Release of transmitters and hormones from neurons plays a critical role in transmission of information between neighboring cells and distant tissues. Revealing mechanisms that control neurosecretion is vital to our understanding of both homeostasis of a wide variety of physiological systems and disease processes that occur when this transmission breaks down. Our long-term goal is to understand neural control of peptide secretion and its impact on physiology and behavior. The broad aim of this proposal is to reveal and study underlying cellular and molecular events regulating synthesis and secretion of a neurohormone (egg laying hormone, ELH) using the bag cell neurons of Aplysia as a model system. We are now able to monitor membrane excitability and ELH secretion from bag cell neurons of freely-behaving animals, from bag cell neurons in excised neural tissue maintained in vitro, and from single bag cell neurons in culture. This gives us a relatively unique ability to move between studies addressing molecular and cell physiological issues and those addressing systems and behavioral questions using a single neuroendocrine system. There are three specific aims of this proposal.
The first aim i s to determine the role of calcium from intracellular stores in regulating the pattern of ELH secretion. We will use electrophysiology, calcium imaging, and RIA measurement of hormone secretion to determine the pattern of intracellular calcium in response to electrical afterdischarge, the effects of manipulating intracellular calcium on ELH secretion, and the source of intracellular calcium controlling ELH secretion in response to afterdischarge.
The second aim i s to investigate protein kinase A (PKA), protein kinase C (PKC), and CaM kinase in bag cell neurons. We plan to monitor kinase activities using both a traditional radiolabeling approach, as well as newly developed optical imaging techniques. In addition, we will investigate the role of CaM kinase II in regulating ELH secretion. The third specific aim is to understand cellular and molecular events that mediate the stimulatory effects of afterdischarge on ELH biosynthesis. We will investigate the effects of afterdischarge on specific steps along the translation pathway, and the roles of the calcium, PKA and PKC signaling pathways in regulating specific translational events leading to alterations in ELH biosynthesis. The outcome of this work should enable us to develop a detailed model of cellular mechanisms that link membrane excitation to neurohormone secretion and neurohormone synthesis.
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