Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B

Abstract

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (I A(glu) ) in rods. Spontaneous I A(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of I A(glu) revealing that multivesicular release constitutes $30% of spontaneous release events. I A(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small I A(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca 2þ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca 2þ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.

Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival

“…However, degeneration in both Stx3 knockout lines was more rapid than in either the Rho −/− or Prph2 −/− (10, 53), suggesting other mechanisms, such as synaptic dysfunction, also contribute to the degeneration. In addition to its role in OS protein trafficking, STX3 is essential for synaptic vesicle exocytosis/release in photoreceptors (23,54). Consistent with a role for STX3 in synaptic release, we find that Stx3 knockout rod and cone synapses are highly abnormal.…”

Resting and stimulated mouse rod photoreceptors show distinct patterns of vesicle release at ribbon synapses

“…Presynaptic recordings of I A(glu) in voltage-clamped rods can provide a reliable presynaptic measure of glutamate release, reflecting the timing and magnitude of exocytotic events ( Grassmeyer et al, 2019 ; Hasegawa et al, 2006 ; Szmajda and Devries, 2011 ). In several experimental systems, including rods and cones, the anionic charge transfer has been shown to be proportional to glutamate concentrations in the cleft and likewise proportional to excitatory postsynaptic currents ( Hays et al, 2020 ; Koch et al, 2007 ; Otis and Jahr, 1998 ; Szmajda and Devries, 2011 ). To measure glutamate release presynaptically, we obtained whole-cell recordings from mouse rods using the permeant SCN − as the principal anion in the patch pipette solution to enhance I A(glu) .…”

Properties of multivesicular release from mouse rod photoreceptors support transmission of single-photon responses

“…Recent studies on mice in which synaptic ribbons have been eliminated by deleting the key ribbon protein, Ribeye, showed surprisingly modest deficits ( Fairless et al, 2020 ; Okawa et al, 2019 ) Multivesicular release in both salamander and mouse rods involves ribbon-associated vesicles ( Hays et al, 2020a ; Hays et al, 2020b ). One role of the ribbon may thus be to help organize vesicles to promote multivesicular release, implementing an Erlang process to improve the encoding of light responses near scotopic threshold ( Schein and Ahmad, 2005 ).…”