Sustaining rapid vesicular release at active zones: potential roles for vesicle tethering

Abstract

Rapid information processing in our nervous system relies on high-frequency fusion of transmitter-filled vesicles at chemical synapses. Some sensory synapses possess prominent electron-dense ribbon structures that provide a scaffold for tethering synaptic vesicles at the active zone (AZ), enabling sustained vesicular release. Here, we review functional data indicating that some central and neuromuscular synapses can also sustain vesicle-fusion rates that are comparable to those of ribbon-type sensory synapses. Comparison of the ultrastructure across these different types of synapses, together with recent work showing that cytomatrix proteins can tether vesicles and speed vesicle reloading, suggests that filamentous structures may play a key role in vesicle supply. We discuss potential mechanisms by which vesicle tethering could contribute to sustained high rates of vesicle fusion across ribbon-type, central, and neuromuscular synapses.

Figure 1

Deletion or mutation of putative vesicle tethering proteins impairs the rapid component of recovery from synaptic depression. (a–c) Examples of a ribbon-type, central, and NMJ synapse with two kinetic components of recovery from synaptic depression (also see Table 1 in main text). Deletion of putative vesicle-tethering proteins or specific interference with vesicle tethering (magenta) resulted in all three examples in a slower recovery from synaptic depression [22,27,29]. The preferentially impaired first component of recovery (magenta arrows) suggests that vesicle tethering is important for activity-dependent speeding of vesicle reloading. (a) Ribbon synapses. Left: presynaptic recordings from a hair cell ribbon synapse. Capacitance increases (upper trace) and Ca²⁺ current (lower trace) elicited by a paired-pulse paradigm (two 15 ms depolarizations). Right: average capacitance increases of the 2nd pulse of paired depolarizations (ΔC_m2) versus inter-pulse interval superimposed with a bi-exponential fit [71]. Inset: capacitance increases of the 2nd pulse of paired depolarizations (20 ms) normalized to the 1st response (ΔC_m2/ ΔC_m1) versus inter-pulse interval for control (black) and Bassoon mutants (magenta) [22]. (b) Central synapses. Left: excitatory postsynaptic currents (EPSCs) recorded at a cerebellar mossy fiber to granule-cell synapse elicited by 300 Hz stimulation followed by test stimuli of increasing intervals. Right: average EPSC amplitude during recovery from depression normalized to the 1st EPSC amplitude for control (black) and Bassoon mutants (magenta) superimposed with bi-exponential fits [29]. Inset: the fast component of recovery on expanded scale. (c) Neuromuscular synapses. Left: EPSCs recorded at the NMJ of 3rd instar Drosophila larvae elicited by 60 Hz stimulation followed by test stimuli of increasing intervals. Right: average EPSC amplitude during recovery from depression normalized to the 1st EPSC amplitude for control (black) and Bruchpilot mutants lacking the last 17 C-terminal amino acids (brp^nude, magenta) superimposed with exponential fits to the slow component [27]. Inset: corresponding fits to the fast component. Adopted, with permission, from [71] and [22] (a), [29] (b), and [27] (c).

Figure 2

Vesicle tethering at ribbon-type, central, and neuromuscular junction (NMJ) synapses. Examples of vesicle tethers (magenta arrows). (a) Ribbon synapses. Top: electron micrograph (EM) of a vesicle tethered at the plasma membrane of a ribbon-type mouse inner hair-cell synapse [22]. Bottom: EM of a cross-fractured synaptic ribbon (horizontal) with tethered vesicles [21]. (b) Central synapses. Top: electron tomographic slice of cerebrocortical synaptosomes [26]. Bottom: 3D reconstruction of vesicles and tethers [119]. (c) Neuromuscular synapses. Left: high-pressure freeze EM with filamentous structures at a Drosophila NMJ AZ (top) [23], consisting most likely of the Drosophila protein Bruchpilot (BRP), that has a funnel-like topology (bottom); SV, synaptic vesicle; adapted from [27], see also [120]. Right: electron tomographic slice of corresponding filamentous structures at the frog NMJ AZ (top), consisting most likely of different classes of AZ macromolecules, as illustrated in the schematic model (bottom) [25]. Adapted, with permission, from [22] and [21] (a), [26] and [119] (b), and [23], [27] and [25] (c).

Figure 3

Schematic illustrations of potential mechanisms underlying vesicle reloading and the putative role of tethers. Vesicular release under resting conditions (top) is followed by vesicle reloading mechanisms (middle) that allow repetitive release during synaptic activity (bottom). Although vesicle reloading during synaptic activity is fast, the complete recovery to the resting condition is slow (gray arrows). Three mechanisms explaining activity-dependent speeding of vesicle reloading are illustrated, although it is important to note that these mechanisms are not mutually exclusive. (a) Ca²⁺-dependent model. Influx of Ca²⁺ through Ca²⁺ channels triggers the fusion of a vesicle which has undergone ‘molecular’ priming (illustrated by v- and t-SNARE assembly) and ‘positional’ priming (illustrated by the proximity of the fusion sensor to the Ca²⁺ channels, ‘coupling distance’) [31,32]. During activity, the Ca²⁺ concentration rises in the terminal, and reloading of vesicles to a molecular and positional primed state is accelerated [48,74]. After activity, Ca²⁺ decays and reloading slows. (b) Two-pools model. Two populations of vesicles with different release probabilities due to different coupling distances. After fusion of both docked vesicles, rapid molecular priming of vesicles is sufficient to sustain release of vesicles loosely coupled to Ca²⁺ channels during activity. The spatial and temporal extent of [Ca²⁺] may increase during sustained activity, augmenting this process. After activity, slower positional priming of a small subset of vesicles resupplies the pool of high release-probability vesicles [78]. (c) Tethering model. Vesicle tethers could accelerate vesicle reloading by (i) providing release-ready vesicles at AZs, (ii) constraining vesicles to dock closer to Ca²⁺ channels (positional priming), and/or (iii) enriching priming factors on the vesicle (molecular priming). After activity, slow positional priming of vesicles closer to Ca²⁺ channels occurs as in panel (b). The inset illustrates vesicle tethering at ribbons, highlighting the similarities between vesicle tethering by ribbons at sensory synapses and by filamentous structures at central and neuromuscular junction (NMJ) synapses.

Figure I

Properties of two central synapses with different RRP sizes. Top: illustration of a synapse with a small release-ready pool (RRP) and rapid vesicle reloading (a), and a larger RRP and slower vesicle reloading (b). Bottom: both synapses exhibit a similar sustained release rate as shown in this simulation. However, to achieve this a threefold faster reloading rate is required for the smaller RRP. The time-constant of recovery reveals the rate of vesicle reloading when there are no other time-dependent processes involved (see Box 1 in main text).