The Regulation of Intracellular Ca2+ and Secretion in the Bag Cell Neurons of Aplysia Californica

Abstract

Neurons enter states of heightened excitability and secretory capacity to initiate fundamental behaviours. This is exemplified by the bag cell neurons of the marine mollusc, Aplysia californica. These neuroendocrine cells undergo an afterdischarge to secrete egg-laying hormone (ELH) and initiate reproduction. I examined the role of two cellular signaling pathways that contribute to the afterdischarge: Ca2+ and protein kinase C (PKC). Investigating these systems provides insight into the cellular mechanisms underlying fundamental behaviours. Intracellular Ca2+ modulates excitability and initiates secretion in the bag cell neurons. I determined how Ca2+ sources and removal systems control intracellular Ca2+. My data revealed that the mitochondria strongly influence Ca2+ signaling in cultured bag cell neurons, as they first buffer voltage-gated Ca2+ influx and subsequently release Ca2+ to the cytosol, in a form of Ca2+-induced Ca2+ release (CICR). Moreover, the degree of mitochondrial Ca2+ uptake and release was shown to be dictated by the function of the plasma membrane Ca2+ ATPase. In addition to voltage-gated Ca2+ influx, I characterized the involvement of Ca2+ handling systems with other distinct Ca2+ sources, including CICR and the Na+/Ca2+ exchanger as well as store-operated Ca2+ influx and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). PKC is implicated in facilitating secretion during the afterdischarge. I tested the impact of PKC on secretion using capacitance tracking under whole-cell voltage-clamp. This technique assays the changes in plasma membrane area that occur during vesicle exocytosis. I demonstrated that PKC activation enhanced Ca2+ influx and potentiated stimulus-evoked secretion. This occurred as a result of the plasma membrane insertion of a covert voltage-gated Ca2+ channel, Apl Cav2, alongside the basal voltage-gated Ca2+ channel, Apl Cav1. iii I provided mechanistic detail of how the interplay between Ca2+ sources and removal systems governs the patterns of free cytosolic Ca2+. These properties have implications for the Ca2+-dependent signaling pathways which mediate long lasting changes in neuronal excitability and secretion. Furthermore, my work demonstrates that protein kinases can dynamically amplify secretory output by rapidly recruiting additional voltage-gated Ca2+ channels to the membrane. This form of facilitation likely enhances secretion during the afterdischarge, and ensures the initiation of reproductive behaviour in Aplysia.