Regulation of Peptidergic Secretory Granule Transport by Neuronal Stimulation

BACKGROUND: Peptidergic neurons store and secrete the contents of secretory granules from axon terminals and dendrites. Secretion of neuropeptides requires not only a highly regulated exocytotic mechanism, but also necessitates coordinated synthesis and transport of granules to release sites. Although these trafficking events are critical, little is known regarding the dynamic behavior of granules and the mechanisms by which transport is regulated in neurons. OBJECTIVE: Since secretory granules cannot be refilled locally after their contents are secreted, our goal was to determine how neurons support regulated release of peptides. Alteration of granule delivery could be used to meet cellular demands; therefore, we assessed changes in granule dynamics in response to neuronal stimulation. METHODS: Vectors encoding a soluble granule protein, peptidylglycine α-hydroxylating monooxygenase (PHM), fused to GFP or pHluorin were constructed to address this question in cultured rat trigeminal ganglion neurons. Time lapse confocal microscopy was used to track individual granules in axons and growth cones, and the effect of high K+ and phorbol ester (PMA) on this behavior was assessed. Exocytosis was visualized by monitoring changes in PHM-pHluorin fluorescence following stimulation. RESULTS: The time course of release differs with these secretagogues; the response to K+ is rapid and terminates within 15 minutes, while PMA-stimulated secretion is maintained over 30 minutes. Our data demonstrate fundamental differences between granule dynamics in axons and growth cones under basal conditions. Moreover, granule behavior in axons is regulated by secretagogue, resulting in changes in velocity, direction and processivity. Release events were observed throughout axons and growth cones. CONCLUSIONS: These results are consistent with an important role for cytoskeletal components and associated motor proteins in sustaining the secretory response. Our findings that peptidergic granules undergo fusion at nonspecific locations in trigeminal ganglion neurons are consistent with a role for non-synaptic peptide release. Supported by DE-017094, DK-032948.