The Munc13 Proteins Differentially Regulate Readily Releasable Pool Dynamics and Calcium-Dependent Recovery at a Central Synapse

Abstract

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

Munc13-3 Superprimes Synaptic Vesicles at Granule Cell-to-Basket Cell Synapses in the Mouse Cerebellum

“…Quantal parameters were estimated from GC-mediated uEPSCs recorded at different extracellular calcium concentrations ([Ca 2ϩ ] o : 1, 2, 5, or 10 mM, Ն80 repetitions at each concentration, 5 s stimulus interval) by a multiple-probability fluctuation analysis (MPFA) well suited for unitary connections (Clements and Silver, 2000). In the MPFA, the variance of charges (corrected for the variance of visually identified failures) is plotted versus the mean charge (including failures), which allows extracting the quantal size q, p r , and the binomial parameter N from a parabolic fit to the data (for details, see Hallermann et al, 2010).…”

“…Identical parameters were applied to analyze both genotypes. The ratio of Munc13-XFP/bassoon signal intensity (Chen et al, 2013) was calculated for each pixel within the delineated active zone for creating ratio images (see Fig. 4 A, B) or averaged per active zone for statistical analysis (see Fig.…”

“…The number of individual colocalized sites was divided by either the total number of Bassoon puncta to estimate the relative proportion of active zones exhibiting colocalization with respective Munc13-XFPs or the total number of Munc13-XFP puncta to estimate the relative specificity with which respective Munc13-XFPs are localized to Bassoon-positive active zones in the cerebellar molecular layer. This analysis was repeated with horizontally flipped Bassoon images to estimate the contribution of incidental colocalization (Lipstein et al, 2013).…”

“…The remaining three Munc13s convey more subtle influences on SV release, which are isoform and apparently also cell-type specific. Munc13-3 is mainly expressed in the cerebellum, pons, brainstem (Augustin et al, 2001), visual cortex (Yang et al, 2002), and the calyx of Held (Chen et al, 2013). It is thought to regulate p r in the cerebellum (Augustin et al, 2001;Beierlein et al, 2007;Bao et al, 2010) and potentially at the calyx of Held (Lee et al, 2013) to slow SV replenishment at the calyx of Held (Chen et al, 2013) and to play a role in network maturation in the visual cortex (Yang et al, 2002).…”

<b>Inhibitory role of Munc13-1 in antigen-induced mast cell </b><b>degranulation </b>

“…On the other hand, Munc13-4 is involved in regulated exocytosis in hematopoietic secretory cells, such as cytotoxic T lymphocytes (CTLs), platelets, neutrophils, and mast cells (7,12,27,30,33,35). It is also known that in neurons, chromaffin cells, and CTLs, multiple Munc13 isoforms are endogenously expressed, and play some redundant roles in the promotion of the docking and priming of secretory vesicles/granules (8,10,24,36). In mast cells, Munc13-4 is shown to be crucial for degranulation (11,35,38); however, the involvement of other Munc13 isoform(s) still remains unknown.…”