HORMONAL REGULATION OF PRESYNAPTIC RELEASE MECHANISMS

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The parent project for this proposal will study the effects of neuromodulatory hormones at a crustacean neuromuscular junction on specific ion channels, intracellular calcium stores and second messengers in the muscle cell. The collaborative proposal will utilize recordings from the parent project and analyze presynaptic is in press in an unidentified journal. From this application, the authors derive from multiple measurements the average quantal content, and from their model calculate the probability of release and the number of docked/releasable quanta. These variables then enable them to test their hypothesis that hormones that act presynaptically at this synapse (such as serotonin and peptide Fl) increase mobilization of vesicles to the release site rather than probability of release per se. In the first aim, data from extracellular recordings at the dactyl opener muscle stimulated at 5 Hz are analyzed and the steady state average quantal content (m) in multiple stimulation trials is derived from an algorithm that determines the shape of inflections in the synaptic responses as individual quanta. Thereafter, this analysis will be repeated during bath application of hormones and again during a wash period. The effect of different doses of hormones on calculated probability of release (p) and number of releasable quanta (n) will be compared early (first 50 ms) and late (second 50 ms) following stimulation with the goal of separating hormonal effects on spontaneous and evoked neurosecretion. To address potential problems with access of the bath-applied test reagents to the release site, experiments will be performed by removing the recording electrode during hormone application and replacing for subsequent recordings. The second aim will use frequency facilitation to investigate whether hormonally induced increase in docked vesicles (n) is due to increased mobilization or decrease demobilization to an undocked pool. The steady state, unstimulated mobilization constant is assumed to be negligible compared to the stimulated mobilization, enabling calculation of these two parameters by reciprocal plot of m vs. fp and determining the hormonal effects on slope and intercept at each frequency. Steady state hormone effects will be established by repeated low frequency stimulation before changing stimulation frequency. The final aim will be to test their expected results in a Monte Carlo simulation. Results of quantal content from different simulation frequencies will serve as input and several parameters varied within this model. This will test whether changing the constant of demobilization or stimulated mobilization of quanta to release sites more closely approximates the experimental results. The possibility of non-uniform effects of hormones on release is considered in this simulation.