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Key Points: During development the giant, auditory calyx of Held forms a one-to-one connection with a principal neuron of the medial nucleus of the trapezoid body. While anatomical studies described that most of the target cells are temporarily contacted by multiple calyces, multi-calyceal innervation was only sporadically observed in in vivo recordings, suggesting a structure-function discrepancy. We correlated synaptic strength of inputs, identified in in vivo recordings, with post hoc labelling of the recorded neuron and synaptic terminals containing vesicular glutamate transporters (VGluT). During development only one input increased to the level of the calyx of Held synapse, and its strength correlated with the large VGluT cluster contacting the postsynaptic soma. As neither competing strong inputs nor multiple large VGluT clusters on a single cell were observed, our findings did not indicate a structure-function discrepancy., Abstract: In adult rodents, a principal neuron in the medial nucleus of the trapezoid (MNTB) is generally contacted by a single, giant axosomatic terminal called the calyx of Held. How this one-on-one relation is established is still unknown, but anatomical evidence suggests that during development principal neurons are innervated by multiple calyces, which may indicate calyceal competition. However, in vivo electrophysiological recordings from principal neurons indicated that only a single strong synaptic connection forms per cell. To test whether a mismatch exists between synaptic strength and terminal size, we compared the strength of synaptic inputs with the morphology of the synaptic terminals. In vivo whole-cell recordings of the MNTB neurons from newborn Wistar rats of either sex were made while stimulating their afferent axons, allowing us to identify multiple inputs. The strength of the strongest input increased to calyceal levels in a few days across cells, while the strength of the second strongest input was stable. The recorded cells were subsequently immunolabelled for vesicular glutamate transporters (VGluT) to reveal axosomatic terminals with structured-illumination microscopy. Synaptic strength of the strongest input was correlated with the contact area of the largest VGluT cluster at the soma (r = 0.8), and no indication of a mismatch between structure and strength was observed. Together, our data agree with a developmental scheme in which one input strengthens and becomes the calyx of Held, but not with multi-calyceal competition., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The shape of the presynaptic action potential (AP) has a strong impact on neurotransmitter release. Because of the small size of most terminals in the central nervous system, little is known about the regulation of their AP shape during natural firing patterns in vivo. The calyx of Held is a giant axosomatic terminal in the auditory brainstem, whose biophysical properties have been well studied in slices. Here, we made whole-cell recordings from calyceal terminals in newborn rat pups. The calyx showed a characteristic burst firing pattern, which has previously been shown to originate from the cochlea. Surprisingly, even for frequencies over 200 Hz, the AP showed little or no depression. Current injections showed that the rate of rise of the AP depended strongly on its onset potential, and that the membrane potential after the AP ( V after ) was close to the value at which no depression would occur during high-frequency activity. Immunolabeling revealed that Na v 1.6 is already present at the calyx shortly after its formation, which was in line with the fast recovery from AP depression that we observed in slice recordings. Our findings thus indicate that fast recovery from depression and an inter-AP membrane potential that minimizes changes on the next AP in vivo, together enable high timing precision of the calyx of Held already shortly after its formation., Competing Interests: The authors declare no conflict of interest.

Key Points: Neurons in the medial nucleus of the trapezoid body of anaesthetized rats of postnatal day (P)2-6 showed burst firing with a preferred interval of about 100 ms, which was stable, and a second preferred interval of 5-30 ms, which shortened during development. In 3 out of 132 cases, evidence for the presence of two large inputs was found. In vivo whole-cell recordings revealed that the excitability of the principal neuron and the size of its largest synaptic inputs were developmentally matched. At P2-4, action potentials were triggered by barrages of small synaptic events that summated to plateau potentials, while at later stages firing depended on a single, large and often prespike-associated input, which is probably the nascent calyx of Held. Simulations with a Hodgkin-Huxley-like model, which was based on fits of the intrinsic postsynaptic properties, suggested an essential role for the low-threshold potassium conductance in this transition., Abstract: In the adult, principal neurons of the medial nucleus of the trapezoid body (MNTB) are typically contacted by a single, giant terminal called the calyx of Held, whereas during early development a principal neuron receives inputs from many axons. How these changes in innervation impact the postsynaptic activity has not yet been studied in vivo. We therefore recorded spontaneous inputs and intrinsic properties of principal neurons in anaesthetized rat pups during the developmental period in which the calyx forms. A characteristic bursting pattern could already be observed at postnatal day (P)2, before formation of the calyx. At this age, action potentials (APs) were triggered by barrages of summating EPSPs causing plateau depolarizations. In contrast, at P5, a single EPSP reliably triggered APs, resulting in a close match between pre- and postsynaptic firing. Postsynaptic excitability and the size of the largest synaptic events were developmentally matched. The developmental changes in intrinsic properties were estimated by fitting in vivo current injections to a Hodgkin-Huxley-type model of the principal neuron. Our simulations indicated that the developmental increases in I h , low-threshold K + channels and leak currents contributed to the reduction in postsynaptic excitability, but that low-threshold K + channels specifically functioned as a dampening influence in the near-threshold range, thus precluding small inputs from triggering APs. Together, these coincident changes help to propagate bursting activity along the auditory brainstem, and are essential steps towards establishing the relay function of the calyx of Held synapse., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Specific missense mutations in the CACNA1A gene, which encodes a subunit of voltage-gated CaV2.1 channels, are associated with familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of common migraine with aura. We used transgenic knock-in (KI) mice harboring the human pathogenic FHM1 mutation S218L to study presynaptic Ca(2+) currents, EPSCs, and in vivo activity at the calyx of Held synapse. Whole-cell patch-clamp recordings of presynaptic terminals from S218L KI mice showed a strong shift of the calcium current I-V curve to more negative potentials, leading to an increase in basal [Ca(2+)]i, increased levels of spontaneous transmitter release, faster recovery from synaptic depression, and enhanced synaptic strength despite smaller action-potential-elicited Ca(2+) currents. The gain-of-function of transmitter release of the S218L mutant was reproduced in vivo, including evidence for an increased release probability, demonstrating its relevance for glutamatergic transmission. This synaptic phenotype may explain the misbalance between excitation and inhibition in neuronal circuits resulting in a persistent hyperexcitability state and other migraine-relevant mechanisms such as an increased susceptibility to cortical spreading depression., (Copyright © 2014 the authors 0270-6474/14/347047-12$15.00/0.)

The calyx of Held synapse of the medial nucleus of the trapezoid body functions as a relay synapse in the auditory brainstem. In vivo recordings have shown that this synapse displays low release probability and that the average size of synaptic potentials does not depend on recent history. We used a ventral approach to make in vivo extracellular recordings from the calyx of Held synapse in rats aged postnatal day 4 (P4) to P29 to study the developmental changes that allow this synapse to function as a relay. Between P4 and P8, we observed evidence for the presence of large short-term depression, which was counteracted by short-term facilitation at short intervals. Major changes occurred in the last few days before the onset of hearing for air-borne sounds, which happened at P13. The bursting pattern changed into a primary-like pattern, the amount of depression and facilitation decreased strongly, and the decay of facilitation became much faster. Whereas short-term plasticity was the most important cause of variability in the size of the synaptic potentials in immature animals, its role became minor around hearing onset and afterward. Similar developmental changes were observed during stimulation experiments both in brain slices and in vivo following cochlear ablation. Our data suggest that the strong reduction in release probability and the speedup of the decay of synaptic facilitation that happen just before hearing onset are important events in the transformation of the calyx of Held synapse into an auditory relay synapse.

The calyx of Held synapse is a giant synapse in the medial nucleus of the trapezoid body (MNTB) of the ventral brainstem, which is involved in sound localization. Although it has many release sites, it can show transmission failures and display an increase in synaptic delay during high-frequency signalling. Its apparent lack of reliability and precision raises the question whether this synapse makes a sizeable contribution to tone adaptation, the decline in response to sustained or repetitive auditory stimuli. We observed evidence for the presence of both ipsilateral and contralateral inhibition, but these effects were already present in the inputs to the MNTB, suggesting that synaptic inhibition within the MNTB does not contribute to tone adaptation. During trains of brief tones at variable intervals, there were no clear changes in reliability or precision at tone intervals of 20 ms or longer. A progressive decrease in the number of spikes measured in the MNTB was observed at shorter tone intervals, but this decrease largely originated upstream from the MNTB. In addition, for tones with short intervals, during the train a progressive increase in first-spike latencies was observed, but much smaller changes were observed in the delay between excitatory postsynaptic potentials and postsynaptic action potentials within the MNTB. We conclude that despite the failures and variability in synaptic delay that are present at the calyx of Held synapse, their contribution to tone adaptation is relatively small compared with upstream factors., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The calyx of Held synapse is a giant axosomatic synapse that acts as a fast relay in the sound localization circuit of the brainstem. In rodents it forms within a relatively brief period starting around the second postnatal day (P2). The relative timing of the formation of its pre- and the postsynaptic compartment are not yet known. By means of fluorescent immunohistochemistry in neonatal rats we therefore compared the developmental expression patterns of the vesicular glutamate transporter (VGLUT)-1 and the postsynaptic density scaffolding proteins Homer-1 and PSD-95 in the medial nucleus of the trapezoid body (MNTB). Before its formation, colocalized punctate staining of VGLUT-1 and Homer-1 or PSD-95 was observed on principal neurons, in agreement with earlier work showing that they are already innervated by fibers from the cochlear nucleus before the calyx forms. The expression of VGLUT-1 clusters within the nascent calyx of Held synapse preceded the expression of Homer-1 and PSD-95 clusters, as indicated by the presence of principal neurons with only a large contiguous cluster (LCC) of VGLUT-1 at P2-3, whereas no neurons with only an LCC for Homer-1 or PSD-95 were seen. At P3 the first principal neurons with both a pre- and a postsynaptic LCC were observed, and at P12 all principal neurons had both a pre- and a postsynaptic LCC. The relatively late appearance of Homer-1 and PSD-95 within the developing calyx of Held synapse suggests that they play a role in its stabilization and the recruitment of additional receptors to its postsynaptic density., (Copyright © 2010 Wiley-Liss, Inc.)

Traditionally, the calyx of Held synapse is viewed as a highly reliable relay in the sound localization circuit of the auditory brainstem, with every presynaptic action potential triggering a postsynaptic action potential in vivo. However, this view is at odds with slice recordings that report large short-term depression (STD). To investigate the reliability and precision of this synapse, we compared slice and in vivo recordings from medial nucleus of the trapezoid body neurons of young adult mice. We show that the extracellularly recorded complex waveform can be used to estimate both presynaptic release and postsynaptic excitability. Whereas under standard slice conditions the synapse underwent large STD, both extracellular and whole-cell recordings indicated that in vivo the size of the EPSPs was independent of recent history. The estimated quantal content was typically <20 in vivo, much lower than in the resting synapse under standard slice conditions. However, due to the large quantal size and summation of EPSPs, the safety factor of this synapse was generally still sufficiently large and postsynaptic failures were observed only infrequently in vivo. When present, failures were typically due to stochastic fluctuations in EPSP size or postsynaptic spike depression. In vivo, the calyx of Held synapse thus functions as a tonic synapse. The price it pays for its low release probability is an increase in jitter and synaptic latency and occasional postsynaptic failures.

The calyx of Held is probably the largest synaptic terminal in the brain, forming a unique one-to-one connection in the auditory ventral brainstem. During early development, calyces have many collaterals, whose function is unknown. Using electrophysiological recordings and fast-calcium imaging in brain slices, we demonstrate that these collaterals are involved in synaptic transmission. We show evidence that the collaterals are pruned and that the pruning already begins 1 week before the onset of hearing. Using two-photon microscopy to image the calyx of Held in neonate rats, we report evidence that both axons and nascent calyces are structurally dynamic, showing the formation, elimination, extension, or retraction of up to 65% of their collaterals within 1 hour. The observed dynamic behavior of axons may add flexibility in the choice of postsynaptic partners and thereby contribute to ensuring that each principal cell eventually is contacted by a single calyx of Held.

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., (J. Comp. Neurol. 496:214-228, 2006. (c) 2006 Wiley-Liss, Inc.)

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 nM. 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.

Vesicle recycling was studied in the rat calyx of Held, a giant brainstem terminal involved in sound localization. Stimulation of brain slices containing the calyx-type synapse with a high extracellular potassium ion concentration in the presence of horseradish peroxidase resulted within several minutes in a reduction of the number of neurotransmitter vesicles and in the appearance of labeled endosome-like structures. After returning to normal solution, the endosome-like structures disappeared over a period of several minutes, whereas simultaneously the number of labeled vesicles increased. A comparison with afferent stimulation suggested that the endosome-like structures normally do not participate in the vesicle cycle. Afferent stimulation at 5 Hz resulted in sustained synaptic transmission, without vesicle depletion but with an estimated endocytotic activity of <0.2 synaptic vesicles per active zone per second. At 20 Hz, the presynaptic action potentials generally failed during prolonged stimulation. In identified synapses, the number of vesicles labeled by photoconversion after stimulation at 5 Hz in the presence of the styryl dye RH414 was much lower than the number of vesicles that were released, as determined by measuring EPSCs. No more than approximately 5% of the vesicles were labeled after 20 min stimulation at 5 Hz, whereas this stimulation protocol was sufficient to largely destain a terminal after previous loading. The results support a scheme for recycling in which two different modes coexist. At physiological demands, a pool of approximately 5% of all vesicles provides sufficient vesicles for release. During intense stimulation, such as occurs in the presence of high extracellular K+, the synapse resorts to bulk endocytosis, a very slow mode of recycling.

The three-dimensional morphology of the axosomatic synaptic structures between a calyx of Held and a principal neuron in the medial nucleus of the trapezoid body (MNTB) in the brainstem of young postnatal day 9 rats was reconstructed from serial ultrathin sections. In the apposition zone between the calyx and the principal neuron two types of membrane specializations were identified: synaptic contacts (SCs) with active zones (AZs) and their associated postsynaptic densities (PSDs) constituted approximately 35% (n = 554) of the specializations; the remaining 65% (n = 1010) were puncta adherentia (PA). Synaptic contacts comprised approximately 5% of the apposition area of presynaptic and postsynaptic membranes. A SC had an average area of 0.100 microm(2), and the nearest neighbors were separated, on average, by 0.59 microm. Approximately one-half of the synaptic vesicles in the calyx were clustered within a distance of 200 nm of the AZ membrane area, a cluster consisting of approximately 60 synaptic vesicles (n = 52 SCs). Approximately two synaptic vesicles per SC were "anatomically docked." Comparing the geometry of the synaptic structure with its previously studied functional properties, we find that during a single presynaptic action potential (AP) (1) approximately 35% of the AZs release a transmitter quantum, (2) the number of SCs and anatomically docked vesicles is comparable with the low estimates of the readily releasable pool (RRP) of quanta, and (3) the broad distribution of PSD areas [coefficient of variation (CV) = 0.9] is likely to contribute to the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and rising phase of the EPSC during a single AP.

Phasic transmitter release at synapses in the mammalian CNS is regulated by local [Ca2+] transients, which control the fusion of readily releasable vesicles docked at active zones (AZs) in the presynaptic membrane. The time course and amplitude of these [Ca2+] transients critically determine the time course and amplitude of the release and thus the frequency and amplitude tuning of the synaptic connection. As yet, the spatiotemporal nature of the [Ca2+] transients and the number and location of release-controlling Ca2+ channels relative to the vesicles, the "topography" of the release sites, have remained elusive. We used a time-dependent model to simulate Ca2+ influx, three-dimensional buffered Ca2+ diffusion, and the binding of Ca2+ to the release sensor. The parameters of the model were constrained by recent anatomical and biophysical data of the calyx of Held. Comparing the predictions of the model with previously measured release probabilities under a variety of experimental conditions, we inferred which release site topography is likely to operate at the calyx: At each AZ one or a few clusters of Ca2+ channels control the release of the vesicles. The distance of a vesicle to the cluster(s) varies across the multiple release sites of a single calyx (ranging from 30 to 300 nm; average approximately 100 nm). Assuming this topography, vesicles in different locations are exposed to different [Ca2+] transients, with peak amplitudes ranging from 0.5 to 40 microm (half-width approximately 400 microsec) during an action potential. Consequently the vesicles have different release probabilities ranging from <0.01 to 1. We demonstrate how this spatially heterogeneous release probability creates functional advantages for synaptic transmission.

Synapses show widely varying degrees of short-term facilitation and depression. Several mechanisms have been proposed to underlie short-term plasticity, but the contributions of presynaptic mechanisms have been particularly difficult to study because of the small size of synaptic boutons in the mammalian brain. Here we review the functional properties of the calyx of Held, a giant nerve terminal that has shed new light on the general mechanisms that control short-term plasticity. The calyx of Held has also provided fresh insights into the strategies used by synapses to extend their dynamic range of operation and preserve the timing of sensory stimuli.

Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca2+]i to approximately 10 micromolar would be sufficient to reproduce the physiological release pattern. Thus, the calcium sensitivity of release at this synapse is high, and the distinction between phasic and delayed release is less pronounced than previously thought.

1. A new form of synaptic depression of excitatory synaptic transmission was observed when making voltage-clamp recordings from large presynaptic terminals, the calyces of Held and postsynaptic cells, the principal cells of the medial nucleus of the trapezoid body (MNTB), in slices of the rat auditory brainstem. 2. A short (100 ms) depolarization of the postsynaptic cell to 0 mV reduced the amplitude of the EPSCs by 35 +/- 5 % (n = 7), measured at 10 ms following the depolarization. Recovery occurred within 0.5 s. 3. The reduction of the EPSCs was most probably due to reduced presynaptic calcium influx, since postsynaptic depolarization reduced presynaptic calcium or barium currents. Conversely, presynaptic depolarization also reduced postsynaptic calcium or barium influx, under conditions where transmitter release was minimal. 4. The calcium currents and the postsynaptic depolarization-induced suppression of synaptic transmission recovered with a similar time course, suggesting that this form of synaptic depression was, most probably, due to depletion of Ca2+ in the synaptic cleft. 5. We conclude that when the Ca2+ influx into the pre- or postsynaptic cell is large, extracellular Ca2+ is depleted. Under these conditions, the Ca2+ concentration in the synaptic cleft is a sensitive indicator of the level of synaptic activity. However, the synaptic cleft is less sensitive to Ca2+ depletion than predicted from its estimated volume.

We studied the relation between the size of presynaptic calcium influx and transmitter release by making simultaneous voltage clamp recordings from presynaptic terminals, the calyces of Held and postsynaptic cells, the principal cells of the medical nucleus of the trapezoid body, in slices of the rat brainstem. Calyces were voltage clamped with different action potential waveforms. The amplitude of the excitatory postsynaptic currents depended supralinearly on the size of the calcium influx, in the absence of changes in the time-course of the calcium influx. This result is in agreement with the view that at this synapse most vesicles are released by the combined action of multiple calcium channels.

1. To study use-dependent changes in the presynaptic Ca2+ influx and their contribution to transmitter release, we made simultaneous voltage clamp recordings from presynaptic terminals (the calyces of Held) and postsynaptic cells (the principal cells of the medial nucleus of the trapezoid body) in slices of the rat auditory brainstem. 2. Following a short (2 ms) prepulse to 0 mV, calcium channels opened faster during steps to negative test potentials. During trains of action potential waveforms the Ca2+ influx per action potential increased. At the same time, however, the amplitude of the EPSCs decreased. 3. The facilitation of the calcium currents appeared to depend on a build-up of intracellular Ca2+, since its magnitude was proportional to the Ca2+ influx and it was reduced in the presence of 10 mM BAPTA. 4. Facilitation of the presynaptic calcium currents may contribute to short-term facilitation of transmitter release, observed when quantal output is low. Alternatively, it may counteract processes that contribute to synaptic depression.

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and gamma-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

1. The calcium current of a 'giant' synaptic terminal (the calyx of Held) was studied using two-electrode voltage clamp in slices of the rat brainstem. 2. In terminals with a long axon (length > 100 microns), the passive current transient decayed biexponentially following voltage steps. In terminals with a short axon (length < 30 microns), the slow component was reduced or absent. These terminals also had small slow calcium tail currents following long depolarizing voltage steps, suggesting that these are largely due to axonal calcium channels. 3. Terminals were voltage clamped with action potential waveform commands. At both 24 and 36 degrees C the calcium current began shortly after the peak of the action potential and ended before the terminal was fully repolarized. 4. The calcium current during the repolarization phase was 69 +/- 1% (n = 3) of maximal, judged from the increase in this current when a plateau phase was added to the action potential waveform. 5. A Hodgkin-Huxley m2 model, based on the measured activation and deactivation of the calcium current, reproduced both the time course and the amplitude increase of the calcium currents during the different action potential waveforms well. 6. The fast gating of the calcium channels in the terminal ensures that they are effectively opened during the repolarization phase of an action potential. This implies that the distance between open calcium channels is minimized, which is in agreement with the view that multiple calcium channels are needed to release a vesicle in this synapse.

Calcium dynamics associated with a single action potential were studied quantitatively in the calyx of Held, a large presynaptic terminal in the rat brainstem. Terminals were loaded with different concentrations of high- or low-affinity Ca2+ indicators via patch pipettes. Spatially averaged Ca2+ signals were measured fluorometrically and analyzed on the basis of a single compartment model. A single action potential led to a total Ca2+ influx of 0.8-1 pC. The accessible volume of the terminal was about 0.4 pl; thus the total calcium concentration increased by 10-13 microM. The Ca(2+)-binding ratio of the endogenous buffer was about 40, as estimated from the competition with Fura-2, indicating that 2.5% of the total calcium remained free. This is consistent with the peak increase in free calcium concentration of about 400 nM, which was measured directly with MagFura-2. The decay of the [Ca2+]i transients was fast, with time constants of 100 ms at 23 degrees C and 45 ms at 35 degrees C, indicating Ca2+ extrusion rates of 400 and 900 s-1, respectively. The combination of the relatively low endogenous Ca(2+)-binding ratio and the high rate of Ca2+ extrusion provides an efficient mechanism for rapidly removing the large Ca2+ load of the terminal evoked by an action potential.

The DBA/2J inbred strain of mice has been used extensively in hearing research as it suffers from earlyonset, progressive hearing loss. Initially, it mostly affects high frequencies, but already at 2 - 3 months hearing loss becomes broad. In search for hearing loss genes other than Cadherin 23 (otocadherin) and fascin-2, which make a large contribution to the high-frequency deficits, we used a large set of the genetic reference population of BXD recombinant inbred strains. For frequencies 4, 8, 16 and 32 kHz, auditory brainstem response hearing thresholds were longitudinally determined from 2 -3 up to 12 weeks of age. Apart from a significant, broad quantitative trait locus (QTL) for high-frequency hearing loss on chromosome 11 containing the fascin-2 gene, we found a novel, small QTL for low-frequency hearing loss on chromosome 18, from hereon called ahl9. Realtime quantitative polymerase chain reaction of organs of Corti, isolated from a subset of strains, showed that a limited number of genes at the QTL were expressed in the organ of Corti. Of those genes, several showed significant expression differences based on the parental line contributing to the allele. Our results may aid in the future identification of genes involved in low-frequency, early-onset hearing loss. [ABSTRACT FROM AUTHOR]

The calyx of Held is an axosomatic terminal in the auditory brainstem that has attracted anatomists because of its giant size and physiologists because of its accessibility to patch-clamp recordings. The calyx allows the principal neurons in the medial nucleus of the trapezoid body (MNTB) to provide inhibition that is both well timed and sustained to many other auditory nuclei. The special adaptations that allow the calyx to drive its principal neuron even when frequencies are high include a large number of release sites with low release probability, a large readily releasable pool, fast presynaptic calcium clearance and little delayed release, a large quantal size, and fast AMPA-type glutamate receptors. The transformation from a synapse that is unremarkable except for its giant size into a fast and reliable auditory relay happens in just a few days. In rodents this transformation is essentially ready when hearing starts. [ABSTRACT FROM AUTHOR]

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