DCVs in mossy fiber synapses

The present study found that DCVs dock directly at the active zone in mossy fiber synapses at rest in acute and slice culture preparations. This finding is in contrast to analogous studies in Schaffer collateral synapses from past and present studies (van de Bospoort et al., 2012;

Farina et al., 2015; Imig et al., 2014; Siksou et al., 2009a). In the absence of Munc13 priming proteins, DCV docking, as well as all vesicle docking, at mossy fiber active zones was completely abolished and a 2.5-fold increase in DCV abundance was observed within 100 nm of the active zone membrane. Although large DCV docking was unaffected by Munc13-deletion in chromaffin cells (Man et al., 2015), these data are consistent with the finding that Munc13-1 is required for synaptic DCV exocytosis in mammalian neuron cultures (van de Bospoort et al., 2012).

While strong stimulation is necessary to trigger DCV release in cultured neurons (van de Bospoort et al., 2012), I found that mossy fiber synapses at rest harbor docked DCVs and, in the absence of Munc13 priming proteins, DCVs accumulated at the active zone which could indicate that DCV fusion (i.e. neuropeptide release) at mossy fiber synapses occurs to some degree at a basal level. Accordingly, bassoon mutant mice exhibit an accumulation of BDNF- and enkephalin-containing DCVs in the presynaptic terminals of mossy fiber synapses (Dieni et al., 2012, 2015), concurrently, in dissociated mouse hippocampal neurons, bassoon mutants exhibit a reduction in the number of fusion-competent vesicles (Altrock et al., 2003).

Both enkephalin and BDNF are involved in synaptic plasticity of the mossy fiber-CA3 synapse (Derrick et al., 1992; Dieni et al., 2012; Li et al., 2010). BDNF contributes to mossy fiber potentiation in a transactivation mechanism that leads to increased synaptic transmission, however the retrograde signaling molecules are still unknown (Huang et al., 2008). Another

121 neuropeptide, enkephalin, binds presynaptic µ-opioid receptors and is involved in frequency-dependent enhancement of mossy fiber-CA3 synaptic transmission during LTP (Derrick et al., 1992). Taken together, these data indicate that synaptic vesicles and neuropeptide-containing DCVs share overlapping molecular mechanisms that operate in steps preceding fusion at mossy fiber active zones.

Alternatively, the accumulation of DCVs may reflect a homeostatic mechanism triggered in response to the loss of network activity in Munc13-deficient slices. In support of this hypothesis, chronic pharmacological silencing of cultured mammalian neurons treated for 48 hours with TTX led to an accumulation of DCVs in both inhibitory and excitatory synapses (Tao et al., 2018a). Furthermore, Tao and colleagues found more membrane-proximal DCVs after chronic TTX treatment (Tao et al., 2018a). Additionally, the researchers found evidence of DCV fusion at the active zone as well as at non-synaptic sites following 48 hours of TTX treatment (Tao et al., 2018a). Tao and colleagues postulated that DCV accumulation in chronically silenced neurons is involved in homeostatic metaplasticity by transporting active zone material to the presynaptic membrane (Tao et al., 2018a). Homeostatic metaplasticity is the compensatory mechanism of a neuron to enhance or diminish synaptic plasticity (i.e. LTP or long-term depression) (Abraham, 2008). In this case, Tao and colleagues propose that additional active zone material is trafficked to chronically silenced synapses to increase the active zone scaffolding material and thus increase the number of release sites at the synapse as has been previously described (Shapira et al., 2003; Sorra et al., 2006; Tao et al., 2018a).

However, the comparable size of active zones reconstructed in Munc13-deficent and control mossy fiber synapses in the present study, and the lack of DCV accumulation in Munc13-deficient Schaffer collateral synapses (Imig et al., 2014) appear inconsistent with this notion.

To address this, future studies should investigate whether BDNF- or enkephalin-positive DCVs accumulate, whether the DCVs contain active zone molecules such as piccolo or bassoon (Maas et al., 2012; Shapira et al., 2003; Tao-Cheng, 2007), or whether active zones areas increase in mossy fiber synapses in Munc13-deficient slices.

My findings indicate Munc13s facilitate DCV docking and priming at mossy fiber active zones.

Past studies show that Munc13-1 is necessary for mossy fiber LTP (Yang and Calakos, 2011).

Neuropeptides released from mossy fiber boutons modulate synaptic transmission by modulation of pre- and postsynaptic targets (Chavkin et al., 1983; McQuiston and Colmers,

122 1996; Salin et al., 1995; Sherwood and Lo, 1999; Weisskopf et al., 1993). I speculate that DCV fusion mediated by Munc13s modulate synaptic transmission in mossy fiber boutons, a potentially novel mechanism for Munc13s in LTP. Further experimentation is needed to better understand the role of Munc13s in neuropeptide signaling in mossy fiber synapses.

Furthermore, I postulate that DCVs likely undergo a basal level of fusion at rest based on the accumulation of DCVs in Munc13-deficient mossy fiber synapses. However, extensive experimentation would be necessary to test whether DCVs fuse in mossy fiber boutons under basal conditions. The speculations could be tested along similar lines as DCV fusion experimentation previously performed in dissociated hippocampal cultures (van de Bospoort et al., 2012; Farina et al., 2015), however be carried out in organotypic slice cultures. By using genetic targeting of granule cells (Kohara et al., 2014), coupled with optogenetic stimulation (Madisen et al., 2012), and phluorin-labeled DCVs (van de Bospoort et al., 2012) one could explore the parameters necessary to evoke DCV fusion in mossy fiber boutons and start examining the molecular machinery regulating synaptic release.

DCVs in mossy fiber synapses have varying morphological characteristics; some DCVs have diameters similar to those of synaptic vesicles, the electron dense material in the center is sometimes segregated to the center of the vesicle with varying halo sizes between the vesicle membrane and the electron dense core, and at times the core is eccentrically located within the DCV lumen. It is possible that the differences in size and dense core opacity are caused by piecemeal degranulation of individual DCVs, as has been described in mouse mast and chromaffin cells, as well as thalamic and hypothalamic neurons (Crivellato et al., 2005). The neuropeptide content of each DCV is not known in mossy fiber boutons. It is also unknown if there is an ultrastructural correlate to neuropeptide content in individual DCVs; if for example, BDNF could be contained in DCVs with a halo, whereas enkephalin or dynorphin are packaged in DCVs with no halo. Both morphologies were observed in mossy fiber boutons.

Further work is needed to correlate the morphological characteristics of DCVs in mossy fiber synapses and the neuropeptide content stored within each DCV. If there is a correlation between DCV morphology and neuropeptide content, one could make further comparisons of DCV accumulation at mossy fiber active zones and functional aspects of the synapse.

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