J. Gerard G. Borst

Short-term plasticity at the calyx of held

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.

The Reduced Release Probability of Releasable Vesicles during Recovery from Short-Term Synaptic Depression

Recovery from synaptic depression is believed to depend mainly on replenishment of the releasable pool of vesicles. We observed that during recovery from depression in a calyx-type synapse, part of the releasable pool was replenished rapidly. Half recovery occurred within 1 s, even in the absence of residual calcium. Vesicles that had recently entered the releasable pool had a 7- to 8-fold lower release probability than those that had been in the pool for more than 30 s. These results suggest that the reduction in the release probability of releasable vesicles contributes greatly to the level of depression. How synapses maintain transmission during repetitive firing is in debate. We propose that during repetitive firing, accumulation of intracellular Ca2+ may facilitate release of the rapidly replenished but reluctant vesicles, making them available for sustaining synaptic transmission.

Doc2b is a High Affinity Ca2+ Sensor for Spontaneous Neurotransmitter Release

“Spontaneous” Release Trigger Synaptic vesicle release occurs in different phases that can be tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events not triggered by action potentials (spontaneous). The vesicle protein synaptotagmin is thought to act as the Ca2+ sensor in the synchronous phase, but for the other two phases, Ca2+ sensors have not been identified. Groffen et al. (p. 1614, published online 11 February) now show that cytoplasmic proteins known as Doc2 (double C2 domain) proteins are required for spontaneous release. Doc2 proteins promote membrane fusion in response to exceptionally low increases in Ca2+, and are several orders of magnitude more sensitive to Ca2+ than synaptotagmin. Doc2 and synaptotagmin compete for SNARE-complex binding during membrane fusion. A mutation that abolishes the Ca2+ dependence of Doc2b also abolishes the Ca2+ dependence of spontaneous release. Thus, Doc2 is a high-affinity Ca2+ sensor for spontaneous release that competes with synaptotagmin for SNARE complex binding. Spontaneous synaptic vesicle fusion is triggered by soluble proteins that compete with synaptotagmins to induce membrane curvature. Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicle-associated Ca2+ sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+ sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+ sensitivity. Doc2 proteins bound to N-ethylmaleimide–sensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain–containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain–containing proteins.

Reliability and Precision of the Mouse Calyx of Held Synapse

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 Synapse : From Model Synapse to Auditory Relay

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.

Calcium action potentials in hair cells pattern auditory neuron activity before hearing onset

We found rat central auditory neurons to fire action potentials in a precise sequence of mini-bursts before the age of hearing onset. This stereotyped pattern was initiated by hair cells in the cochlea, which trigger brief bursts of action potentials in auditory neurons each time they fire a Ca2+ spike. By generating theta-like activity, hair cells may limit the influence of synaptic depression in developing auditory circuits and promote consolidation of synapses.

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 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.

The low synaptic release probability in vivo

The release probability, the average probability that an active zone of a presynaptic terminal releases one or more vesicles following an action potential, is tightly regulated. Measurements in cultured neurons or in slices indicate that this probability can vary greatly between synapses, but on average it is estimated to be as high as 0.5. In vivo, however, the size of synaptic potentials is relatively independent of recent history, suggesting that release probability is much lower. Possible causes for this discrepancy include maturational differences, a higher spontaneous activity, a lower extracellular calcium concentration and more prominent tonic inhibition by ambient neurotransmitters during in vivo recordings. Existing evidence thus suggests that under physiological conditions in vivo, presynaptic action potentials trigger the release of neurotransmitter much less frequently than what is observed in in vitro preparations.

Factors Controlling the Input–Output Relationship of Spherical Bushy Cells in the Gerbil Cochlear Nucleus

Despite the presence of large endbulb inputs, the spherical bushy cells (SBCs) of the rostral anteroventral cochlear nucleus do not function as simple auditory relays. We used the good signal-to-noise ratio of juxtacellular recordings to dissect the intrinsic and network mechanisms controlling the input–output relationship of SBCs in anesthetized gerbils. The SBCs generally operated close to action potential (AP) threshold and showed no evidence for synaptic depression, suggesting that the endbulbs of Held have low release probability in vivo. Analysis of the complex waveforms suggested that in the absence of auditory stimulation, postsynaptic spike depression and stochastic fluctuations in EPSP size were the main factors determining jitter and reliability of the endbulb synapse. During auditory stimulation, progressively larger EPSPs were needed to trigger APs at increasing sound intensities. Simulations suggested hyperpolarizing inhibition could explain the observed decrease in EPSP efficacy. Synaptic inhibition showed a delayed onset and generally had a higher threshold than excitatory inputs, but otherwise inhibition and excitation showed mostly overlapping frequency–response areas. The recruitment of synaptic inhibition caused postsynaptic spikes to be preferentially triggered by well-timed, large EPSPs, resulting in improved phase locking despite more variable EPSP–AP latencies. Our results suggest that the lack of synaptic depression, caused by low release probability, and the apparent absence of sound-evoked synaptic inhibition at low sound intensity maximize sensitivity of SBCs. At higher sound intensities, the recruitment of synaptic inhibition constrains their firing rate and optimizes their temporal precision.

Dynamic development of the calyx of Held synapse.

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.