EPSCs at the synapses of sensory receptors and of some CNS neurons include large events thought to represent the synchronous release of the neurotransmitter contained in several synaptic vesicles by a process known as multiquantal release. However, determination of the unitary, quantal size underlying such putatively multiquantal events has proven difficult at hair cell synapses, hindering confirmation that large EPSCs are in fact multiquantal. Here, we address this issue by performing presynaptic membrane capacitance measurements together with paired recordings at the ribbon synapses of adult hair cells. These simultaneous presynaptic and postsynaptic assays of exocytosis, together with electron microscopic estimates of single vesicle capacitance, allow us to estimate a single vesicle EPSC charge of approximately Ϫ45 fC, a value in close agreement with the mean postsynaptic charge transfer of uniformly small EPSCs recorded during periods of presynaptic hyperpolarization. By thus establishing the magnitude of the fundamental quantal event at this peripheral sensory synapse, we provide evidence that the majority of spontaneous and evoked EPSCs are multiquantal. Furthermore, we show that the prevalence of uniquantal versus multiquantal events is Ca 2ϩ dependent. Paired recordings also reveal a tight correlation between membrane capacitance increase and evoked EPSC charge, indicating that glutamate release during prolonged hair cell depolarization does not significantly saturate or desensitize postsynaptic AMPA receptors. We propose that the large EPSCs reflect the highly synchronized release of multiple vesicles at single presynaptic ribbon-type active zones through a compound or coordinated vesicle fusion mechanism.

The sense of hearing depends on fast, finely graded neurotransmission at the ribbon synapses connecting hair cells to afferent nerve fibers. The processing that occurs at this first chemical synapse in the auditory pathway determines the quality and extent of the information conveyed to the central nervous system. Knowledge of the synapse's input-output function is therefore essential for understanding how auditory stimuli are encoded. To investigate the transfer function at the hair cell's synapse, we developed a preparation of the bullfrog's amphibian papilla. In the portion of this receptor organ representing stimuli of 400 -800 Hz, each afferent nerve fiber forms several synaptic terminals onto one to three hair cells. By performing simultaneous voltage-clamp recordings from presynaptic hair cells and postsynaptic afferent fibers, we established that the rate of evoked vesicle release, as determined from the average postsynaptic current, depends linearly on the amplitude of the presynaptic Ca 2؉ current. This result implies that, for receptor potentials in the physiological range, the hair cell's synapse transmits information with high fidelity.auditory system ͉ exocytosis ͉ glutamate ͉ ribbon synapse ͉ synaptic vesicle T he remarkable acuity and temporal precision of the auditory system are contingent on exocytosis at the hair cell's ribbon synapses, whose transfer characteristics are largely determined by the dependence of vesicle fusion on Ca 2ϩ influx through voltage-gated channels. The relation of presynaptic voltage and Ca 2ϩ influx to transmitter release at ribbon synapses is of particular interest in light of several unusual features of the hair cell's synaptic signaling. Afferent synapses encode inputs with graded membrane potentials (reviewed in ref. 1), transmit information with microsecond temporal fidelity (2), demonstrate multivesicular release (3-5), sustain high rates of exocytosis for prolonged periods (6-8), and display frequency tuning (9). Transmitter release is triggered by an atypical class of voltage-gated Ca 2ϩ channels [noninactivating, L-type channels with ␣ 1D principal subunits (10)] clustered at the presynaptic active zones (11)(12)(13)(14). Finally, synaptotagmins I and II, the putative Ca 2ϩ sensors for vesicle fusion in the brain, have not yet been detected at hair-cell synapses (15); instead, an alternative Ca 2ϩ sensor, such as otoferlin (16), may effect exocytosis at these sites.The operation of the hair cell's synapse has been investigated only indirectly by recording signals independently from either its presynaptic or postsynaptic element. Presynaptic measurements of membrane capacitance, an index of synaptic vesicle fusion, have provided information about the relation between presynaptic Ca 2ϩ signals and vesicle release. Flash photolysis of caged Ca 2ϩ indicates that the rate of exocytosis displays a fifth-order dependence on the cytoplasmic Ca 2ϩ concentration (17), whereas hair-cell depolarization elicits vesicle release that is directly proportional to the presynapt...

Records of excitatory postsynaptic currents (EPSCs) are often complex, with overlapping signals that display a large range of amplitudes. Statistical analysis of the kinetics and amplitudes of such complex EPSCs is nonetheless essential to the understanding of transmitter release. We therefore developed a maximum-likelihood blind deconvolution algorithm to detect exocytotic events in complex EPSC records. The algorithm is capable of characterizing the kinetics of the prototypical EPSC as well as delineating individual release events at higher temporal resolution than other extant methods. The approach also accommodates data with low signal-to-noise ratios and those with substantial overlaps between events. We demonstrated the algorithm’s efficacy on paired whole-cell electrode recordings and synthetic data of high complexity. Using the algorithm to align EPSCs, we characterized their kinetics in a parameter-free way. Combining this approach with maximum-entropy deconvolution, we were able to identify independent release events in complex records at a temporal resolution of less than 250 µs. We determined that the increase in total postsynaptic current associated with depolarization of the presynaptic cell stems primarily from an increase in the rate of EPSCs rather than an increase in their amplitude. Finally, we found that fluctuations owing to postsynaptic receptor kinetics and experimental noise, as well as the model dependence of the deconvolution process, explain our inability to observe quantized peaks in histograms of EPSC amplitudes from physiological recordings.