Abstract
Purpose::
Synaptic vesicle protein 2 (SV2) comprises a family of synaptic vesicle proteins that regulate calcium mediated synaptic transmission via a poorly understood mechanism. The three isoforms of SV2 are differentially distributed in the vertebrate retina, with the rod bipolar cell (RBC) containing only the SV2B isoform. To determine the role of SV2B in the regulation of glutamate release from RBCs, we compared synaptic vesicle dynamics and calcium responses of RBCs isolated from the retinas of SV2B knock-out mice (KO) and their wild-type litter mates (WT).
Methods::
Intraterminal calcium of acutely dissociated mouse RBCs was measured using a fluorescent calcium indicator dye. Capacitance measurements were used to track changes in membrane surface area indicative of exocytosis (Zhou et al., 2006). SV2B knock-out mice and wild-type litter mates were generated as described (Janz et al., 1999).
Results::
The resting [Ca2+]i in RBC terminals of KO mice was significantly higher than that of WT RBCs (KO: 105±24 nM, n=16; WT: 43±9 nM, n=19; P<0.02). In response to a 500 ms depolarization, the increase in [Ca2+]i was similar between KO and WT terminals, as was the magnitude of exocytosis and the time course of endocytosis. However, in response to a short train of ten 100 ms depolarizations from -70 to 0 mV, KO terminals exhibited a a larger cumulative [Ca2+]i increase than WT terminals (KO: 326±51 nM, n=8; WT: 154±46 nM, n=6; P<0.05). Although the total amount of membrane added following a train was similar, the secretory response to the first stimulus in a train was significantly diminished in the KO RBCs (KO: 3±2 fF, n=9; WT: 11±2 fF, n=6; P<0.05). In addition, the fast component of endocytosis was slower in KO RBCs than in WT RBCs (KO: τfast =10±1 s, n=7; WT: τfast=3±0.5 s, n=6; P<0.01). To separate direct effects of the loss of SV2B from indirect effects attributable to elevated [Ca2+]i, we redesigned the pipette solution so that the basal [Ca2+]i of WT RBCs mimicked the higher level of KO RBCs. With this solution, the calcium responses of WT RBCs evoked by a stimulus train approximated those of KO RBCs, and the fast time constant of endocytosis was similar to that of KO RBCs. Interestingly, the secretory phenotype was not reproduced, suggesting a dual role for SV2B.
Conclusions::
SV2B is an important regulator of synaptic vesicle dynamics in mouse RBCs. Regulation by SV2B occurs by two distinct mechanisms. The first is a direct action on the secretory apparatus. In addition, SV2B governs RBC synaptic vesicle dynamics indirectly via the regulation of presynaptic calcium.
Keywords: synapse • bipolar cells • calcium