lanes 5-7), and ACTB (lanes 8-10). Band intensity and density represent gene expression. 1.5% agarose gel run at 150V for 30 minutes. Introduction Schizophrenia is a psychotic disorder that affects about 1% of the population and is mainly characterized by an over-activity of dopamine in the brain. Dopamine is a neurotransmitter, stored in synaptic vesicles in the axon terminal of nerves, which is known to be involved in regulating mood and behavior. Haloperidol is a typical antipsychotic. It acts as a dopamine antagonist by blocking dopamine receptors and decreasing abnormal excitement in the brain. Synapsin IIa is a gene that regulates synaptic vesicle mobilization during synaptic activity and also maintains reserve pools for neurotransmitters, such as dopamine. Schizophrenic post-mortem brains have shown a decrease in synapsin IIa expression; however, previous studies have shown that synapsin II mRNA is up-regulated after haloperidol treatment in rodents (Gitler et al. 2008, Chong et al. 2002). The mechanism of how haloperidol affects synapsin IIa expression is still unclear. Zebrafish make an excellent model for this study because of the similarities between zebrafish and human nervous systems. Drugs can be directly added into the water with the zebrafish and previous studies have shown abnormal locomotor activity after haloperidol treatment, indicating the zebrafish responded to the treatment. Objective Test the effects of increasing haloperidol concentrations on synapsin IIa gene expression in zebrafish embryos. http://www.csuchico.edu/~pmccaffrey/syllabi/CMSD%20320/362unit12.html 100bp 1000bp 500bp Zebrafish breeding Methods Haloperidol Treatments RNA Extraction Reverse Transcriptase PCR Reactions Gel Electrophoresis quant-PCR http:// neurosciencenews.com/healing-thalamus-strokes-genetic-lhx2-lhx9 / Acknowledgements I would like to thank Dr. Kaltreider for his help analyzing results and his assistance completing the qRT-PCR analysis, Dr. Hagerty for her help reviewing my results and graphs, and finally Dr. Boehmler for her patience, instruction, and helpfulness in completing this study. References Chong, V.Z.; Young, L.T.; Mishra, R.K. 2002. cDNA array reveals differential gene expression following chronic neuroleptic administration: implications of synapsin II in haloperidol treatment. Journal of Neurochemistry 82: 1533-1539. Fan, C.Y.; Cowden, J.; Simmons, S.O.; Padilla, S.; Ramabhadran, R. 2010. Gene expression changes in developing zebrafish as potential markers for rapid developmental neurotoxicity screening. Neurotoxicology and Teratology 32: 91-98. Gitler, D.; Cheng, Q.; Greengard, P.; Augustine, G.J. 2008. Synapsin IIa controls the reserve pool of glutamatergic synaptic vesicles. The Journal of Neuroscience 28(43):10835- 10843. Vawter, M.P.; Thatcher, L.; Usen, N.; Hyde, T.M.; Kleinman, J.E.; Freed, W.J. 2002. Reduction of synapsin in the hippocampus of patients with bipolar disorder and schizophrenia. Molecular Psychiatry 7: 571-578. Future Studies 1) Repeat haloperidol treatments multiple times to be able to statistically test differences in synapsin IIa gene expression. 2)Test the effects of chronic antipsychotic treatment on synapsin IIa gene expression from the juvenile stage to adulthood. 3) Compare synapsin IIa gene expression using different antipsychotic treatments 4) Test the change in spatial expression of synapsin IIa using in situ hybridization in zebrafish embryos. Conclusions The semi-quantitative PCR analysis showed a trend of decreasing gene expression in the haloperidol treatment groups. The relative quantitative PCR analysis also showed a possible trend of decreasing gene expression in the 4.5µM and 9µM haloperidol treatment groups. Figure 4. qRT-PCR gene expression differences showing relative quantification among three haloperidol concentrations: 0µM, 4.5µM, and 9µM. Error bars show RQ minimum and RQ maximum calculated by the 95% Confidence Interval. Figure 2. Mean PCR band intensities for gene expression among three haloperidol concentrations: 0 mM, 4.5 mM, and 9 mM. Band intensities correlate to gene expression of synapsin IIa, GAPDH, ACTB. Error bars show standard error of the mean. Figure 3. Mean PCR band density for synapsin IIa expression among three different haloperidol concentrations: 0mM, 4.5mM, and 9mM (n=3 PCR reactions per group). Band densities were normalized to ACTB. Error bars show standard error of the mean. 0mM was the control, 4.5mM showed a 30% decrease in expression, and 9mM showed a 14% decrease in expression. Primer SequenceSize Synapsin IIaFor 5’-AGT CTG CCA TCA TGA ACT-3’ Rev 5’-TAA GCA TCC ATG GCT ATG-3’ 1420 bp GAPDHFor 5’-GTGTAGGCGTGGACTGTGGT-3’ Rev 5’-TGGGAGTCAACCAGGACAAATA-3’ 121 bp ACTBFor 5’-TCACCACCACAGCCGAAAG-3’ Rev 5’-AGAGGCAGCGGTTCCCAT-3’ 98 bp Table 1. Summary table of primer sequences and amplicon size for synapsin IIa, GAPDH, and ACTB.