Hair cell ribbon synapses exhibit several distinguishing features. Structurally, a dense body, or ribbon, is anchored to the presynaptic membrane and tethers synaptic vesicles; functionally, neurotransmitter release is dominated by large EPSC events produced by seemingly synchronous multivesicular release. However, the specific role of the synaptic ribbon in promoting this form of release remains elusive. Using complete ultrastructural reconstructions and capacitance measurements of bullfrog amphibian papilla hair cells dialyzed with high concentrations of a slow Ca2+ buffer (10 mM EGTA), we found that the number of synaptic vesicles at the base of the ribbon correlated closely to those vesicles that released most rapidly and efficiently, while the rest of the ribbon-tethered vesicles correlated to a second, slower pool of vesicles. Combined with the persistence of multivesicular release in extreme Ca2+ buffering conditions (10 mM BAPTA), our data argues against the Ca2+-dependent compound fusion of ribbon-tethered vesicles at hair cell synapses. Moreover, during hair cell depolarization, our results suggest that elevated Ca2+ levels enhance vesicle pool replenishment rates. Finally, using Ca2+ diffusion simulations, we propose that the ribbon and its vesicles define a small cytoplasmic volume where Ca2+ buffer is saturated, despite 10 mM BAPTA conditions. This local buffer saturation permits fast and large Ca2+ rises near release sites beneath the synaptic ribbon that can trigger multiquantal EPSCs. We conclude that, by restricting the available presynaptic volume, the ribbon may be creating conditions for the synchronous release of a small cohort of docked vesicles.

Sound-evoked spikes in the auditory nerve can phase-lock with submillisecond precision for prolonged periods of time. However, the synaptic mechanisms that enable this accurate spike firing remain poorly understood. Using paired recordings from adult frog hair cells and their afferent fibers we show here that during sinewave stimuli EPSC failures occur even during strong stimuli. However, exclusion of EPSC failures leads to mean EPSC amplitudes that are independent of Ca2+ current. Given the intrinsic jitter in spike triggering, evoked EPSPs and spikes had surprisingly similar degrees of synchronization to a sinewave stimulus. This similarity was explained by an unexpected finding: large-amplitude multiquantal EPSCs have a significantly larger synchronization index than smaller evoked EPSCs. Large EPSPs therefore enhance the precision of spike timing. The hair cells’ unique capacity for continuous, large-amplitude, and highly synchronous multiquantal release thus underlies its ability to trigger phase-locked spikes in afferent fibers.

Short-term facilitation and depression coexist at many CNS synapses. Facilitation, however, has not been fully characterized at hair cell synapses. Using paired recordings and membrane capacitance measurements we find that paired-pulse plasticity at an adult frog auditory hair cell synapse depends on pulse duration and inter-pulse intervals. For short 20 ms depolarizing pulses, and inter-pulse intervals between 15 to 50 ms, facilitation occurred when hair cells were held at −90 mV. However, hair cells held at −60 mV displayed only paired-pulse depression. Facilitation was dependent on residual free Ca2+ levels because it was greatly reduced by the Ca2+ buffers EGTA and BAPTA. Furthermore, low external Ca2+ augmented facilitation, whereas depression was augmented by high external Ca2+, consistent with depletion of a small pool of fast releasing synaptic vesicles. Recovery from depression had a double-exponential time course with a fast component that may reflect the rapid replenishment of a depleted vesicle pool. We suggest that hair cells held at more depolarized in vivo-like resting membrane potentials have a tonic influx of Ca2+; they are thus in a dynamic state of continuous vesicle release, pool depletion and replenishment. Further Ca2+ influx during paired-pulse stimuli then leads to depression. However, at membrane potentials of −90 mV ongoing release and pool depletion are minimized, so facilitation is revealed at time intervals when rapid vesicle pool replenishment occurs. Finally, we propose that vesicle pool replenishment kinetics is not rate-limited by vesicle endocytosis, which is too slow to influence the rapid pool replenishment process.

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