Fig 2: Munc18-1a is expressed in the auditory brainstem of mice.(A) Munc18-1a immunostaining shows specific Munc18-1a expression in the Superior olivary complex (SOC), Facial motor nucleus (VII), and Lateral reticular nucleus (LRN) of the brainstem of wild-type (WT) mice. Scale bar 200 µm. (B) Munc18-1b-Venus is not expressed in nuclei of the brainstem of homozygous Munc18-1-Venus knock-in (M18V KI) mice. (C and D) Higher magnification of indicated regions in A and B, respectively, shows that Munc18-1a is highly expressed in the SOC, which lacks Munc18-1b-Venus expression. Scale bar 100 µm. (E) Bright field (bf) image of a coronal section of P21 wild-type mouse brain (images E-J all from same section), showing part of the brainstem including MNTB (white oval) and LSO. (F) Fluorescence image of immunostaining against Munc18-1a (M18-1a), showing relative high expression in the MNTB and LSO. Scale bar 100 µm. (G) Fluorescence image of immunostaining against Munc18-1b (M18-1b), showing relative low expression in the MNTB and LSO. (H) Composite image of E, F, G showing non-complementary expression of Munc18-1a (red) and Munc18-1b (green). (I and J) Confocal zoom from image in F and G of the MNTB region showing (I) Munc18-1a expression in presynaptic Calyces (arrowheads) and post-synaptic principal cells (*). In contrast, expression of Munc18-1b is punctate but unspecific for Calyces (J). Scale bar 10 µm.

Fig 3: Alternative splicing of Munc18-1 does not affect basal evoked release.Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal munc18-1 null neurons cultured on glia islands. (A) Typical examples of paired pulse traces, traces from different intervals are plotted superimposed. (B) Both splice variants support evoked release to the same extent. Initial EPSC size (M18-1b: 3.21 ± 0.31 nA, n = 45; M18-1a: 3.50 ± 0.35 nA, n = 38, Unpaired t test, p = 0.54). (C) Quantification of the RRP estimate derived from back-extrapolation of the cumulative charge released during a RRP depleting train (100 pulses at 40 Hz) (M18-1b: 377.8 ± 52.7 pC, n = 35; M18-1a: 287.3 ± 33.3, n = 29, Unpaired t test with Welch correction, p = 0.15). (D) Quantification of the paired pulse ratio for different intervals (20 ms interval: M18-1b, 0.89 ± 0.04 EPSC2nd/EPSC1st; M18-1a, 0.79 ± 0.03 EPSC2nd/EPSC1st, Unpaired t test, p = 0.060).

Fig 4: Alternative splicing of Munc18-1 does not affect spontaneous release.Munc18-1a or -1b was reintroduced using lentivirus in autaptic hippocampal munc18-1 null neurons cultured on glia islands. (A) Example traces of spontaneous release of synaptic vesicles in the absence of stimulation. (B) Quantification of the mEPSC size and decay kinetics. mEPSC size (M18-1b: 18.11 ± 0.88 pA, n = 46; M18-1a: 16.29 ± 0.71 pA, n = 39, Unpaired t test with Welch correction, p = 0.11). mEPSC decay (M18-1b: 3.18 ± 0.15, n = 46; M18-1a: 2.84 ± 0.12, n = 39, Unpaired t test with Welch correction, p = 0.08). (C) Both splice variants support spontaneous release at similar rate. mEPSC frequency (M18-1b: 5.60 ± 1.29 Hz, n = 46; M18-1a: 6.21 ± 1.74 Hz, n = 39, Mann-Whitney Test, p = 0.5665.

Fig 5: Morphology of hippocampal neurons expressing a single splice variant of Munc18-1.(A) Amino acid sequences of the C-terminus of mouse Munc18-1a and 1b starting at site 562 (adapted from [10]). Phosphorylation sites identified in high-throughput screens are highlighted (Fig 1b, M18-1a: S590, S594; M18-1b: T581, T588, S593, S594) [16]. Arrow points to a predicted CaMKII phosphorylation site in Munc18-1a identified using NetPhos 2.0 [40] and Phosida [38, 39]. (B) Representative immunoblot of Munc18-1 protein levels in rescued munc18-1 null neurons. Neurons were analysed at DIV13 with western blot analysis using double immuno-fluorescent labelling for Munc18-1 (red) and a-Tubulin (green). (C-E) Munc18-1a or -1b was reintroduced using lentivirus in hippocampal munc18-1 null neurons cultured in the absence of glia. (C) Examples of MAP2 (dendritic marker) and VAMP2 (synaptic marker) immunostainings to visualize neuronal morphology. Scale bar = 100 µm. (D) Quantification of neuronal morphology. Number of synapses (M18-1b: 212.1 ± 8.9 synapses, n = 79; M18-1a: 241.3 ± 10.0 synapses, n = 78, Unpaired t test, p = 0.0309). Synapse density (M18-1b: 0.248 ± 0.009 synapses/µm dendrite, n = 79; M18-1a: 0.233 ± 0.006 synapses/µm dendrite, n = 78, Mann-Whitney Test, p = 0.1466). Total dendritic length (M18-1b: 0.895 ± 0.040 mm, n = 79; M18-1a: 1.057 ± 0.041 mm, n = 78, Mann-Whitney Test, p = 0.009). Number of branches (M18-1b: 127.2 ± 5.6 branches, n = 79; M18-1a: 147.0 ± 6.0 branches, n = 78, Mann-Whitney Test, p = 0.0043). (E) Sholl analysis suggests that dendritic branching is more elaborate in Munc18-1a expressing neurons. Shown are of the number of dendrite crossings per ring. Rings are placed with increasing radius around the soma (increments of 5 µm).