Abstract
Rods are capable of greater slow release than cones contributing to overall slower release kinetics. Slow release in rods involves Ca2+-induced Ca2+ release (CICR). By impairing release from ribbons, we found that unlike cones where release occurs entirely at ribbon-style active zones, slow release from rods occurs mostly at ectopic, non-ribbon sites. To investigate the role of CICR in ribbon and non-ribbon release from rods, we used total internal reflection fluorescence microscopy as a tool for visualizing terminals of isolated rods loaded with fluorescent Ca2+ indicator dyes and synaptic vesicles loaded with dextran-conjugated pH-sensitive rhodamine. We found that rather than simply facilitating release, activation of CICR by ryanodine triggered release directly in rods, independent of plasma membrane Ca2+ channel activation. Ryanodine-evoked release occurred mostly at non-ribbon sites and release evoked by sustained depolarization at non-ribbon sites was mostly due to CICR. Unlike release at ribbon-style active zones, non-ribbon release did not occur at fixed locations. Fluorescence recovery after photobleaching of endoplasmic reticulum (ER)-tracker dye in rod terminals showed that ER extends continuously from synapse to soma. Release of Ca2+ from terminal ER by lengthy depolarization did not significantly deplete Ca2+ from ER in the perikaryon. Collectively, these results indicate that CICR-triggered release at non-ribbon sites is a major mechanism for maintaining vesicle release from rods and that CICR in terminals may be sustained by diffusion of Ca2+ through ER from other parts of the cell.
Highlights
Light-evoked voltage changes in photoreceptor cells are transmitted to second-order retinal neurons by changing the rate of glutamate release
Using total internal reflection fluorescence microscopy (TIRFM) to visualize fusion of synaptic vesicles in rods loaded with dextran-conjugated pHsensitive rhodamine, many of the vesicle fusion events evoked by long depolarizing steps were found to occur at sites >1 μm from ribbons (Chen et al, 2013)
To further assess the contribution of ribbons to slow release from rods, we studied the effects of damaging ribbons by fluorophore-assisted laser inactivation (FALI) on glutamatergic excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to simultaneously voltage-clamped rods (−70 to −10 mV, 200 ms; Figure 1A)
Summary
Light-evoked voltage changes in photoreceptor cells are transmitted to second-order retinal neurons by changing the rate of glutamate release. Damaging ribbons by fluorophore-assisted laser inactivation (FALI) of the ribbon protein Ribeye selectively diminished exocytotic increases in membrane capacitance evoked by short test steps but not capacitance increases evoked by longer steps (Chen et al, 2013). These results suggest that fast release from rods involves the ribbon but slow release involves non-ribbon release sites. Consistent with contributions from non-ribbon release sites in rods are the presence of putative fusion events at non-ribbon sites revealed by electron microscopic (EM) tomography (Zampighi et al, 2011)
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