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

Abstract

The vertebrate visual system can detect and transmit signals from single photons. To understand how single-photon responses are transmitted, we characterized voltage-dependent properties of glutamate release in mouse rods. We measured presynaptic glutamate transporter anion current and found that rates of synaptic vesicle release increased with voltage-dependent Ca2+ current. Ca2+ influx and release rate also rose with temperature, attaining a rate of ∼11 vesicles/s/ribbon at −40 mV (35°C). By contrast, spontaneous release events at hyperpolarized potentials (−60 to −70 mV) were univesicular and occurred at random intervals. However, when rods were voltage clamped at −40 mV for many seconds to simulate maintained darkness, release occurred in coordinated bursts of 17 ± 7 quanta (mean ± SD; n = 22). Like fast release evoked by brief depolarizing stimuli, these bursts involved vesicles in the readily releasable pool of vesicles and were triggered by the opening of nearby ribbon-associated Ca2+ channels. Spontaneous release rates were elevated and bursts were absent after genetic elimination of the Ca2+ sensor synaptotagmin 1 (Syt1). This study shows that at the resting potential in darkness, rods release glutamate-filled vesicles from a pool at the base of synaptic ribbons at low rates but in Syt1-dependent bursts. The absence of bursting in cones suggests that this behavior may have a role in transmitting scotopic responses.

Properties of multi-vesicular release from rod photoreceptors support transmission of single photon responses

“…1) (Grassmeyer et al, 2019). As described earlier (Hays et al, 2020b), these inward currents had waveforms typical of quantal post-synaptic currents but with a slow time course (10-90% rise time of 9-10 ms with decay time constants of ∼40 ms). Spontaneous inward currents in rods voltage-clamped at -70 mV showed a unimodal amplitude distribution suggesting they consisted entirely of uniquantal events (Fig.…”

“…Thus, dark adaptation might influence the statistics of vesicle release. However, when holding dark-adapted rods at -40 mV, we found that the number of vesicles in each multivesicular release event (16.7 ± 5.8, n=7) and the intervals between multivesicular event start times (2.0 ± 0.54 s, n=6, room temperature) did not differ significantly from those measured in light-adapted retinas (17 ± 7 v/burst, n=22, p=0.74, unpaired t-test; 2.4 ± 0.62 s intervals, n=14, p=0.18, unpaired t-test) (Hays et al, 2020b). Thus, recording from rods in light-adapted versus dark-adapted states did not strongly alter release statistics.…”

“…1C). As described previously (Hays et al, 2020b), multivesicular release events involved the rapid sequential release of 10-20 vesicles triggered by Ca 2+ influx and derived from a pool of ribbon-associated vesicles shared with evoked release.…”

“…Spontaneous release can be Ca 2+ -independent or due to chance openings of voltage-gated Ca 2+ channels (Cork et al, 2016; Kavalali, 2015, 2019). The persistence of spontaneous events in rods held at -70 mV, even after blocking Ca 2+ channels with extracellular Cd 2+ or strongly buffering intracellular Ca 2+ with 10 mM BAPTA, showed evidence for Ca 2+ -independent release (Hays et al, 2020b). To analyze frequency distributions, only rods in which we observed >50 events were included in the data set.…”

“…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, 2020b)(Hays et al, 2020a). 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).…”