Synaptic vesicle exocytosis in primary cultures of baroreceptor neurons is reduced during high-frequency stimulation. Calcium influx through voltage-gated calcium channels (VGCC) is a key step in neurotransmitter release. With the help of FM2-10, a marker of synaptic vesicle recycling, the present study investigates the differential contribution of several VGCC subtypes to exocytosis in neuronal processes and how this contribution is altered at high frequencies. In control experiments, field stimulation at 0.5 Hz evoked about 66 +/- 5% destaining. Combined blockade of N- and P/Q-subtypes with Ctx-MVIIC was far more effective in reducing exocytosis (11 +/- 8%) than blocking N-type (49 +/- 5%, Ctx-GVIA) or P-type (46 +/- 1%, Agatoxin) alone. The effectiveness of the blockers also varied with the duration of stimulation: Ctx-GVIA attenuating exocytosis significantly in the first 60 s and Agatoxin affecting exocytosis only towards the end of 180 s stimulation period. Field stimulation at 10 Hz evoked exocytosis (36 +/- 18%) comparable to that evoked by 0.5 Hz in the presence of Ctx-GVIA. While blockade with Agatoxin had no effects, Ctx-GVIA, Ctx-MVIIC and L-type blocker Nifedepine had small but similar inhibitory effects on exocytosis at 10 Hz. The data suggest that N-type is the major contributor to exocytosis at 0.5 Hz, and this contribution is reduced during prolonged stimulation periods and at high frequencies.
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