Ralf Schneggenburger

Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse.

The size of a pool of readily releasable vesicles at a giant brainstem synapse, the calyx of Held, was probed with three independent approaches. Using simultaneous pre- and postsynaptic whole-cell recordings, two forms of presynaptic Ca2+ stimuli were applied in rapid succession: uncaging of Ca2+ by flash photolysis and the opening of voltage-gated Ca2+ channels. The ensuing transmitter release showed a nearly complete cross-inhibition between the two stimuli, indicating the depletion of a limited pool of about 700 transmitter quanta. The pool size was confirmed in experiments using enhanced extracellular Ca2+ concentrations, as well as short, high-frequency stimulus trains. The results reveal a surprisingly large pool of functionally available vesicles, of which a fraction of about 0.2 is released by a single presynaptic action potential under physiological conditions.

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.

RIM Determines Ca2+ Channel Density and Vesicle Docking at the Presynaptic Active Zone

At presynaptic active zones, neurotransmitter release is initiated by the opening of voltage-gated Ca²+ channels close to docked vesicles. The mechanisms that enrich Ca²+ channels at active zones are, however, largely unknown, possibly because of the limited presynaptic accessibility of most synapses. Here, we have established a Cre-lox based conditional knockout approach at a presynaptically accessible central nervous system synapse, the calyx of Held, to directly study the functions of RIM proteins. Removal of all RIM1/2 isoforms strongly reduced the presynaptic Ca²+ channel density, revealing a role of RIM proteins in Ca²+ channel targeting. Removal of RIMs also reduced the readily releasable pool, paralleled by a similar reduction of the number of docked vesicles, and the Ca²+ channel-vesicle coupling was decreased. Thus, RIM proteins co-ordinately regulate key functions for fast transmitter release, enabling a high presynaptic Ca²+ channel density and vesicle docking at the active zone.

Allosteric modulation of the presynaptic Ca2+ sensor for vesicle fusion.

Neurotransmitter release is triggered by an increase in the cytosolic Ca2+ concentration ([Ca2+]i), but it is unknown whether the Ca2+-sensitivity of vesicle fusion is modulated during synaptic plasticity. We investigated whether the potentiation of neurotransmitter release by phorbol esters, which target presynaptic protein kinase C (PKC)/munc-13 signalling cascades, exerts a direct effect on the Ca2+-sensitivity of vesicle fusion. Using direct presynaptic Ca2+-manipulation and Ca2+ uncaging at a giant presynaptic terminal, the calyx of Held, we show that phorbol esters potentiate transmitter release by increasing the apparent Ca2+-sensitivity of vesicle fusion. Phorbol esters potentiate Ca2+-evoked release as well as the spontaneous release rate. We explain both effects by an increased fusion ‘willingness’ in a new allosteric model of Ca2+-activation of vesicle fusion. In agreement with an allosteric mechanism, we observe that the classically high Ca2+ cooperativity in triggering vesicle fusion (∼ 4) is gradually reduced below 3 µM [Ca2+]i, reaching a value of <1 at basal [Ca2+]i. Our data indicate that spontaneous transmitter release close to resting [Ca2+]i is a consequence of an intrinsic property of the molecular machinery that mediates synaptic vesicle fusion.

The synaptic vesicle protein CSPα prevents presynaptic degeneration

Abstract Cysteine string protein α (CSPα)—an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40 chaperones—is thought to regulate Ca 2+ channels and/or synaptic vesicle exocytosis. We now show that, in young mice, deletion of CSPα does not impair survival and causes no significant changes in presynaptic Ca 2+ currents or synaptic vesicle exocytosis as measured in the Calyx of Held synapse. At 2–4 weeks of age, however, CSPα-deficient mice develop a progressive, fatal sensorimotor disorder. The neuromuscular junctions and Calyx synapses of CSPα-deficient mice exhibit increasing neurodegenerative changes, synaptic transmission becomes severely impaired, and the mutant mice die at ∼2 months of age. Our data suggest that CSPα is not essential for the normal operation of Ca 2+ channels or exocytosis but acts as a presynaptic chaperone that maintains continued synaptic function, raising the possibility that enhanced CSPα function could attenuate neurodegenerative diseases.

The calyx of Held

The calyx of Held is a large glutamatergic synapse in the mammalian auditory brainstem. By using brain slice preparations, direct patch-clamp recordings can be made from the nerve terminal and its postsynaptic target (principal neurons of the medial nucleus of the trapezoid body). Over the last decade, this preparation has been increasingly employed to investigate basic presynaptic mechanisms of transmission in the central nervous system. We review here the background to this preparation and summarise key findings concerning voltage-gated ion channels of the nerve terminal and the ionic mechanisms involved in exocytosis and modulation of transmitter release. The accessibility of this giant terminal has also permitted Ca2+-imaging and -uncaging studies combined with electrophysiological recording and capacitance measurements of exocytosis. Together, these studies convey the panopoly of presynaptic regulatory processes underlying the regulation of transmitter release, its modulatory control and short-term plasticity within one identified synaptic terminal.

A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity?

α‐RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N‐terminal sequences. The α‐RIM N‐terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that α‐RIMs contain adjacent but separate Munc13‐ and Rab3‐binding sites, allowing formation of a tripartite Rab3/RIM/Munc13 complex. Munc13 binding is mediated by the α‐RIM zinc‐finger domain. Elucidation of the three‐dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes α‐RIM/Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of α‐RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the α‐RIM N‐terminal sequence.

Probing the Intracellular Calcium Sensitivity of Transmitter Release during Synaptic Facilitation

In nerve terminals, residual Ca(2+) remaining from previous activity can cause facilitation of transmitter release by a mechanism that is still under debate. Here we show that the intracellular Ca(2+) sensitivity of transmitter release at the calyx of Held is largely unchanged during facilitation, which leaves an increased microdomain Ca(2+) signal as a possible mechanism for facilitation. We measured the Ca(2+) dependencies of facilitation, as well as of transmitter release, to estimate the required increment in microdomain Ca(2+). These measurements show that linear summation of residual and microdomain Ca(2+) accounts for only 30% of the observed facilitation. However, a small degree of supralinearity in the summation of intracellular Ca(2+) signals, which might be caused by saturation of cytosolic Ca(2+) buffer(s), is sufficient to explain facilitation at this CNS synapse.

Parvalbumin Is a Mobile Presynaptic Ca2+ Buffer in the Calyx of Held that Accelerates the Decay of Ca2+ and Short-Term Facilitation

Presynaptic Ca2+ signaling plays a crucial role in short-term plasticity of synaptic transmission. Here, we studied the role of mobile endogenous presynaptic Ca2+ buffer(s) in modulating paired-pulse facilitation at a large excitatory nerve terminal in the auditory brainstem, the calyx of Held. To do so, we assessed the effect of presynaptic whole-cell recording, which should lead to the diffusional loss of endogenous mobile Ca2+ buffers, on paired-pulse facilitation and on intracellular Ca2+ concentration ([Ca2+]i) transients evoked by action potentials. In unperturbed calyces briefly preloaded with the Ca2+ indicator fura-6F, the [Ca2+]i transient decayed surprisingly fast (τfast, ∼30 ms). Presynaptic whole-cell recordings made without additional Ca2+ buffers slowed the decay kinetics of [Ca2+]i and paired-pulse facilitation (twofold to threefold), but the amplitude of the [Ca2+]i transient was changed only marginally. The fast [Ca2+]i decay was restored by adding the slow Ca2+ buffer EGTA (50–100 μm) or parvalbumin (100 μm), a Ca2+-binding protein with slow Ca2+-binding kinetics, to the presynaptic pipette solution. In contrast, the fast Ca2+ buffer fura-2 strongly reduced the amplitude of the [Ca2+]i transient and slowed its decay, suggesting that the mobile endogenous buffer in calyces of Held has slow, rather than fast, binding kinetics. In parvalbumin knock-out mice, the decay of [Ca2+]i and facilitation was slowed approximately twofold compared with wild-type mice, similar to what is observed during whole-cell recordings in rat calyces of Held. Thus, in young calyces of Held, a mobile Ca2+ buffer with slow binding kinetics, primarily represented by parvalbumin, accelerates the decay of spatially averaged [Ca2+]i and paired-pulse facilitation.

Interplay between Na+/Ca2+ Exchangers and Mitochondria in Ca2+ Clearance at the Calyx of Held

The clearance of Ca2+ from nerve terminals is critical for determining the build-up of residual Ca2+ after repetitive presynaptic activity. We found previously that K+-dependent Na+/Ca2+ exchangers (NCKXs) show polarized distributions in axon terminals of supraoptic magnocellular neurons and play a major role in Ca2+ clearance. The role of NCKXs in presynaptic terminals, however, has not been studied. We investigated the contribution of NCKX in conjunction with other Ca2+ clearance mechanisms at the calyx of Held by analyzing the decay of Ca2+ transients evoked by depolarizing pulses. Inhibition of Na+/Ca2+ exchange by replacing external Na+ with Li+ decreased the Ca2+ decay rate by 68%. Selective inhibition of NCKX by replacing internal K+ with TEA+ (tetraethylammonium) or Li+ decreased the Ca2+ decay rate by 42%, and the additional inhibition of the K+-independent form of Na+/Ca2+ exchanger (NCX) by reducing external [Na+] caused an additional decrease by 26%. Inhibition of plasma membrane Ca2+-ATPase (PMCA) decreased the Ca2+ decay rate by 23%, whereas inhibition of SERCA (smooth endoplasmic reticulum Ca2+-ATPase) had no effect. The contribution of mitochondria was negligible for small Ca2+ transients but became apparent at [Ca2+]i > 2.5 μm, when Na+/Ca2+ exchange became saturated. Mitochondrial contribution was also observed when the duration of Ca2+ transients was prolonged by inhibiting Na+/Ca2+ exchangers or by increasing Ca2+ buffers. These results suggest that, in response to small Ca2+ transients (<2 μm), Ca2+ loads are cleared from the calyx of Held by NCKX (42%), NCX (26%), and PMCA (23%), and that mitochondria participate when the Ca2+ load is larger or prolonged.