Abstract
Purpose::
To test whether synaptic release from photoreceptors is regulated by spatially averaged levels of Ca2+ in the synaptic terminal or by Ca2+ microdomains close to Ca2+ channels.
Methods::
Exocytosis from rods and cones was triggered by flash photolysis of the caged Ca2+ compound, DM-nitrophen. Release was monitored by measuring increases in post-synaptic currents (PSCs) in OFF bipolar and horizontal cells while recording simultaneously from photoreceptors in salamander retinal slice.
Results::
PSCs were evoked by elevating intraterminal Ca2+ by flash photolysis of DM-nitrophen to submicromolar levels in both rod and cone terminals. The ability of submicromolar Ca2+ to stimulate exocytosis is consistent with earlier capacitance measurements which raised the possibility that synaptic release from photoreceptors may be regulated by spatially averaged Ca2+ levels in the terminal (Rieke & Schwartz, 1996; Thoreson et al, 2004). Instantaneously elevating the intracellular Ca2+-concentration to the same level in either rods or cones produced equally fast PSCs indicating that the faster kinetics of release from cones compared to rods (Rabl et al, 2005) is not due to the presence of different Ca2+ sensors or other intrinsic differences in the release machinery. Ca2+changes in rod and cone terminals produced by membrane depolarization were visualized by confocal microscopy using low and high affinity Ca2+ sensitive dyes. Brief depolarizing steps (50-100 ms) stimulated localized, punctuate increases in Ca2+ that co-localized with a fluorescently-labeled ribeye peptide suggesting that Ca2+channels cluster near the synaptic ribbon. We further analyzed the distance between Ca2+ channels and release sites by comparing effects of BAPTA vs. EGTA in the patch pipette and exploiting differences in rates of Ca2+ buffering by the two buffers. In rods and cones, the faster buffer BAPTA (5 mM) but not the slower buffer EGTA (0.5 and 5 mM) inhibited the sustained component of release evoked by maintained depolarization. However, BAPTA did not significantly alter the initial fast component suggesting that vesicles involved in the initial burst of release are within ~100 nm of Ca2+ channels whereas sustained release involves vesicles further from the channels.
Conclusions::
Despite the presence of a high affinity release mechanism in both rod and cone terminals, synaptic release is regulated by elevated Ca2+ levels in microdomains close to channels, not the spatially averaged intraterminal Ca2+ concentration.
Keywords: photoreceptors • calcium • electrophysiology: non-clinical