The Development of an Embryonic Zebrafish Neuromuscular Co-culture System to Evaluate the Effects of Cone Snail Venom Peptides on the Synapse
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The venom of fish-hunting cone snails is composed of potent bioactive peptides that send prey into an immediate paralysis through continuous firing of action potentials resulting in rapid tetanus of the musculature. In this study we are developing both in vivo and in vitro techniques to identify the distribution of specific molecular targets of neurotoxins present in the venom of these predatory marine cone snails. As these targets are not necessarily restricted to the cell soma of neurons it is necessary to investigate other sub-cellular regions. To further investigate the locations of these peptide targets we are developing an embryonic zebrafish neuromuscular co-culture system that can be utilized in various electrical recording techniques to evaluate the activity of the synapse before and after application of C4 toxin. Zebrafish are particularly useful in our investigations because teleost fish are the primary targets of piscivorous cone snails in the wild, ensuring maximal effects of the toxins on our model system. Single muscle fibers and motorneurons were isolated from embryonic zebrafish to create a model synapse emulating the actual neuromuscular junctions formed in animals. Varicosities or swellings of the neural axon are visual indicators of potential synapses existing in culture. We plan to confirm the functionality of a synaptically coupled cell pair using labeled alpha-bungarotoxin. In addition to developing a neuromuscular co-culture system we have created an in situ larval spinal motility assay to investigate the effects of topically applied purified venom peptides on coordinated swimming behavior of larval spines. The combination of both in vivo and in vitro investigations is a powerful approach to identifying specific molecular targets of cone snail venom.