Synaptic vesicle exocytosis measured in presynaptic boutons of baroreceptor neurons

Release of neurotransmitter in both peripheral and central neurons depends on the exocytosis of vesicles in presynaptic nerve terminals. We used the fluorescent membrane probe FM1-43 to measure exocytosis from living synaptic boutons of Dil labeled aortic baroreceptor and unlabeled nodose neurons in culture in=231. Superfusion of these neurons with a depolarizing solution (60 mM KCI) containing 10 μM FM 1-43 resulted in an array of fluorescent punctae along neurites with the greatest staining along that part of the neurite which made contact with other nearby neuntes. This staining pattern was persistent following washing in normal saline and was colocalized with synapsin I protein, suggesting that these fluorescent regions are synaptic terminals. Intensity of FMI-43 fluorescent punctae decreased consistently and repeatably following KCl stimulation in the absence of dye. The destaining of these terminal regions is consistent with neurotransmitter vesicle exocytosis as reported by others. Over the four minute time course that the terminal regions were imaged, KCl depolarization decreased FM 1-43 fluorescent intensity by approximately 50% within 90 sec. and reached near steady-state levels within 240 seconds. The role of specific voltage-gated calcium channels during the KCl evoked response was evaluated with application of the N-type blocker ω-conotoxin GVIA. Within a terminal region, ω-conotoxin GVIA completely inhibited the release of FMI-43 in some boutons, and was ineffective in others. This preparation will allow for the optical study of synaptic vesicle release in visceral sensory neurons and should serve as a useful model for the study of activity dependent neurotransmitter vesicle release.