1. Introduction Development of Xenopus visual system well characterized: Title Name Biology Department & Program in Neuroscience, Davidson College, Davidson, NC 28035-7118 Abstract Topographical mapping of the Xenopus retinal axons requires guidance cues throughout its visual system. When the retinal axons exit the Xenopus eye, they follow molecular cues to find their target, the optic tectum. Some retinal ganglion cells (RGCs) express acetylcholine receptors (AChRs), thus cholinergic input may play a crucial role in the initial mapping of the RGCs. Malathion is a widely used pesticide applied to bodies of water that inhibits ACh esterase (AChE). Malathion prevents AChE’s normal decomposition of ACh released into the synaptic cleft. The use of malathion has been shown to cause morphological changes in non-target aquatic vertebrates such as zebrafishes and tadpoles. This study investigated the effect of malathion on the mapping of tadpole retinal axons. Xenopus RGCs mapped appropriately to the tectum even with application of the AChE inhibitor malathion. No significant changes in optic tract lengths, widths, curvature, or area were detected as the result of malathion exposure. Thus, AChE levels may be independent or play a minor role in the initial mapping of Xenopus RGCs to the tectum. Methods Stage 29/30 tadpoles were put in 1 mL of 2.5 mg/L of malathion. A control group of tadpoles at the same stage were put in 1 mL of 2.5 mg/L of acetone. The acetone acted as vehicle control. Tadpoles were allowed to grow until they reached Stage 40, when RGCs have reached the tectum. Results No significant difference detected; p value of all parameters were greater than 0.05. Conclusion When AChE is inhibited by malathion, RGC axons map correctly to the optic tectum. AChE levels in the development of visual system may not be important for RGCs to map to the tectum. Future studies Investigate the rate of axon growth with time-lapse video. Use immunostaining rather than HRP to visual the axons. 24 well plate Stage 29/30 1-2 days later Stage 40 Tadpole lateral view Tadpole lateral view Brain and eye transverse view Brain lateral view The tadpoles were then anesthetized, their lens removed, and filled with horseradish peroxidase (HRP). The HRP will anterogradely label the RGC axons. The tadpoles were then fixed in 4% gluteraldehyde overnight. After rinsing, the tadpole brains were extracted and reacted with DAB (diaminobenzidine), an HRP detection substrate to visualize the RGC axons in the brain. Images were taken using ImagePro and measured according to various parameters: a. Area b. Curvature – ratio of b/c c. Length of middle tract d. Width of optic tract e. Width of tectum aborization f. Optic tract to hindbrain length FIX Remove lens Anterograde transport of HRPDissect brain and detect HRP via DAB substrate a b c d e f Acknowledgements We thank Cyndi Payne, Amy Becton, NSF, and Davidson College for their help and support. lens vs. Cholinergic input important in mapping of visual system ( Schmidt 1995 ). Nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus visual system ( Sargent et al. 1989, Edwards and Cline, 1999 ). Cholinergic neurons exist in Xenopus visual system ( Titmus et al. 1999 ). Malathion shown to affect morphology of zebrafish embryos and tadpoles ( Chemotti et al. 2005, Cook et al. 2005, Davis et al. 2005 ). Malathion is a AChE inhibitor. Experimental question: Does malathion affect the mapping of RGC axons? Add HRP 200 μm Malathion Acetone Stage 40