Assessing the suitability of embryonic chick sympathetic ganglia for botulinum toxin study
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Botulinum neurotoxin (BoNT), a potent neurotoxin produced under anaerobic conditions by the bacteria Clostridium botulinum, is the most lethal toxin known to man. The primary target site for BoNT action is the cholinergic nerve terminal that innervates the neuromuscular junction. Botulinum neurotoxin inhibits the formation of the SNARE complex which is critical to the release of vesicular acetylcholine (ACh). Without the release of ACh, flaccid paralysis (botulism) ensues. Because of the potential usefulness of toxin in clinical conditions involving cholinergic terminals in the autonomic nervous system, we are testing the suitability of embryonic chick sympathetic ganglia for BoNT study. To achieve this, it was first necessary to develop a protocol for processing ganglia. We tested two different ganglia preparations. The first, a synaptosome-enriched fraction and the second, sympathetic explants plated in poly-lysine coated plates. These protocols were then tested to characterize the neurochemistry, as well as the capacity for vesicular release in sympathetic ganglia isolated from 9-10 day old chick embryos. Using protein immunochemistry we have identified SNAP-25, VAMP and syntaxin - all components of the SNARE complex; synaptotagmin - the putative calcium sensor in both the synaptosome-enriched fraction and in the explant preparations. Choline acetyltransferase - an enzyme involved in the synthesis of ACh in this embryonic preparation and the vesicular acetylcholine transporter protein were only identified in the explant preparation. To determine the sensitivity of the sympathetic ganglia preparations to BoNT/A and BoNT/C substrate cleavage assays were utilized. The synaptosome-enriched fraction did not result in BoNT/A induced cleavage of SNAP-25. The isolated explants were grown for a minimum of 4 days, and then exposed to toxin for 2 hours. The toxin was then removed and media was returned for variable incubation periods. Based on our findings we can conclude that the day 10 embryonic chick sympathetic ganglia explants are susceptible to BoNT/A and C binding, internalization and protein targeting.