Takeshi Sakaba

Multiple Roles of Calcium Ions in the Regulation of Neurotransmitter Release

The intracellular calcium concentration ([Ca(2+)]) has important roles in the triggering of neurotransmitter release and the regulation of short-term plasticity (STP). Transmitter release is initiated by quite high concentrations within microdomains, while short-term facilitation is strongly influenced by the global buildup of residual calcium. A global rise in [Ca(2+)] also accelerates the recruitment of release-ready vesicles, thereby controlling the degree of short-term depression (STD) during sustained activity, as well as the recovery of the vesicle pool in periods of rest. We survey data that lead us to propose two distinct roles of [Ca(2+)] in vesicle recruitment: one accelerating molecular priming (vesicle docking and the buildup of a release machinery), the other promoting the tight coupling between releasable vesicles and Ca(2+) channels. Such coupling is essential for rendering vesicles sensitive to short [Ca(2+)] transients, generated during action potentials.

Vesicle pools and short-term synaptic depression: lessons from a large synapse

Depletion of a pool of readily releasable vesicles during repetitive presynaptic activity is a candidate mechanism for the induction of short-term synaptic depression. The large, calyx-type synaptic terminals in the brainstem auditory pathway, and especially the calyx of Held, offer unique possibilities for studying the cellular mechanisms leading to synaptic depression. Recent work at these synapses using presynaptic whole-cell patch-clamp recordings has revealed a large pool of readily releasable vesicles. During prolonged presynaptic depolarization, vesicles are released in kinetically distinct phases, indicating heterogeneity of release probability between vesicles. Heterogeneity might endow synapses with a rapid phase of depression at the onset of activity, followed by sustained and surprisingly large synaptic strength during the steady-state phase of depression. By influencing the synaptic output during repetitive activity, vesicle pool dynamics are expected to modulate information processing in neuronal networks of the CNS.

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high-frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin-dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles.

Direct modulation of synaptic vesicle priming by GABA(B) receptor activation at a glutamatergic synapse

Second messenger cascades involving G proteins and calcium are known to modulate neurotransmitter release. A prominent effect of such a cascade is the downmodulation of presynaptic calcium influx, which markedly reduces evoked neurotransmitter release. Here we show that G-protein-mediated signalling, such as through GABA (γ-amino butyric acid) subtype B (GABAB) receptors, retards the recruitment of synaptic vesicles during sustained activity and after short-term depression. This retardation occurs through a lowering of cyclic AMP, which blocks the stimulatory effect of increased calcium concentration on vesicle recruitment. In this signalling pathway, cAMP (functioning through the cAMP-dependent guanine nucleotide exchange factor) and calcium/calmodulin cooperate to enhance vesicle priming. The differential modulation of the two forms of synaptic plasticity, presynaptic inhibition and calcium-dependent recovery from synaptic depression, is expected to have interesting consequences for the dynamic behaviour of neural networks.

The Coupling between Synaptic Vesicles and Ca2+ Channels Determines Fast Neurotransmitter Release

In order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials.

Calcium dependence of exo- and endocytotic coupling at a glutamatergic synapse.

The mechanism coupling exocytosis and endocytosis remains to be elucidated at central synapses. Here, we show that the mechanism linking these two processes is dependent on microdomain-[Ca2+](i) similar to that which triggers exocytosis, as well as the exocytotic protein synaptobrevin/VAMP. Furthermore, block of endocytosis has a limited, retrograde action on exocytosis, delaying recruitment of release-ready vesicles and enhancing short-term depression. This effect sets in so rapidly that it cannot be explained by the nonavailability of recycled vesicles. Rather, we postulate that perturbation of a step linking exocytosis and endocytosis temporarily prevents new vesicles from docking at specialized sites for exocytosis.

Quantitative Analysis of Calcium-Dependent Vesicle Recruitment and Its Functional Role at the Calyx of Held Synapse

Recruitment of release-ready vesicles at synapses is one of the important factors, which determine dynamic properties of signaling between neurons in the brain. It has been shown that the rate of vesicle recruitment is accelerated by strong synaptic activity. An elevated concentration of calcium ions in the presynaptic terminal ([Ca2+]i) has been proposed to be responsible for this effect. However, the precise relationship between [Ca2+]i and recruitment has not been established yet, and the functional consequences of accelerated recruitment during synaptic activity have not been quantified experimentally. To probe the intracellular Ca2+ dependence of vesicle recruitment and to examine its functional role during trains of action potential (AP)-like stimuli, we monitored [Ca2+]i and synaptic responses simultaneously with paired recordings at the calyx of Held synapse. We found that a distinct, rapidly releasing vesicle pool is replenished with a rate that increases linearly with [Ca2+]i, without any apparent cooperativity. The slope factor for this increase is ∼1 pool/(μm·s). Blocking Ca2+-dependent recruitment specifically with a calmodulin binding peptide revealed that the steady-state EPSCs during 100 Hz AP-like trains were maintained through this Ca2+-dependent recruitment mechanism. Using a simple model of vesicle dynamics, we estimated that the recruitment rate accelerated 10-fold during the steady-state compared with the rate at resting [Ca2+]i. We could also demonstrate an approximate sixfold increase in release probability (facilitation) during the initial 5–15 AP-like stimuli of such trains in our experimental condition, regardless of EPSC depression.

Roles of the Fast-Releasing and the Slowly Releasing Vesicles in Synaptic Transmission at the Calyx of Held

In the calyx of Held, fast and slow components of neurotransmitter release can be distinguished during a step depolarization. The two components show different sensitivity to molecular/pharmacological manipulations. Here, their roles during a high-frequency train of action potential (AP)-like stimuli were examined by using both deconvolution of EPSCs and presynaptic capacitance measurements. During a 100 Hz train of AP-like stimuli, synchronous release showed a pronounced depression within the 20 stimuli. Asynchronous release persisted during the train, was variable in its amount, and was more prominent during a 300 Hz train. We have shown previously that slowly releasing vesicles were recruited faster than fast-releasing vesicles after depletion. By further slowing recovery of the fast-releasing vesicles by inhibiting calmodulin-dependent processes (Sakaba and Neher, 2001b), the slowly releasing vesicles were isolated during recovery from vesicle depletion. When a high-frequency train was applied, the isolated slowly releasing vesicles were released predominantly asynchronously. In contrast, synchronous release was mediated mainly by the fast-releasing vesicles. The results suggest that fast-releasing vesicles contribute mainly to synchronous release and that depletion of fast-releasing vesicles shape the synaptic depression of the synchronous phase of EPSCs, whereas slowly releasing vesicles are released mainly asynchronously during high-frequency stimulation. The latter is less subject to depression presumably because of a rapid vesicular recruitment process, which is a characteristic of this component.

Involvement of actin polymerization in vesicle recruitment at the calyx of Held synapse

Depletion and replenishment of pools of synaptic vesicles are important determinants of short-term synaptic plasticity, but the underlying molecular mechanisms are not yet clear. As a first step toward understanding the process of vesicle recruitment, we have applied various specific agents directly to the presynaptic terminal of the calyx of Held synapse. Here we show that the nonhydrolyzable ATP analog ATP-γS retards the recovery from vesicle pool depletion, as does latrunculin A. Phalloidin has no effects on recovery, suggesting that dynamic actin reorganization is not necessary. Unexpectedly, neither N-ethylmaleimide nor staurosporine affected the recovery, calling into question the role ofN-ethylmaleimide-sensitive factor and protein kinases. The results suggest that intact actin polymerization is involved in vesicle recruitment.

Dynamic Control of Synaptic Vesicle Replenishment and Short-Term Plasticity by Ca2+-Calmodulin-Munc13-1 Signaling

Short-term synaptic plasticity, the dynamic alteration of synaptic strength during high-frequency activity, is a fundamental characteristic of all synapses. At the calyx of Held, repetitive activity eventually results in short-term synaptic depression, which is in part due to the gradual exhaustion of releasable synaptic vesicles. This is counterbalanced by Ca(2+)-dependent vesicle replenishment, but the molecular mechanisms of this replenishment are largely unknown. We studied calyces of Held in knockin mice that express a Ca(2+)-Calmodulin insensitive Munc13-1(W464R) variant of the synaptic vesicle priming protein Munc13-1. Calyces of these mice exhibit a slower rate of synaptic vesicle replenishment, aberrant short-term depression and reduced recovery from synaptic depression after high-frequency stimulation. Our data establish Munc13-1 as a major presynaptic target of Ca(2+)-Calmodulin signaling and show that the Ca(2+)-Calmodulin-Munc13-1 complex is a pivotal component of the molecular machinery that determines short-term synaptic plasticity characteristics.