4. Discussion
4.4. Morphological correlates of mossy fiber facilitation
Release probability and short-term plasticity are dependent upon multiple factors, including calcium channel-sensor coupling distance (Vyleta and Jonas, 2014), shape of the action potential (Geiger and Jonas, 2000), calcium buffers (Blatow et al., 2003; Dumas et al., 2004;
Vyleta and Jonas, 2014), type of calcium sensor (Jackman et al., 2016), and accessibility to energy sources (i.e. mitochondria) (Kwon et al., 2016).
Despite comparable numbers of membrane-proximal synaptic vesicles within 40 nm of the active zone membrane, I found fewer morphologically docked and primed synaptic vesicles at individual mossy fiber active zones compared to Schaffer collateral synapses from the same slice. These data indicate that differences in the availability of docked and primed vesicles could, in addition to other factors, co-determine initial release probability. Past studies examined whether release probability is dependent on the availability of morphologically docked synaptic vesicles, however the conclusions did not lead to a general consensus (Eltes et al., 2017; Holderith et al., 2012; Millar et al., 2002; Schikorski and Stevens, 1997; Xu-Friedman et al., 2001). This question was worth revisiting in light of some limitations from past studies that can be circumvented with different techniques (i.e. cryo-fixation and electron tomography).
My findings are consistent with those reported by Eltes and colleagues that synapses with a low release probability (facilitating, tonic) harbor fewer docked synaptic vesicles than synapses with higher release probability (depressing, phasic) (Eltes et al., 2017). Along the same lines, Schikorski and Stevens found that the variability in the number of docked synaptic vesicles in the rat CA1 accounted for the heterogeneity in synaptic release probability and concluded that the number of docked vesicles contributes to synaptic release probability (Schikorski and Stevens, 1997). Conversely, my results differ from studies in the rodent cerebellum where synapses from parallel fibers (low release probability, facilitating) and synapses from climbing fibers (high release probability, depressing) harbored similar numbers of docked synaptic vesicles (Xu-Friedman et al., 2001). Further, in hippocampal associational/commissural synapses the number of docked synaptic vesicles and the release probability positively correlated with active zone area (Holderith et al., 2012).
112 In contrast to Schaffer collateral synapses, mossy fiber synapses at rest had a second pool of proximal vesicles 5-20 nm from the active zone. I interpret the membrane-proximal accumulation of vesicles in mossy fibers as structural evidence of a tethering step preceding synaptic vesicle docking/priming. Although undetected in wild-type Schaffer collaterals in the present study, the membrane-proximal accumulation of vesicles is highly reminiscent of the synaptic vesicle organization observed in Schaffer collateral synapses lacking key priming proteins, such as Munc13s, and core components of the neuronal SNARE complex, such as SNAP-25 and synaptobrevin-2 (Imig et al., 2014). This indicates that membrane-proximal vesicle tethering is not unique to mossy fibers per se, but implies that molecular processes responsible for forming the tethered pool operate differently in Schaffer collateral and mossy fiber synapses. Differences in molecular processes could involve a limited copy number of any of the aforementioned priming and SNARE proteins that, upon genetic deletion, manifest a membrane-proximal accumulation of synaptic vesicles.
Specialized vesicle tethering mechanisms have evolved to support the transmitter release properties, and behaviors of distinct synapse types have been shown in specialized synapses such as invertebrate neuromuscular junctions and mammalian ribbon synapses (Hallermann and Silver, 2013).
My findings support a previously proposed vesicle tethering mechanism (Hallermann and Silver, 2013) for the first time in mossy fiber synapses. Synaptic vesicles are thought to loosely tether to the plasma membrane close enough for interaction to occur between v-SNAREs and t-SNARE (Neher and Brose, 2018). A tightening of the SNARE complex assembly results in morphological docking of synaptic vesicles to the plasma membrane mediated by Munc13 and CAPS priming molecules (Neher and Brose, 2018). These steps represent a loose and then tight synaptic vesicle docking state (Neher and Brose, 2018). The generation of a LS synaptic vesicle pool has been shown in dissociated mouse hippocampal neurons to rely on the calcium sensor synaptotagmin-1 (Chang et al., 2018). My finding of a membrane proximal pool of synaptic vesicles in mossy fiber synapses indicates the morphological correlate of tethered and therefore LS synaptic vesicles. The relationship between these tethered vesicles and the hypothesized LS state in tonic mossy fiber synapses requires further investigation, both in terms of its reliance of synaptotagmin-1, and in terms of how quickly the tethered pool is formed or depleted. These questions could be addressed by performing flash-and-freeze
113 experiments-coupled with 3D ultrastructural analysis in synaptotagmin-1 mutant hippocampal slice cultures analogous to the study in dissociated neuron culture by Chang and colleagues (Chang et al., 2018).
Although I did not directly quantify filamentous material in my tomograms, the distance at which the membrane-proximal pool accumulated in wild-type mossy fiber synapses in the present study, and in priming-deficient Schaffer collateral synapses (Imig et al., 2014) is highly comparable to the length of long, single tethers described in cryo-electron tomographic reconstructions of frozen-hydrated synaptosomes (Fernández-Busnadiego et al., 2010, 2013).
Since the molecular identity of vesicle tethers remains to be clarified, it is difficult to speculate which proteins are specifically responsible for generating a prominent membrane-proximal pool in wild-type mossy fibers at rest. Whereas synaptosomes isolated from RIM1α-deficient mice exhibited a perturbation of filamentous active zone material, it is unlikely that tethering is mediated by RIM1α alone. Another candidate is bassoon, an active zone protein that has been implicated in rapid RRP replenishment in the cerebellum during high synaptic activity (Hallermann et al., 2010) and is necessary for the proper maturation of active zones in hippocampal mossy fiber synapses (Lanore et al., 2010).
I hypothesize that the tethered vesicle pool I observed at mossy fiber active zones is ideally situated to resupply the docked and primed pool of vesicles during sustained activity.
Moreover, I propose that a low ratio of docked to tethered vesicles, as I observed in hippocampal mossy fibers, may serve as a structural feature distinguishing facilitating (“tonic”) synapses. This view is compatible with recently proposed theories postulating that
“tonic” and “phasic” synapses have different ratios of tightly (TS) and loosely (LS) docked vesicle states (Neher and Brose, 2018). In the future, additional experiments examining the time course of docked and tethered pool depletion during induced short- and long-term plasticity regimes are necessary to test these hypotheses.
A direct visualization and quantification of mossy fiber tethers by cryo-electron tomography would be informative, particularly in combination with genetic perturbations. Nevertheless, to perform such experiments in a tissue context would be exceptionally technically challenging, requiring a combination of HPF and the subsequent generation of lift-out FIB lamella and ultimately tomographically reconstructed under cryo conditions (Mahamid et al., 2015; Schaffer et al., 2019). Additional technical challenges would need to be addressed,
114 including how to identify mossy fiber boutons in the vitrified sample, and how to identify active zones in the ideal orientation for tomography.
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