Calyceal Synapses Research Articles

Evidence That Ultrafast Nonquantal Transmission Underlies Synchronized Vestibular Action Potential Generation.

Amniotes evolved a unique postsynaptic terminal in the inner ear vestibular organs called the calyx that receives both quantal and nonquantal (NQ) synaptic inputs from Type I sensory hair cells. The nonquantal synaptic current includes an ultrafast component that has been hypothesized to underlie the exceptionally high synchronization index (vector strength) of vestibular afferent neurons in response to sound and vibration. Here, we present three lines of evidence supporting the hypothesis that nonquantal transmission is responsible for synchronized vestibular action potentials of short latency in the guinea pig utricle of either sex. First, synchronized vestibular nerve responses are unchanged after administration of the AMPA receptor antagonist CNQX, while auditory nerve responses are completely abolished. Second, stimulus evoked vestibular nerve compound action potentials (vCAP) are shown to occur without measurable synaptic delay and three times shorter than the latency of auditory nerve compound action potentials (cCAP), relative to the generation of extracellular receptor potentials. Third, paired-pulse stimuli designed to deplete the readily releasable pool (RRP) of synaptic vesicles in hair cells reveal forward masking in guinea pig auditory cCAPs, but a complete lack of forward masking in vestibular vCAPs. Results support the conclusion that the fast component of nonquantal transmission at calyceal synapses is indefatigable and responsible for ultrafast responses of vestibular organs evoked by transient stimuli.SIGNIFICANCE STATEMENT The mammalian vestibular system drives some of the fastest reflex pathways in the nervous system, ensuring stable gaze and postural control for locomotion on land. To achieve this, terrestrial amniotes evolved a large, unique calyx afferent terminal which completely envelopes one or more presynaptic vestibular hair cells, which transmits mechanosensory signals mediated by quantal and nonquantal (NQ) synaptic transmission. We present several lines of evidence in the guinea pig which reveals the most sensitive vestibular afferents are remarkably fast, much faster than their auditory nerve counterparts. Here, we present neurophysiological and pharmacological evidence that demonstrates this vestibular speed advantage arises from ultrafast NQ electrical synaptic transmission from Type I hair cells to their calyx partners.

Assessment of Possible Contributions of Hyaluronan and Proteoglycan Binding Link Protein 4 to Differential Perineuronal Net Formation at the Calyx of Held.

The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of Hapln4 exhibited a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the neuropil surrounding the whole calyx of Held terminals. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil, together with HAPLN1 and tenascin-R, in both gene knockout (KO) and wild-type (WT) mice. An in situ proximity ligation assay demonstrated the molecular association of brevican with HAPLN4 in WT and HAPLN1 in gene KO mice. Further elucidation of the roles of HAPLN4 may highlight the developmental and physiological importance of PNN formation in the calyx of Held.

Ca2+ Regulates the Kinetics of Synaptic Vesicle Fusion at the Afferent Inner Hair Cell Synapse.

The early auditory pathway processes information at high rates and with utmost temporal fidelity. Consequently, the synapses in the auditory pathway are highly specialized to meet the extraordinary requirements on signal transmission. The calyceal synapses in the auditory brainstem feature more than a hundred active zones (AZs) with thousands of releasable synaptic vesicles (SVs). In contrast, the first auditory synapse, the afferent synapse of inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs), typically exhibits a single ribbon-type AZ tethering only tens of SVs resulting in a highly stochastic pattern of transmitter release. Spontaneous excitatory postsynaptic currents (sEPSCs), besides more conventional EPSCs with a single peak, fast rise and decay (compact), also include EPSCs with multiple peaks, variable rise and decay times (non-compact). The strong heterogeneity in size and shape of spontaneous EPSCs has led to the hypothesis of multivesicular release (MVR) that is more (compact) or less (non-compact) synchronized by coordination of release sites. Alternatively, univesicular release (UVR), potentially involving glutamate release through a flickering fusion pore for non-compact EPSCs, has been suggested to underlie IHC exocytosis. Here, we further investigated the mode of release by recording sEPSCs from SGNs of hearing rats while manipulating presynaptic IHC Ca2+ influx by changes in extracellular [Ca2+] ([Ca2+]e) and by application of the Ca2+ channel antagonist, isradipine, or the Ca2+ channel agonist, BayK8644 (BayK). Our data reveal that Ca2+ influx manipulation leaves the distributions of sEPSC amplitude and charge largely unchanged. Regardless the type of manipulation, the rate of sEPSC decreased with the reduction in Ca2+ influx. The fraction of compact sEPSCs was increased in the presence of BayK, an effect that was abolished when combined with decreased [Ca2+]e. In conclusion, we propose that UVR is the prevailing mode of exocytosis at cochlear IHCs of hearing rats, whereby the rate of exocytosis and the kinetics of SV fusion are regulated by Ca2+ influx.

SK Channels Regulate Resting Properties and Signaling Reliability of a Developing Fast-Spiking Neuron.

Reliable and precise signal transmission is essential in circuits of the auditory brainstem to encode timing with submillisecond accuracy. Globular bushy cells reliably and faithfully transfer spike signals to the principal neurons of the medial nucleus of the trapezoid body (MNTB) through the giant glutamatergic synapse, the calyx of Held. Thus, the MNTB works as a relay nucleus that preserves the temporal pattern of firing at high frequency. Using whole-cell patch-clamp recordings, we observed a K+ conductance mediated by small-conductance calcium-activated potassium (SK) channels in the MNTB neurons from rats of either sex. SK channels were activated by intracellular Ca2+ sparks and mediated spontaneous transient outward currents in developing MNTB neurons. SK channels were also activated by Ca2+ influx through voltage-gated Ca2+ channels and synaptically activated NMDA receptors. Blocking SK channels with apamin depolarized the resting membrane potential, reduced resting conductance, and affected the responsiveness of MNTB neurons to signal inputs. Moreover, SK channels were activated by action potentials and affected the spike afterhyperpolarization. Blocking SK channels disrupted the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons and induced extra postsynaptic action potentials in response to presynaptic firing. These data reveal that SK channels play crucial roles in regulating the resting properties and maintaining reliable signal transmission of MNTB neurons.SIGNIFICANCE STATEMENT Reliable and precise signal transmission is required in auditory brainstem circuits to localize the sound source. The calyx of Held synapse in the mammalian medial nucleus of the trapezoid body (MNTB) plays an important role in sound localization. We investigated the potassium channels that shape the reliability of signal transfer across the calyceal synapse and observed a potassium conductance mediated by small-conductance calcium-activated potassium (SK) channels in rat MNTB principal neurons. We found that SK channels are tonically activated and contribute to the resting membrane properties of MNTB neurons. Interestingly, SK channels are transiently activated by calcium sparks and calcium influx during action potentials and control the one-to-one signal transmission from presynaptic calyces to postsynaptic MNTB neurons.

Tuning and Timing in Mammalian Type I Hair Cells and Calyceal Synapses

Afferent nerve fibers in the central zones of vestibular epithelia form calyceal endings around type I hair cells and have phasic response properties that emphasize fast head motions. We investigated how stages from hair-cell transduction to calyceal spiking contribute tuning and timing to central (striolar)-zone afferents of the rat saccular epithelium. In an excised preparation, we deflected individual hair bundles with rigid probes driven with steps and sinusoids (0.5-500 Hz) and recorded whole-cell responses from hair cells and calyces at room temperature and body temperature. In immature hair cells and calyces (postnatal days (P)1-P4), tuning sharpened at each stage. Transducer adaptation and membrane-charging time produced bandpass filtering of the receptor potential with best frequencies of 10-30 Hz and phase leads below 10 Hz. For small stimuli, electrical resonances sharply tuned the hair-cell membrane in the frequency range of 5-40 Hz. The synaptic delay of quantal transmission added a phase lag at frequencies above 10 Hz. The influence of spike thresholds at the calyceal spike initiation stage sharpened tuning and advanced response phase. Two additional mechanisms strongly advanced response phase above 10 Hz when present: (1) maturing (P7-P9) type I hair cells acquired low-voltage-activated channels that shortened the rise time of the receptor potential and (2) some calyces had nonquantal transmission with little synaptic delay. By reducing response time, the identified inner-ear mechanisms (transducer adaptation, low-voltage-activated channels, nonquantal transmission, and spike triggering) may compensate for transmission delays in vestibular reflex pathways and help stabilize posture and gaze during rapid head motions.

GluA4 sustains sensing of sounds through stable, speedy, sumptuous, spineless synapses

Glutamatergic synapses are of immense importance in that they mediate most excitation in the vertebrate brain. They are remarkably diverse. Some produce large, precisely timed synaptic currents, while others produce small, slow currents. Some are indefatigable, signalling robustly and with high fidelity, independent of how rapidly they are driven while others are plastic, constantly strengthening or weakening as a function of whether they are driven rapidly or slowly. α-Amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors report the presynaptic release of neurotransmitter to postsynaptic neurons. They are tetrameric ion channels formed as combinations of four α subunits, GluA1–4, each of which has the alternatively spliced forms, flip and flop. The subunits differ in the length and in the sequence of their cytoplasmic, carboxy terminal tails which mediate interactions with other molecular components of synapses that regulate receptor trafficking and tethering (Jackson & Nicoll, 2011). The receptor subunit composition determines the speed of synaptic signalling; its receptor number determines the strength of a synapse. It is essential for some functions of the brain that glutamatergic synapses not be altered by synaptic traffic. At the early stages of the auditory pathway, for example, acoustic information must be transmitted faithfully, without distortion by previous sounds, for that information to be useful for computing the location and meaning of sounds. Acoustic information from the cochlea is relayed through the cochlear nucleus and the contralateral medial nucleus of the trapezoid body (MNTB) to the contralateral lateral superior olive. The lateral superior olive compares the intensity of sounds at the two ears for localizing sound sources in the horizontal plane. MNTB neurons are excited through a single sumptuous, calyceal synapse that contacts the smooth surface of the target soma, the calyx of Held (Fig. 1A). This synapse has become a darling of synaptic physiologists because the presynaptic terminals are accessible to electrodes but has been largely ignored by cell biologists because it is boringly stable and not experimentally tractable. Figure 1 Glutamatergic, excitatory synapses are diverse As at upstream synapses, AMPA receptors in mature MNTB neurons are formed from GluA3 flop and/or GluA4 flop subunits in which GluA1 and GluA2 are not functionally evident (Joshi et al. 2004; Koike-Tani et al. 2005). In this issue of The Journal of Physiology, Yang et al. (2011) compare EPSCs in the MNTB of wild type mice with those in mice lacking GluA3 (GluA3 KO) and in mice lacking GluA4 (GluA4 KO). They find that in the MNTB of GluA4 KO mice, EPSCs are dramatically diminished in size and slowed whereas in GluA3 KO mice, EPSCs are only subtly slowed. GluA2 substituted to a small extent for the loss of GluA3 but not detectably for the loss of GluA4. The calyceal synapse does not rely entirely on GluA4, however, because the removal of GluA3 results in greater synaptic depression and desensitization than in the wild type. Subtle but detectable differences in presynaptic functions in KO mice serve as a reminder that differences between wild type and GluA4 KO mice reflect not only the removal of GluA4 at this synapse but can also reflect changes upstream in the auditory pathway as well as compensatory changes at all levels. These findings leave no doubt, however, that GluA4 is essential in mature, excitatory synapses in the MNTB, that GluA3 plays a minor role, and that GluA1 and 2 are almost undetectable in the MNTB. Cell biologists have been attracted by the dynamic glutamatergic synapses on spines in hippocampal pyramidal cells (Fig. 1B). They have studied the trafficking of AMPA receptors in hippocampal synapses where endogenous AMPA receptors are formed from either GluA1/2 or GluA2/3 subunits. Spines give neighbouring synapses biochemical privacy. The long, cytoplasmic, carboxy terminal tails of GluA1 subunits interact with proteins that drive AMPA receptors into synapses as a function of activity (Hayashi et al. 2000). GluA2/3 AMPA receptors cycle constitutively, replacing GluA1/2 receptors. The short cytoplasmic tails of GluA2 subunits regulate the interaction with a different group of synaptic proteins that promote the removal of GluA2-containing AMPA receptors (Kim et al. 2001). The relative rates of insertion and excision of receptors determines how many AMPA receptors are at a synapse and how much current it delivers at any moment. The findings of Yang et al. show that calyceal synapses of MNTB neurons manage without either GluA1 or GluA2, which are so important in the hippocampus, and do pretty well in the absence of GluA3. At these synapses fleets of the speedy GluA4 flop subunits seem to be coordinated by synaptic proteins in the communal postsynaptic environment of the soma.

Vesicular glutamate filling and AMPA receptor occupancy at the calyx of Held synapse of immature rats

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded L-glutamate (1-100 mM) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36-37 degrees C), the qEPSC amplitude increased when intraterminal L-glutamate concentration was elevated from 1 mM to 10 mM, but it reached a plateau at 10 mM. This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal L-glutamate from 1 mM to 100 mM , even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 microM). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nM; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Branching of calyceal afferents during postnatal development in the rat auditory brainstem

Cells in the anteroventral cochlear nucleus (aVCN) send out calyceal axons that form large excitatory somatic terminals, the calyces of Held, onto principal cells of the contralateral medial nucleus of the trapezoid body (MNTB). It is unclear which fraction of these axons might form more than one calyx and whether this fraction changes during development. We combined in vitro anterograde tracing, stereological cell counts, analysis of apoptosis, and immunohistochemistry to study the development of calyceal afferents in rats of different postnatal ages. We found that some principal cells were contacted by multiple large axosomatic inputs, but these invariably originated from the same axon. Conversely, at least 18% of traced afferents branched to form multiple calyces, independently of age. Calyces from the same axon generally innervated nearby principal cells, and most of these branch points were <50 microm away from the synaptic terminals. Our results show that the projection from the aVCN to the MNTB is divergent, both when calyces have just been formed and in the adult. Cell counts did not provide evidence for principal cell loss during development, although analysis of apoptosis showed a large increase in nonneuronal cell death around the onset of hearing. Our data suggest that, once a calyceal synapse forms in the MNTB, it stays.

Role of Ca(2+) in the synchronization of transmitter release at calyceal synapses in the auditory system of rat.

The synchronization of transmitter release in the synapse of the medial nucleus of the trapezoid body (MNTB) is achieved during early postnatal development as a consequence of elimination of delayed asynchronous releases and appears to reflect changes in the dynamics of Ca(2+) entry and clearance. To examine the role of Ca(2+) in regulating synchronization of transmitter release in the mature synapse (after postnatal day 9, P9), we perturbed Ca(2+) dynamics systematically. Replacement of external Ca(2+) (2 mM) with Sr(2+) induced delayed asynchronous release following the major EPSC. We tried to reproduce asynchronous releases without using Sr(2+) and instead by manipulating the time course and the size of Ca(2+) transient in the presynaptic terminal, under the assumption that replacement of external Na(+) with Li(+) or application of eosin-Y would prolong the lifetime of Ca(2+) transient by reducing the rate of Ca(2+) extrusion from the terminal. With application of Li(+), Ca(2+) transient in the terminal was prolonged, the EPSC decay time course was prolonged, and the EPSC amplitude increased. However, these EPSCs were not followed by delayed asynchronous release. When Ca(2+) influx was reduced, either by partial Ca(2+) channel blockade with a low concentration of Cd(2+) or omega-agatoxin IVA, a marked asynchronous release resulted. This was further enhanced by the combined application of Li(+) or eosin-Y. These results suggest that cooperative increases of both Ca(2+) influx and Ca(2+) clearance capacities leading to a sharper Ca(2+) spike in the presynaptic terminal underlie synchronized transmitter release in the presynaptic terminal of the MNTB.

The Role of Perisynaptic Glial Sheaths in Glutamate Spillover and Extracellular Ca 2+ Depletion

Recent findings suggest that rapid activation of extrasynaptic receptors and transient depletion of extracellular Ca 2+ may represent an important component of glutamatergic synaptic transmission. These phenomena imply a previously unrecognized role for synaptic glial sheaths: to retard extracellular diffusion in the synaptic vicinity. The present study is an attempt to assess the extent and physiological implications of this retardation using a detailed compartmental model of the typical synaptic environment. The model allows reconstruction of a partial (asymmetric) glial sheath covered with transporter molecules, which gives a more realistic representation of the vicinity of central synapses. Simulations show to what extent, in conditions compatible with physiology, the occupancy of synaptic receptors and the depletion of Ca 2+ in the cleft increase with increased glial coverage. The impact of glial sheaths on synaptic transmission is shown to become greater with smaller synapses and with slower kinetics of perisynaptic ion transients. At a calyceal synapse, a profound temporal filtering of fast Ca 2+ influx is found, and similar phenomena are predicted to occur following simultaneous activation of multiple synapses in the neuropil. The results provide a quantitative guidance for interpretation of physiological experiments that address fast transients of neurotransmitters and small ions in the brain tissue.

Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse.

Glycine and GABAA (gamma-aminobutyric acid A) receptors are inhibitory neurotransmitter-gated Cl- channels localized in postsynaptic membranes. In some cases, GABAA receptors are also found presynaptically, but they retain their inhibitory effect as their activation reduces excitatory transmitter release. Here we report evidence for presynaptic ionotropic glycine receptors, using pre- and postsynaptic recordings of a calyceal synapse in the medial nucleus of the trapezoid body (MNTB). Unlike the classical action of glycine, presynaptic glycine receptors triggered a weakly depolarizing Cl- current in the nerve terminal. The depolarization enhanced transmitter release by activating Ca2+ channels and increasing resting intraterminal Ca2+ concentrations. Repetitive activation of glycinergic synapses on MNTB neurons also enhanced glutamatergic synaptic currents, indicating that presynaptic glycine receptors are activated by glycine spillover. These results reveal a novel site of action of the transmitter glycine, and indicate that under certain conditions presynaptic Cl- channels may increase transmitter release.

Release probability modulates short-term plasticity at a rat giant terminal.

1. Modulation of release probability is a major factor underlying short-term synaptic plasticity in the central nervous system. We have investigated the relationship between release probability ((Pr) and paired-pulse modulation at a large auditory calyceal synapse containing many transmitter release sites. Whole-cell patch electrode recordings were made of excitatory postsynaptic currents (EPSCs), evoked by stimulation of auditory nerve fibres giving rise to the endbulbs of Held. 2. Quantitative estimates of Pr and quantal amplitude were obtained using the recently developed variance-mean analysis technique. Release probability conditions were modulated by bath application of cadmium, elevated calcium and protein kinase C activation by phorbol esters. 3. Our results show that, under physiological conditions, most sites released neurotransmitter following a single presynaptic nerve impulse, with a mean Pr of 0.6. The mean quantal amplitude was 44 pA, which was consistent with the mean amplitude of miniature EPSCs (47 pA). 4. Under high release probability conditions with elevated calcium or phorbol esters, Pr at all sites approached 1.0. At these high Pr values, variance-mean analysis indicated a significant postsynaptic contribution to paired-pulse depression. The miniature EPSC amplitudes were decreased following stimulation in elevated calcium, confirming a postsynaptic component of paired-pulse depression at this glutamatergic connection. 5. A notable feature was the large variability between neurons in the relationship between paired-pulse ratio and Pr. Based on current models of vesicle release and ultrastructural evidence, we suggest that this variability may be partly due to morphological differences between endbulb specializations, particularly in the ratio of fusion-ready to reserve populations of vesicles at endbulb release sites.

A novel presynaptic inhibitory mechanism underlies paired pulse depression at a fast central synapse.

Several distinct mechanisms may cause synaptic depression, a common form of short-term synaptic plasticity. These include postsynaptic receptor desensitization, presynaptic depletion of releasable vesicles, or other presynaptic mechanisms depressing vesicle release. At the endbulb of Held, a fast central calyceal synapse in the auditory pathway, cyclothiazide (CTZ) abolished marked paired pulse depression (PPD) by acting presynaptically to enhance transmitter release, rather than by blocking postsynaptic receptor desensitization. PPD and its response to CTZ were not altered by prior depletion of the releasable vesicle pool but were blocked by lowering external calcium concentration, while raising external calcium enhanced PPD. We conclude that a major component of PPD at the endbulb is due to a novel, transient depression of release, which is dependent on the level of presynaptic calcium entry and is CTZ sensitive.

Inhibition of transmitter release shortens the duration of the excitatory synaptic current at a calyceal synapse.

1. We investigated the effect of reducing transmitter release on the time course of multiquantal, evoked synaptic currents to test for transmitter "cross talk" between neighboring synaptic release sites within a calyceal synapse. By using a brain slice preparation, neurons in the chick nucleus magnocellularis (nMAG) were voltage clamped and individual presynaptic axons were stimulated to evoke excitatory postsynaptic currents (EPSCs). 2. Application of 100-microM baclofen or 50-microM GABA in the presence of a gamma-aminobutyric acid-A (GABAA) receptor antagonist produced an 85% reduction of EPSCs, consistent with the activation of presynaptic gamma-aminobutyric acid-B (GABAB) receptors. In parallel with the reduction in the amplitude of the EPSC by GABAB receptor activation, the normally strong paired pulse depression (PPD) of the EPSC was converted to facilitation. The reduction in EPSC amplitude by gamma-aminobutyric acid (GABA) or baclofen was accompanied by a 20% reduction in the exponential time constant of decay of the EPSC. Weaker effects on the EPSC time course were observed for synapses with the least PPD. 3. Cd2+ (5 microM), which inhibits presynaptic calcium current, also reduced EPSC amplitude by 85% and converted PPD to facilitation. EPSCs were narrower in Cd2+, though less so than in baclofen. 4. The time course of the EPSC was longer than that of miniature synaptic currents, even after significant block by baclofen, GABA or Cd2+, indicating that dispersion of quantal release may help shape the synaptic waveform. However, the narrowing of the EPSC by baclofen, GABA, and Cd2+ suggests that high levels of quantal release at the calyceal synapse may delay the removal of transmitter, further slowing the EPSC.

Delayed clearance of transmitter and the role of glutamate transporters at synapses with multiple release sites

The roles of glutamate diffusion, uptake, and channel kinetics in shaping the AMPA receptor EPSC were examined at a calyceal synapse. The EPSC decay was described by three exponential components: two matching desensitizing channel kinetics, and a third component at least 10 times slower. The slowest component had identical voltage dependence to the steady-state AMPA current and was selectively increased and prolonged by blockade of glutamate uptake, indicating that the slow EPSC represented rebinding of glutamate at partially desensitized AMPA receptors. The data were in strong agreement with the predictions of a model of transmitter diffusion from multiple release sites into a large synaptic cleft. Within the first millisecond after release, transmitter concentrations in the cleft fell below millimolar levels, causing the fastest parts of the EPSC to be shaped by channel kinetics. The slowest component was determined by the removal over tens of milliseconds of the final 10-100 microM glutamate by diffusion and uptake. The data and modeling indicate that transmitter uptake and cooperation between release sites are significant determinants of a slow "tail" of glutamate in the synaptic cleft. This slow clearance of glutamate is likely to limit postsynaptic receptor availability through desensitization.

Desensitization of AMPA receptors upon multiquantal neurotransmitter release

We have investigated the role of AMPA receptor desensitization during transmission at a calyceal synapse. Cyclothiazide blocked the rapid desensitization of AMPA receptors and markedly prolonged the decay time of the evoked excitatory postsynaptic current (PSC). This effect was greater than what would be expected from a simple prolongation of channel open time. Additionally, the drug reduced the depression of PSCs evoked at brief intervals. The effects of cyclothiazide on the PSC were reduced when the level of transmitter release was lowered. These data indicate that AMPA receptors are desensitized by neurotransmitter and that this desensitization depends on the number of quanta in the synaptic cleft. We propose that release of transmitter from many closely spaced sites prolongs the time of receptor-transmitter contact and thereby promotes desensitization. Desensitization may therefore contribute to synaptic depression and prevent the interaction of transmitter quanta within the synaptic cleft.

The calyceal synapse of type I vestibular hair cells

The type I vestibular hair cell, except as its apex, is entirely surrounded by a nerve calyx. In the basal region of the hair cell two distinct junctional complexes exist. In one, the intercellular space is wide (250–350 ) and contains finely granular material. Synaptic ribbons are distributed more or less randomly along this junction. In the other, fusion, or very close apposition, of membranes occurs. It is proposed that junctional complexes containing synaptic ribbons are chemical synapses and that the areas of membrane fusion are electrical synapses. Synaptic modifications are absent along the neck of the hair cell.