Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse.

We report functional and structural evidence for GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss.SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca2+-permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca2+ accumulation and swelling that can be prevented by blocking CP-AMPARs. We demonstrate that CP-AMPARs mediate transmission at this first-order sensory synapse and that limiting Ca2+ accumulation in the terminal may protect against hearing loss.

The Coupling between Ca2+ Channels and the Exocytotic Ca2+ Sensor at Hair Cell Ribbon Synapses Varies Tonotopically along the Mature Cochlea.

The cochlea processes auditory signals over a wide range of frequencies and intensities. However, the transfer characteristics at hair cell ribbon synapses are still poorly understood at different frequency locations along the cochlea. Using recordings from mature gerbils, we report here a surprisingly strong block of exocytosis by the slow Ca2+ buffer EGTA (10 mM) in basal hair cells tuned to high frequencies (∼30 kHz). In addition, using recordings from gerbil, mouse, and bullfrog auditory organs, we find that the spatial coupling between Ca2+ influx and exocytosis changes from nanodomain in low-frequency tuned hair cells (∼<2 kHz) to progressively more microdomain in high-frequency cells (∼>2 kHz). Hair cell synapses have thus developed remarkable frequency-dependent tuning of exocytosis: accurate low-latency encoding of onset and offset of sound intensity in the cochlea's base and submillisecond encoding of membrane receptor potential fluctuations in the apex for precise phase-locking to sound signals. We also found that synaptic vesicle pool recovery from depletion was sensitive to high concentrations of EGTA, suggesting that intracellular Ca2+ buffers play an important role in vesicle recruitment in both low- and high-frequency hair cells. In conclusion, our results indicate that microdomain coupling is important for exocytosis in high-frequency hair cells, suggesting a novel hypothesis for why these cells are more susceptible to sound-induced damage than low-frequency cells; high-frequency inner hair cells must have a low Ca2+ buffer capacity to sustain exocytosis, thus making them more prone to Ca2+-induced cytotoxicity.SIGNIFICANCE STATEMENT In the inner ear, sensory hair cells signal reception of sound. They do this by converting the sound-induced movement of their hair bundles present at the top of these cells, into an electrical current. This current depolarizes the hair cell and triggers the calcium-induced release of the neurotransmitter glutamate that activates the postsynaptic auditory fibers. The speed and precision of this process enables the brain to perceive the vital components of sound, such as frequency and intensity. We show that the coupling strength between calcium channels and the exocytosis calcium sensor at inner hair cell synapses changes along the mammalian cochlea such that the timing and/or intensity of sound is encoded with high precision.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Proton-mediated block of Ca2+ channels during multivesicular release regulates short-term plasticity at an auditory hair cell synapse.

Synaptic vesicles release both neurotransmitter and protons during exocytosis, which may result in a transient acidification of the synaptic cleft that can block Ca(2+) channels located close to the sites of exocytosis. Evidence for this effect has been reported for retinal ribbon-type synapses, but not for hair cell ribbon synapses. Here, we report evidence for proton release from bullfrog auditory hair cells when they are held at more physiological, in vivo-like holding potentials (Vh = -60 mV) that facilitate multivesicular release. During paired recordings of hair cells and afferent fibers, L-type voltage-gated Ca(2+) currents showed a transient block, which was highly correlated with the EPSC amplitude (or the amount of glutamate release). This effect was masked at Vh = -90 mV due to the presence of a T-type Ca(2+) current and blocked by strong pH buffering with HEPES or TABS. Increasing vesicular pH with internal methylamine in hair cells also abolished the transient block. High concentrations of intracellular Ca(2+) buffer (10 mm BAPTA) greatly reduced exocytosis and abolished the transient block of the Ca(2+) current. We estimate that this transient block is due to the rapid multivesicular release of ∼600-1300 H(+) ions per synaptic ribbon. Finally, during a train of depolarizing pulses, paired pulse plasticity was significantly changed by using 40 mm HEPES in addition to bicarbonate buffer. We propose that this transient block of Ca(2+) current leads to more efficient exocytosis per Ca(2+) ion influx and it may contribute to spike adaptation at the auditory nerve.

Phase-locking precision is enhanced by multiquantal release at an auditory hair cell ribbon synapse.

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 sine-wave stimuli, synaptic failures occur even during strong stimuli. However, exclusion of these failures leads to mean excitatory postsynaptic current (EPSC) amplitudes that are independent of Ca(2+) current. Given the intrinsic jitter in spike triggering, evoked synaptic potentials and spikes had surprisingly similar degrees of synchronization to a sine-wave stimulus. This similarity was explained by an unexpected finding: large-amplitude evoked EPSCs have a significantly larger synchronization index than smaller evoked EPSCs. Large EPSCs 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.

Specialized postsynaptic morphology enhances neurotransmitter dilution and high-frequency signaling at an auditory synapse.

Sensory processing in the auditory system requires that synapses, neurons, and circuits encode information with particularly high temporal and spectral precision. In the amphibian papillia, sound frequencies up to 1 kHz are encoded along a tonotopic array of hair cells and transmitted to afferent fibers via fast, repetitive synaptic transmission, thereby promoting phase locking between the presynaptic and postsynaptic cells. Here, we have combined serial section electron microscopy, paired electrophysiological recordings, and Monte Carlo diffusion simulations to examine novel mechanisms that facilitate fast synaptic transmission in the inner ear of frogs (Rana catesbeiana and Rana pipiens). Three-dimensional anatomical reconstructions reveal specialized spine-like contacts between individual afferent fibers and hair cells that are surrounded by large, open regions of extracellular space. Morphologically realistic diffusion simulations suggest that these local enlargements in extracellular space speed transmitter clearance and reduce spillover between neighboring synapses, thereby minimizing postsynaptic receptor desensitization and improving sensitivity during prolonged signal transmission. Additionally, evoked EPSCs in afferent fibers are unaffected by glutamate transporter blockade, suggesting that transmitter diffusion and dilution, and not uptake, play a primary role in speeding neurotransmission and ensuring fidelity at these synapses.

An excess-calcium-binding-site model predicts neurotransmitter release at the neuromuscular junction.

Despite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca(2+)-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20-40 independent Ca(2+)-binding sites on synaptic vesicles, only a fraction of which need to bind Ca(2+) to trigger fusion, are sufficient to predict physiological release. Our excess-Ca(2+)-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca(2+)-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca(2+) dynamics during synapse activation.

Ca(2+) influx and neurotransmitter release at ribbon synapses.

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.

Sharp Ca²âº nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses.

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 Ca²âº 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 Ca²âº buffering conditions (10 mM BAPTA), our data argue against the Ca²âº-dependent compound fusion of ribbon-tethered vesicles at hair cell synapses. Moreover, during hair cell depolarization, our results suggest that elevated Ca²âº levels enhance vesicle pool replenishment rates. Finally, using Ca²âº diffusion simulations, we propose that the ribbon and its vesicles define a small cytoplasmic volume where Ca²âº buffer is saturated, despite 10 mM BAPTA conditions. This local buffer saturation permits fast and large Ca²âº 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.

Recovery from short-term depression and facilitation is ultrafast and Ca2+ dependent at auditory hair cell synapses.

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 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.

The effects of presynaptic calcium channel modulation by roscovitine on transmitter release at the adult frog neuromuscular junction.

Calcium (Ca2+) influx through presynaptic calcium channels triggers transmitter release, and any alterations in the gating of these calcium channels results in changes in the magnitude of transmitter released. We used (R)-roscovitine, a cyclin-dependent kinase inhibitor that also appears to act directly on calcium channels, as a tool to modulate presynaptic calcium influx and study effects on transmitter release. We show that this compound increased the quantal content of acetylcholine released from the Rana frog motor nerve terminal (by 149%) without changing paired-pulse facilitation (under low calcium conditions). In contrast, exposure to 3,4-diaminopyridine (DAP; which similarly affects transmitter release by partially blocking potassium channels, altering the shape of the presynaptic action potential, and indirectly increasing calcium entry) increased paired-pulse facilitation (by 23%). In addition, we show that (R)-roscovitine predominately slowed deactivation kinetics of calcium current (by 427%) recorded from Xenopus frog motoneurons, and as a result, increased the integral of calcium channel current evoked by a physiological action potential waveform (by 44%). Because we did not observe any significant effects of structurally related cyclin-dependent kinase inhibitors [(S)-roscovitine or olomoucine] on evoked transmitter release or calcium current kinetics, it appears that these effects of (R)-roscovitine are independent of cyclin-dependent kinases (cdks). In summary, we hypothesize that (R)-roscovitine effects on transmitter release at the adult frog neuromuscular junction (NMJ) are mediated by its effects on calcium channel gating, and these effects increase our understanding of calcium triggered secretion at this synapse.