Colocalization of FM1-43, Bassoon, and GnRH-1: GnRH-1 Release from Cell Bodies and Their Neuroprocesses

Abstract

Pulsatile release of GnRH-1 is critical for reproductive function. However, the cellular mechanism of GnRH-1 neurosecretion is still elusive. In this study, we examined the neurosecretory process of GnRH-1 neurons using time-lapse image acquisition followed by immunocytochemistry with confocal microscopy. To monitor exocytotic processes, cultured GnRH-1 neurons derived from monkey embryos were labeled with the lipophilic dye, FM1-43, or its fixable form FM1-43Fx, in the presence or absence of depolarization signals, and changes in vesicles labeled with FM1-43 were analyzed. The results show FM1-43 was taken up into the cell and labeled puncta in the soma and neuroprocesses in the absence of depolarization signals, indicating that GnRH-1 neurons were spontaneously active. Depolarization of GnRH-1 neurons with high K+ or veratridine challenge increased the intensity and size of puncta in both soma and neuroprocesses, and the veratridine-induced changes in puncta were blocked by tetrodotoxin, indicating that changes in the puncta intensity and size reflect neurosecretory activity. Subsequent double immunocytochemistry for GnRH-1 and the synaptic vesicle marker, vesicle-associated membrane protein, demonstrated that the FM1-43Fx-labeled puncta were synaptic vesicles with the GnRH-1 peptide. Additional double immunocytochemistry for GnRH-1 and the marker of the neurosecretory active zone, Bassoon, indicated that the FM1-43Fx-labeled puncta were located at the sites of neurosecretory active zones in GnRH-1 neurons. These results suggest that GnRH-1 neurons have the capacity to release the peptide from the soma and dendrites. Collectively, we hypothesize that soma-dendritic release of the peptide may be a mechanism of synchronized activity among GnRH-1 neurons.