Presynaptic plasticity at two giant auditory synapses in normal and deaf mice

Large calyceal synapses are often regarded as simple relay points, built for high-fidelity and high-frequency synaptic transmission and a minimal requirement for synaptic plasticity, but this view is oversimplified. Calyceal synapses can exhibit surprising activity-dependent developmental plasticity. Here we compare basal synaptic transmission and activity-dependent plasticity at two stereotypical calyceal synapses in the auditory pathway, the endbulb and the calyx of Held. Basal synaptic transmission was more powerful at the calyx than the endbulb synapse: the amplitude of evoked AMPA receptor-mediated excitatory postsynaptic currents (eEPSCs) was significantly greater at the calyx, as were the release probability, and the number of release sites. The quantal amplitude was smaller at the calyx, consistent with the smaller amplitude of spontaneous miniature EPSCs at this synapse. High-frequency trains of stimuli revealed that the calyx had a larger readily releasable pool of vesicles (RRP), less tetanic depression and less asynchronous transmitter release. Activity-dependent synaptic plasticity was assessed in congenitally deaf mutant mice ( dn/dn ). Previously we showed that a lack of synaptic activity in deaf mice increases synaptic strength at the endbulb of Held via presynaptic mechanisms. In contrast, we have now found that deafness does not affect synaptic transmission at the calyx synapse, as eEPSC and mEPSC amplitude, release probability, number of release sites, size of RRP, tetanic depression and asynchronous release were unchanged compared to normal mice. Synaptic transmission at the calyx synapse is more powerful and has less capacity for developmental plasticity compared to the endbulb synapse.     

Effects of congenital deafness in the cochlear nuclei of Shaker-2 mice: an ultrastructural analysis of synapse morphology in the endbulbs of Held

It is well established that manipulation of the sensory environment can significantly alter central auditory system development. For example, congenitally deaf white cats exhibit synaptic alterations in the cochlear nucleus distinct from age-matched, normal hearing controls. The large, axosomatic endings of auditory nerve fibers, called endbulbs of Held, display reduced size and branching, loss of synaptic vesicles, and a hypertrophy of the associated postsynaptic densities on the target spherical bushy cells. Such alterations, however, could arise from the cat's genetic syndrome rather than from deafness. In order to examine further the role of hearing on synapse development, we have studied endbulbs of Held in the shaker-2 ( sh2 ) mouse. These mice carry a point mutation on chromosome 11, affecting myosin 15 and producing abnormally short stereocilia in hair cells of the inner ear. The homozygous mutant mice are born deaf and develop perpetual circling behavior, although receptor cells and primary neurons remain intact at least for the initial 100 days of postnatal life. Endbulbs of Held in 7-month old, deaf sh2 mice exhibited fewer synaptic vesicles in the presynaptic ending, the loss of intercellular cisternae, and a hypertrophy of associated postsynaptic densities. On average, postsynaptic density area for sh2 endbulbs was 0.23 +/- 0.19 microm(2) compared to 0.07 +/- 0.04 microm(2) ( p < 0.001) for age-matched, hearing littermates. These changes at the endbulb synapse in sh2 mice resemble those of the congenitally deaf white cat and are consistent with the idea that they represent a generalized response to deafness.     

Synaptic transmission at the cochlear nucleus endbulb synapse during age-related hearing loss in mice

Age-related hearing loss (AHL) typically starts from high-frequency regions of the cochlea and over time invades lower-frequency regions. During this progressive hearing loss, sound-evoked activity in spiral ganglion cells is reduced. DBA mice have an early onset of AHL. In this study, we examined synaptic transmission at the endbulb of Held synapse between auditory nerve fibers and bushy cells in the anterior ventral cochlear nucleus (AVCN). Synaptic transmission in hearing-impaired high-frequency areas of the AVCN was altered in old DBA mice. The spontaneous miniature excitatory postsynaptic current (mEPSC) frequency was substantially reduced (about 60%), and mEPSCs were significantly slower (about 115%) and smaller (about 70%) in high-frequency regions of old (average age 45 days) DBA mice compared with tonotopically matched regions of young (average age 22 days) DBA mice. Moreover, synaptic release probability was about 30% higher in high-frequency regions of young DBA than that in old DBA mice. Auditory nerve-evoked EPSCs showed less rectification in old DBA mice, suggesting recruitment of GluR2 subunits into the AMPA receptor complex. No similar age-related changes in synaptic release or EPSCs were found in age-matched, normal hearing young and old CBA mice. Taken together, our results suggest that auditory nerve activity plays a critical role in maintaining normal synaptic function at the endbulb of Held synapse after the onset of hearing. Auditory nerve activity regulates both presynaptic (release probability) and postsynaptic (receptor composition and kinetics) function at the endbulb synapse after the onset of hearing.     

Post-tetanic potentiation in the rat calyx of Held synapse

We studied synaptic plasticity in the calyx of Held synapse, an axosomatic synapse in the auditory brainstem, by making whole-cell patch clamp recordings of the principal cells innervated by the calyces in a slice preparation of 7- to 10-day-old rats. A 5 min 20 Hz stimulus train increased the amplitude of excitatory postsynaptic currents (EPSCs) on average more than twofold. The amplitude of the synaptic currents took several minutes to return to control values. The post-tetanic potentiation (PTP) was accompanied by a clear increase in the frequency, but not the amplitude, of spontaneous EPSCs, which returned to baseline more rapidly than the potentiation of evoked release. The size of the readily releasable pool of vesicles was increased by about 30%. In experiments in which presynaptic measurements of the intracellular calcium concentration were combined with postsynaptic voltage clamp recordings, PTP was accompanied by an increase in the presynaptic calcium concentration to about 210 n m . The decay of the PTP matched the decay of this increase. When the decay of the calcium transient was shortened by dialysing the terminal with EGTA, the PTP decay sped up in parallel. Our experiments suggest that PTP at the calyx of Held synapse is due to a long-lasting increase in the presynaptic calcium concentration following a tetanus, which results in an increase in the release probability of the vesicles of the readily releasable pool. Although part of the PTP can be explained by a direct activation of the calcium sensor for phasic release, other mechanisms are likely to contribute as well.     

An increase in calcium influx contributes to post-tetanic potentiation at the rat calyx of Held synapse

We studied the contribution of a change in presynaptic calcium influx to posttetanic potentiation (PTP) in the calyx of Held synapse, an axosomatic synapse in the auditory brain stem. We made whole cell patch-clamp recordings of a principal cell after loading of the presynaptic terminal with a calcium dye. After induction of PTP by a high-frequency train of afferent stimuli, the Fluo-4 fluorescence transients evoked by an action potential became on average 15 +/- 4% larger (n = 7). Model predictions did not match the fluorescence transients evoked by trains of brief calcium currents unless the endogenous calcium buffer had low affinity for calcium, making a contribution of saturation of the endogenous buffer to the synaptic potentiation we observed in the present experiments less likely. Our data therefore suggest that the increase of release probability during PTP at the calyx of Held synapse is largely explained by an increase in the calcium influx per action potential.     

Endogenous activation of adenosine A1 receptors, but not P2X receptors, during high-frequency synaptic transmission at the calyx of Held

Activation of presynaptic receptors plays an important role in modulation of transmission at many synapses, particularly during high-frequency trains of stimulation. Adenosine-triphosphate (ATP) is coreleased with several neurotransmitters and acts at presynaptic sites to reduce transmitter release; such presynaptic P2X receptors occur at inhibitory and excitatory terminals in the medial nucleus of the trapezoid body (MNTB). We have investigated the mechanism of purinergic modulation during high-frequency repetitive stimulation at the calyx of Held synapse. Suppression of calyceal excitatory postsynaptic currents (EPSCs) by ATP and ATPgammaS (100 microM) was mimicked by adenosine application and was blocked by DPCPX (10 microM), indicating mediation by adenosine A1 receptors. DPCPX enhanced EPSC amplitudes during high-frequency synaptic stimulation, suggesting that adenosine has a physiological role in modulating transmission at the calyx. The Luciferin-Luciferase method was used to probe for endogenous ATP release (at 37 degrees C), but no release was detected. Blockers of ectonucleotidases also had no effect on endogenous synaptic depression, suggesting that it is adenosine acting on A1 receptors, rather than degradation of released ATP, which accounts for presynaptic purinergic suppression of synaptic transmission during physiological stimulus trains at this glutamatergic synapse.     

Estimation of quantal parameters at the calyx of Held synapse

The calyx of Held has recently emerged as a convenient model system to study CNS synapses. In order to understand the mechanisms of synaptic transmission and short-term synaptic plasticity, quantal parameters and their changes should be estimated precisely. For this purpose, various methods have been applied to the calyx of Held synapse. The results confirm many aspects of the early findings on transmission at the neuromuscular junction. On the other hand, the simplest quantal hypothesis does not work at the calyx of Held, because of additional factors such as heterogeneous release probability of synaptic vesicles, intra- and intersite quantal variability, an overlap of facilitation and depression of transmitter release, changes in quantal sizes due to desensitization and saturation of postsynaptic receptors, and delayed clearance of transmitter from the synaptic cleft. These factors should always be taken into account for fully understanding the mechanisms of synaptic transmission and plasticity.     

Developmental profiles of glutamate receptors and synaptic transmission at a single synapse in the mouse auditory brainstem

Using whole-cell recordings from presynaptic terminals and postsynaptic principal neurons in the mouse medial nucleus of the trapezoid body (MNTB), we have characterized properties of the calyx of Held synapse during the first three postnatal weeks. We observed that evoked excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors (NMDAR) increased until postnatal day 11/12 (P11/12) after which they declined to very low or undetectable levels at P16. Meanwhile, EPSCs mediated by AMPA receptors (AMPAR) showed an approximate three-fold increase in amplitude. These changes were paralleled by NMDAR and AMPAR currents evoked by exogenous NMDA and kainate to MNTB neurons except that whole-cell kainate currents remained constant after P7/8 while AMPAR-EPSCs continued to increase. We found that the decay time constant τ for NMDAR-EPSCs and AMPAR-EPSCs declined by about 30 % and 70 %, respectively. Analyses of NMDAR-EPSCs with subunit-specific pharmacological agents including ifenprodil, N,N,N',N' -tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), zinc and Mg²⁺ revealed subtle developmental changes in subunit composition. As maturation progressed, this synapse displayed a reduction in the number of presynaptic spike failures and the extent of synaptic depression in response to trains of stimuli (50–300 Hz) while the recovery rate from depression accelerated. These results demonstrate profound changes in the size and kinetics of postsynaptic glutamate receptors and in the spike-firing capability of presynaptic terminals at the calyx of Held-MNTB synapse during early development. We suggest that these concurrent presynaptic and postsynaptic adaptations represent important steps for synapse consolidation and refinement and ultimately for the development of fast high-fidelity transmission at this synapse.     

The pool of fast releasing vesicles is augmented by myosin light chain kinase inhibition at the calyx of Held synapse

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca²⁺ currents or action potential waveform. Moreover, Ca²⁺ imaging experiments showed that [Ca²⁺]_i transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca²⁺ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.     

Noradrenaline increases high-frequency firing at the calyx of held synapse during development by inhibiting glutamate release

The mammalian auditory brain stem receives profuse adrenergic innervation, whose function is poorly understood. Here we investigate, during postnatal development, the effect of noradrenaline (NA) at the calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB). We observed that NA inhibits the large glutamatergic EPSC, evoked by afferent fiber stimulation, in a dose-dependent manner. The inhibition was maximal (approximately 48%) at the concentration of 2 microM. It was antagonized by yohimbine and mimicked by the alpha2-adrenergic specific agonist UK14304. Both AMPA and NMDA receptor-mediated EPSCs were inhibited in parallel by NA, suggesting a presynaptic effect. Presynaptic recordings showed that NA inhibits the action potential (AP) generated Ca current by about 20%; however, NA did not significantly affect the presynaptic AP waveform. We thus conclude that the calyx of Held presynaptic terminal expresses alpha2-adrenergic receptors that inhibit its Ca current and thus glutamate release. Noradrenaline was effective in all cells tested from postnatal days 6 to 7 (P6-P7), and thereafter the number of responsive cells diminished, although half of the P14 cells tested still had EPSCs that were inhibited by NA. By contrast, activation by L-2-amino-5-phosphonovaleric acid-sensitive metabotropic glutamate receptors strongly inhibited the EPSCs of all cells tested from P6 to P14. The effect of NA on postsynaptic action potential firing was dependent on the stimulus frequency. At 10 Hz, NA had no effect on firing probability; however, NA helped MNTB cells fire more action potentials during a 100-Hz train of stimuli, even though it did not increase the steady-state depressed EPSC, because it produced a smaller N-methyl-D-aspartate (NMDA) receptor-activated depolarizing plateau. We therefore suggest that the reduction by NA of the first few EPSCs in a train leads to a smaller NMDA depolarizing plateau and thus to increased firing probability at 100 Hz in young synapses. Surprisingly, the inhibition of glutamate release by NA can thus actually increase the excitability of MNTB neurons during early postnatal development.     

Synaptic transmission at the calyx of Held under in vivo like activity levels

One of the hallmarks of auditory neurons in vivo is spontaneous activity that occurs even in the absence of any sensory stimuli. Sound-evoked bursts of discharges are thus embedded within this background of random firing. The calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) has been characterized in vitro as a fast relay that reliably fires at high stimulus frequencies (< or =800 Hz). However, inherently due to the preparation method, spontaneous activity is absent in studies using brain stem slices. Here we first determine in vivo spontaneous firing rates of MNTB principal cells from Mongolian gerbils and then reintroduce this random firing to in vitro gerbil brain stem synapses at near-physiological temperature. After conditioning synapses with afferent fiber stimulation for 2 min at Poisson averaged rates of 20, 40, and 60 Hz, we observed a number of differences in the properties of synaptic transmission between conditioned and unconditioned synapses. Foremost, we observed reduced steady-state EPSC amplitudes that depressed even further during an embedded short-stimulation train of 100, 300, or 600 Hz (a protocol that thus simulates in vitro what probably occurs at the in vivo MNTB after a short sound stimulus in a silent background). Accordingly, current-clamp, dynamic-clamp, and loose-patch recordings revealed a number of action potential failures at the postsynaptic cell during high-frequency-stimulation trains, although the initial onset of evoked activity was still transmitted with higher fidelity. We thus propose that some in vivo auditory synapses are in a tonic state of reduced EPSC amplitudes as a consequence of high spontaneous spiking and this in vivo-like conditioning has important consequences for the encoding of signals throughout the auditory pathway.     

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 m m ) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36–37°C), the qEPSC amplitude increased when intraterminal l -glutamate concentration was elevated from 1 m m to 10 m m , but it reached a plateau at 10 m m . 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 m m to 100 m m , even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 μ m ). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 n m ; 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.     

Adenosine A1 receptor-mediated presynaptic inhibition at the calyx of Held of immature rats

At the calyx of Held synapse in brainstem slices of 5- to 7-day-old (P5–7) rats, adenosine, or the type 1 adenosine (A₁) receptor agonist N ⁶-cyclopentyladenosine (CPA), inhibited excitatory postsynaptic currents (EPSCs) without affecting the amplitude of miniature EPSCs. The A₁ receptor antagonist 8-cyclopentyltheophylline (CPT) had no effect on the amplitude of EPSCs evoked at a low frequency, but significantly reduced the magnitude of synaptic depression caused by repetitive stimulation at 10 Hz, suggesting that endogenous adenosine is involved in the regulation of transmitter release. Adenosine inhibited presynaptic Ca²⁺ currents ( I _pCa) recorded directly from calyceal terminals, but had no effect on presynaptic K⁺ currents. When EPSCs were evoked by I _pCa during simultaneous pre- and postsynaptic recordings, the magnitude of the adenosine-induced inhibition of I _pCa fully explained that of EPSCs, suggesting that the presynaptic Ca²⁺ channel is the main target of A₁ receptors. Whereas the N-type Ca²⁺ channel blocker ω-conotoxin attenuated EPSCs, it had no effect on the magnitude of adenosine-induced inhibition of EPSCs. During postnatal development, in parallel with a decrease in the A₁ receptor immunoreactivity at the calyceal terminal, the inhibitory effect of adenosine became weaker. We conclude that presynaptic A₁ receptors at the immature calyx of Held synapse play a regulatory role in transmitter release during high frequency transmission, by inhibiting multiple types of presynaptic Ca²⁺ channels.     

Presynaptic Rat Kv1.2 Channels Suppress Synaptic Terminal Hyperexcitability Following Action Potential Invasion

Voltage-gated K⁺ channels activating close to resting membrane potentials are widely expressed and differentially located in axons, presynaptic terminals and cell bodies. There is extensive evidence for localisation of Kv1 subunits at many central synaptic terminals but few clues to their presynaptic function. We have used the calyx of Held to investigate the role of presynaptic Kv1 channels in the rat by selectively blocking Kv1.1 and Kv1.2 containing channels with dendrotoxin-K (DTX-K) and tityustoxin-Kα (TsTX-Kα) respectively. We show that Kv1.2 homomers are responsible for two-thirds of presynaptic low threshold current, whilst Kv1.1/Kv1.2 heteromers contribute the remaining current. These channels are located in the transition zone between the axon and synaptic terminal, contrasting with the high threshold K⁺ channel subunit Kv3.1 which is located on the synaptic terminal itself. Kv1 homomers were absent from bushy cell somata (from which the calyx axons arise); instead somatic low threshold channels consisted of heteromers containing Kv1.1, Kv1.2 and Kv1.6 subunits. Current-clamp recording from the calyx showed that each presynaptic action potential (AP) was followed by a depolarising after-potential (DAP) lasting around 50 ms. Kv1.1/Kv1.2 heteromers had little influence on terminal excitability, since DTX-K did not alter AP firing. However TsTX-Kα increased DAP amplitude, bringing the terminal closer to threshold for generating an additional AP. Paired pre- and postsynaptic recordings confirmed that this aberrant AP evoked an excitatory postsynaptic current (EPSC). We conclude that Kv1.2 channels have a general presynaptic function in suppressing terminal hyperexcitability during the depolarising after-potential.     

Release kinetics, quantal parameters and their modulation during short-term depression at a developing synapse in the rat CNS

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size ( q ) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5–7 synapses by 10–100 Hz stimulation, q declined rapidly to 40–12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12–14), but quite severe for frequencies ≥ 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of ∼480 ms in P5–7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8–10 quanta s⁻¹ per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of ∼50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.     

Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.     

Kinetics of both synchronous and asynchronous quantal release during trains of action potential-evoked EPSCs at the rat calyx of Held

We studied the kinetics of transmitter release during trains of action potential (AP)-evoked excitatory postsynaptic currents (EPSCs) at the calyx of Held synapse of juvenile rats. Using a new quantitative method based on a combination of ensemble fluctuation analysis and deconvolution, we were able to analyse mean quantal size ( q ) and release rate (ξ) continuously in a time-resolved manner. Estimates derived this way agreed well with values of q and quantal content ( M ) calculated for each EPSC within the train from ensemble means of peak amplitudes and their variances. Separate analysis of synchronous and asynchronous quantal release during long stimulus trains (200 ms, 100 Hz) revealed that the latter component was highly variable among different synapses but it was unequivocally identified in 18 out of 37 synapses analysed. Peak rates of asynchronous release ranged from 0.2 to 15.2 vesicles ms⁻¹ (ves ms⁻¹) with a mean of 2.3 ± 0.6 ves ms⁻¹. On average, asynchronous release accounted for less than 14% of the total number of about 3670 ± 350 vesicles released during 200 ms trains. Following such trains, asynchronous release decayed with several time constants, the fastest one being in the order of 15 ms. The short duration of asynchronous release at the calyx of Held synapse may aid in generating brief postsynaptic depolarizations, avoiding temporal summation and preserving action potential timing during high frequency bursts.     

Multiple large inputs to principal cells in the mouse medial nucleus of the trapezoid body

The calyx of Held is a giant nerve terminal that forms a synapse directly onto the principal cells of the medial nucleus of the trapezoid body (MNTB) in the mammalian auditory brain stem. This central synapse, which is involved in sound localization, has become widely used for studying synaptic transmission. Anatomical studies of this nucleus have indicated that each principal cell is innervated by only one calyx. Here we use previously established electrophysiological criteria of excitatory postsynaptic current amplitude, kinetics, and transmitter type, as well as other characteristics commonly reported for this synapse, to examine the input properties of principal neurons. Our findings indicate that some principal cells receive more than one strong excitatory input. These inputs meet previously established electrophysiological criteria for identification as calyceal nerve terminals. Implications for the execution and analysis of experiments to avoid errors due to such multiple inputs are discussed.     

Interactions between multiple sources of short-term plasticity during evoked and spontaneous activity at the rat calyx of Held

Sustained activity at most central synapses is accompanied by a number of short-term changes in synaptic strength which act over a range of time scales. Here we examine experimental data and develop a model of synaptic depression at the calyx of Held synaptic terminal that combines many of these mechanisms (acting at differing sites and across a range of time scales). This new model incorporates vesicle recycling, facilitation, activity-dependent vesicle retrieval and multiple mechanisms affecting calcium channel activity and release probability. It can accurately reproduce the time course of experimentally measured short-term depression across different stimulus frequencies and exhibits a slow decay in EPSC amplitude during sustained stimulation. We show that the slow decay is a consequence of vesicle release inhibition by multiple mechanisms and is accompanied by a partial recovery of the releasable vesicle pool. This prediction is supported by patch-clamp data, using long duration repetitive EPSC stimulation at up to 400 Hz. The model also explains the recovery from depression in terms of interaction between these multiple processes, which together generate a stimulus-history-dependent recovery after repetitive stimulation. Given the high rates of spontaneous activity in the auditory pathway, the model also demonstrates how these multiple interactions cause chronic synaptic depression under in vivo conditions. While the magnitude of the depression converges to the same steady state for a given frequency, the time courses of onset and recovery are faster in the presence of spontaneous activity. We conclude that interactions between multiple sources of short-term plasticity can account for the complex kinetics during high frequency stimulation and cause stimulus-history-dependent recovery at this relay synapse.