Ca2+ channels and transmitter release at the active zone
Introduction
At most synapses in the central nervous system (CNS), transmitter release is triggered by brief action potentials (AP) with duration of a millisecond or less, which can occur repeatedly up to frequencies of several hundred Hz. In order to faithfully transmit the information contained in trains of APs, transmitter release must occur rapidly. At the same time, a single AP typically leads to the fusion of only a small percentage (∼10%) of the readily releasable pool (RRP) of vesicles [1] – leaving a large fraction of RRP vesicles behind for subsequent APs. There are two key requirements for tight temporal regulation of Ca2+-evoked release: first, vesides must have an intrinsic release apparatus [2], [3], [4], allowing them to fuse extremely rapidly (<1 ms) in response to an intracellular Ca2+ elevation. Second, docked vesicles must be placed at close, less than 100 nanometer distance from voltage-gated Ca2+ channels, to guarantee high and brief “local” [Ca2+]i transients. The fact that release at CNS synapses occurs at active zones with sub-micrometer dimensions [5], [6], [7], [8] seems a key adaptation to allow for short diffusional distances of Ca2+ ions.
Here, we will review some aspects of our current understanding of Ca2+ channel–vesicle co-localization at CNS synapses. We will first summarize anatomical features of active zones relevant for release mechanisms and Ca2+ channel–vesicle co-localization. Detailed insights into how intracellular Ca2+ ions, and Ca2+ channels regulate vesicle fusion has been obtained at the calyx of Held, a CNS synapse with a large nerve terminal at which direct presynaptic patch-clamp recordings can be obtained [9], [10]. We will review how simultaneous measurements of presynaptic Ca2+ currents and transmitter release has been used to infer Ca2+ channel–vesicle co-localization, and we will discuss kinetic release components observed at the calyx of Held, and their mechanistic interpretation.
Section snippets
Ultrastructural constraints for transmitter release at the synapse
In EM images of CNS synapses, active zones are seen to oppose the postsynaptic density and contain docked vesicles and vesicle clusters [5], [6], [7], [8] (see also Fig. 1). The calyx of Held is a glutamatergic relay synapse in the auditory system that is distinguished by a large, cup-shaped nerve terminal which forms onto the soma of its postsynaptic partner neuron [9], [10]. Early ultrastructural analysis has indicated that the calyx contains conventional active zones, with vesicles of ∼45 nm
Spatial relation between Ca2+ channels and docked vesicles
Release is triggered by Ca2+ channel opening. In order to understand how Ca2+ influx through Ca2+ channels drives release, it is instructive to first think about the microdomain [Ca2+]i (from Greek, micro = small) which builds up around a single open Ca2+ channel (Fig. 2a). Pioneering modeling studies have shown that the amplitude and spatial profile of the microdomain [Ca2+]i strongly depends on the distance to the Ca2+ channel, on the single-channel Ca2+ current, and on the on-rate,
Inferences on the number of Ca2+ channels that control release
If the Ca2+ channel open probability can be varied by voltage-clamp stimuli of the nerve terminal or following (partial) block with slow Ca2+ channel blockers, and if presynaptic Ca2+ influx can be measured, then inferences can be made on the number of Ca2+ channels in release control [34]. Consider two limiting cases, in which either a large number of Ca2+ channels (“domain overlap”), or else, only 1–2 Ca2+ channels control vesicle release (“single channel control”; Fig. 2B). In the limit of
Distinct kinetic components of release: intrinsic or positional differences of RRP vesicles?
A second type of experiment which can potentially inform about the proximity of readily releasable vesicles to Ca2+ channels involves a kinetic analysis of release in response to step-like voltage-clamp depolarizations in the nerve terminal [48], [49]. Step-like Ca2+ currents evoked release in a fast- and a slow phase, which are attributed to two subpopulations of the readily releasable pool, FRP and SRP for fast- and slowly releasable, respectively (Fig. 3A; [48]). It can be assumed that both
Inferences from partial pool depleting stimuli
How could the apparently contradictory conclusions of the two studies [49], [50], “intrinsic” versus “positional” heterogeneity of release kinetics, be consolidated? An important assumption made in the Ca2+ uncaging study by Wölfel et al., who favor the “intrinsic” model, is that Ca2+ uncaging indeed produces a spatially homogeneous [Ca2+]i signal. Although homogeneity was shown within the spatial resolution (∼1 μm) of their fast imaging measurements, it cannot be completely excluded that local
Limiting cases of Ca2+ channel–vesicle maps at the active zone
What can we conclude about the topology of Ca2+ channels and docked vesicles at the active zone, and about possible mechanisms explaining the heterogeneity of release kinetics? Fig. 4 depicts a few models, using a schematic active zone of 0.06 μm2 surface with 6 docked vesicles of 45 nm diameter drawn to scale – representative for a middle-sized active zone (see Fig. 1E). In the “intrinsic” model (Fig. 4A), vesicles might be surrounded by many Ca2+ channels homogenously creating similar [Ca2+]i
An alternative explanation for heterogeneous release kinetics
Considering that release takes place at active zones with a high density of docked vesicles, it is likely that even release of the FRP alone (∼half of all readily releasable vesicles) leads to the fusion of several vesicles at each active zone, and would therefore cause multivesicular release. The release of ∼half of all docked vesicles would lead to a membrane surface increase representing as much as one third of the active zone membrane (Fig. 4C), considering a vesicle diameter of 45 nm and a
Concluding remarks, and new functions for presynaptic proteins
Which molecular factors determine intrinsic and positional influences on Ca2+-dependent transmitter release? Some recent progress has been made by combining the presynaptic accessibility of the calyx of Held synapse, with novel methods for the molecular targeting of synapses in vivo [65]. Synaptotagmin-2 (Syt2) is the major Syt isoform at the calyx and clearly mediates the intrinsically fast Ca2+-dependent release at the calyx, as demonstrated in Ca2+ uncaging experiments made in Syt2 knock-out
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2016, Journal of Neuroscience MethodsCitation Excerpt :In contrast, the EQ and decay methods assume uniform p that remains constant throughout a stimulus train. However, p does not remain constant at facilitating synapses, and p may be non-uniform at many synapses (Dobrunz and Stevens, 1997; Sakaba and Neher, 2001b; Meinrenken et al., 2002; Trommershäuser et al., 2003; Moulder and Mennerick, 2005; Schneggenburger et al., 2012). In this study, we compare estimates of synaptic parameters at the calyx of Held synapse based on four approaches: the train method (Schneggenburger et al., 1999), the EQ method (Elmqvist and Quastel, 1965), the decay method (Ruiz et al., 2011), and fitting to a depletion-based model.