Investigations will be carried out on the inaccessible subcellular compartment underlying guantal transmitter release at motor nerve terminals. This will involve a new methodology, specifically developed to answer criticisms of the previous technique. Significant advances provided by this approach are the ability to (a) obtain unbiased estimates of the quantal release paramters m, n and p, (b) calculate variance of p (varsp) which can be used to monitor real-time changes in (Ca2+)i distribution, and (c) utilize miniature potentials (mepps) alone to compute release parameters (rather than mepps and nerve-evoked potentials). This should allow studies of subcellular processes and drug actions which were not previously possible. Among these are the: (1) effect of mitochondrial and Na pump inhibitors on var p ( to distinguish direct vs. indirect actions of Na+ to promote transmitter release); (2) effect of (Ca2+) on release parameters with time and concentration (to provide data from the case of a uniform (Ca2+)i distribution at transmitter release sites); (3) effect of Sr2+ and Ba2+ on var Sp to see if they act at intracellular organelles or at release sites (La3+ and Mg2+ will be examined to determine effects of valency and a conventional antagonist); (4) computation of release parameters for mepps and sub-mepps recorded at the same junction, to see if mepps are comprised of sub-mepps; (5) to examine the Ca dependence (cooperativity) of n, m, p and var p before and after BoTX poisoning to elucidate the mechanism of toxicity; (6) effect of temperature on each release parameter-- if it reflects a biochemical (high Q10) or a biophysical (low Q10) process (results to be compared to a theoretical model); (7) effect of hyperosmotic pressure on release parameters, using non-permeters of uptake of acetylcholinesterase r horseradish peroxidase into synaptic vesicles (effect of hemicholinium-3 and physostigmine on the cytoplasmic pool of ACh and on vesicle loading will also be tested); (9) effects of specific pharmacological toxicologic agents e.g., methylmercury; others may include tetraethyllead, tetrahydroaminoacridine, A13+, Cu2+ and heavy metal chelators. Results of these physiologic studies should provide insight into the mechanisms involved in Ca2+ regulation at the subcellular level and document the usefulness of the new method for carrying out subsequent investigations on the actions of various pharmacologic/toxicologic agents.
NIH ExPorter
NIH ExPorter