Ca2+ influx and release in Aplysia bag cell neurons
Geiger, Julia Elizabeth
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Intracellular Ca2+ is influenced by extracellular Ca2+ influx and release from internal stores. Depending on the initial influx, a range of responses can result. To better understand the processes surrounding Ca2+ handling, I examined intracellular Ca2+ following receptor-operated or voltage-gated Ca2+ influx in bag cell neurons of the marine mollusc, Aplysia californica. Following a brief synaptic input, these neuroendocrine cells undergo a 30 min period of firing, known as the afterdischarge, triggering secretion of neuropeptides that initiate egg-laying. I hypothesize that Ca2+ influx occurs via receptor-mediated activation of a non-selective cation channel and activity-dependent elevation of Ca2+ produces Ca2+ release from intracellular stores. To test this, cultured bag cell neurons were injected with Ca2+ indicator, fura-PE3, and subjected to simultaneous imaging and electrophysiology. Pharmacological activation of a non-selective cation channel that drives the afterdischarge, produced inward current and, as assessed by Mn2+-quench of fura, Ca2+ entry. This receptor-operated Ca2+ influx pathway may contribute to plasticity or secretion. Voltage-gated Ca2+ influx was investigated using trains of action potentials delivered at 5 Hz, 10 s (mimicking the synaptic input that initiates the afterdischarge) or 5 Hz, 1 min (mimicking the fast phase of the afterdischarge). While both trains transiently elevated intracellular Ca2+, only the 5 Hz, 1 min stimulus produced a Ca2+ rise that markedly outlasted the initial influx, consistent with Ca2+-induced Ca2+ release (CICR). Thus, robust Ca2+ influx is required to trigger bag cell neuron CICR. Depletion of the smooth endoplasmic reticulum store, with Ca2+-ATPase blocker, cyclopiazonic acid, or collapse of the mitochondrial membrane potential, with protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, significantly attenuated CICR. Furthermore, application of tetraphenylphosphonium, a blocker of mitochondrial Ca2+ release, also reduced CICR. Dual depletion of the endoplasmic reticulum and mitochondria did not reduce CICR further than depletion of mitochondria alone. Thus, in departure from the traditional mechanism of CICR, bag cell neuron CICR relies ultimately on mitochondrial Ca2+ release. Finally, intracellular Ca2+ changes during, and after, a stimulus mimicking the full afterdischarge were enhanced by PKC activation. Activity-dependent induction of CICR in bag cell neurons has the potential to influence secretion, membrane currents, or gene expression.