Research will be continued on the in vitro crab sinus gland neurosecretory system. The biophysical bases of electrical responses recorded intracellularly from the peptide neurohormone secreting terminals will be studied, and changes of electrical responses resulting from all other manipulations to be employed monitored. Fine-structural changes in terminals will also be monitored in parallel electron-microscope studies. Stimulus-secretion coupling is to be studied with improved time resolution to correlate intracellular changes during repetitive stimulation with 'facilitation' of erythrophore concentrating hormone (ECH) release. The rapid, semi-quantitative crab leg segment assay, sensitive to 5 times 10 to the minus 12th power molar synthetic ECH, will be used to determine ECH released into the perfusate. Involvement of metabolically dependent processes in acute release and in subsequent replenishment of hormone in terminals will be examined. Organ culture of the sinus gland system will be undertaken for studies employing isotopically labelled ECH precursor amino acids. Selective application of amino acids or drugs to somata or terminals is possible and will be exploited to localize processes involved in replenishment of hormone released from the terminals. The localization and time course of isotope distribution will be followed, not only by counting of perfusate collected during stimulation, but also by autoradiography of whole mounts, and of light and electron microscope sections. Experiments will test the effect on replenishment processes of secretory activity and seek the mechanism by which stimulation of these processes occurs. Electron-microscope examination of the terminals marked with Procion rubine injected through the intracellular recording electrode will permit correlation of electrophysiological characteristics of individual terminals with the type of neurosecretory granules each contains. Identification of the terminal type releasing ECH will be attempted.
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