Cellular Physiological analysis of peptidergic exocytotic events using primary culture system of isolated GnRH neurons

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To understand the excitation-secretion dynamics of peptidergic neuron from various cellular compartments, we recently established a primary culture system of isolated fish terminal nerve (TN)-gonadotropin releasing hormone (GnRH) neuron. In this study, we examined the electrophysiological properties of the cultured TN-GnRH neurons. They showed subthreshold spontaneous membrane potential oscillations and could be induced to fire in phasic or tonic patterns. Then, we applied a single cell electroporation method for introducing exogenous genes to visually identified TN-GnRH neurons. We successfully introduced plasmids encoding fusion proteins of GFP and exocytotic vesicle components to identified TN-GnRH neurons. Exogenous expression of those genes constructs showed distributions of punctate fluorescence in the soma and neurites. Time-lapse imaging of those fluorescence enabled visualization of movements and exocytosis of peptides in a peptidergic neuron.