The accumulation and extrusion of Ca²⁺ in the pre- and postsynaptic compartments play a critical role in initiating plastic changes in biological synapses. To emulate this fundamental process in electronic devices, we developed diffusive Ag-in-oxide memristors with a temporal response during and after stimulation similar to that of the synaptic Ca²⁺ dynamics. In situ high-resolution transmission electron microscopy and nanoparticle dynamics simulations both demonstrate that Ag atoms disperse under electrical bias and regroup spontaneously under zero bias because of interfacial energy minimization, closely resembling synaptic influx and extrusion of Ca²⁺, respectively. The diffusive memristor and its dynamics enable a direct emulation of both short- and long-term plasticity of biological synapses, representing an advance in hardware implementation of neuromorphic functionalities.

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 mmEGTA), 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 mmBAPTA), our data argue 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 mmBAPTA 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.

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

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 −45 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 Ca²⁺ 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.

Na⁺channel recovery from inactivation limits the maximal rate of neuronal firing. However, the properties of presynaptic Na⁺channels are not well established because of the small size of most CNS boutons. Here we study the Na⁺currents of the rat calyx of Held terminal and compare them with those of postsynaptic cells. We find that presynaptic Na⁺currents recover from inactivation with a fast, single-exponential time constant (24°C, τ of 1.4-1.8 ms; 35°C, τ of 0.5 ms), and their inactivation rate accelerates twofold during development, which may contribute to the shortening of the action potential as the terminal matures. In contrast, recordings from postsynaptic cells in brainstem slices, and acutely dissociated, reveal that their Na⁺currents recover from inactivation with a double-exponential time course (τ_fastof 1.2-1.6 ms; τ_slowof 80-125 ms; 24°C). Surprisingly, confocal immunofluorescence revealed that Na⁺channels are mostly absent from the calyx terminal but are instead highly concentrated in an unusually long (≈20-40 μm) unmyelinated axonal heminode. Outside-out patch recordings confirmed this segregation. Expression of Na_v1.6 α-subunit increased during development, whereas the Na_v1.2α-subunit was not present. Serial EM reconstructions also revealed a long pre-calyx heminode, and biophysical modeling showed that exclusion of Na⁺channels from the calyx terminal produces an action potential waveform with a shorter half-width. We propose that the high density and polarized locus of Na⁺channels on a long heminode are critical design features that allow the mature calyx of Held terminal to fire reliably at frequencies near 1 kHz.

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 interpulse intervals. For short 20 ms depolarizing pulses, and interpulse intervals between 15 and 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 depolarizedin 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.