We employed homologous recombination in mouse embryonic stem (ES) cells to generate KI mutant mice with a T to C substitution in the codon sequence

of Munc13-1 residue 464 in exon 11 of the Munc13-1 (Unc13a) gene ( Figures 1A–1E; see Experimental Procedures for details). This substitution causes a tryptophan to arginine exchange (Munc13-1W464R), which abolishes Ca2+-CaM binding ( Junge et al., 2004), and introduces an AgeI restriction site. Homozygous, cre-recombined Munc13-1W464R mice were viable and fertile, had a normal life expectancy, and were indistinguishable from WT littermates in the cage environment. The FRAX597 supplier size and cytoarchitecture of Munc13-1W464R and WT brains were identical (data not shown). Importantly, PARP inhibitor whole brain Munc13-1 expression levels did not differ between Munc13-1W464R and WT mice at postnatal day (P) 9 or 28 (Figure 1F). Likewise, the expression levels of Munc13-2, Munc13-3, and key interaction partners of Munc13-1 such as RIM1 and Syntaxin-1 were similar in Munc13-1W464R and WT mice, and none of the other synaptic markers tested showed altered expression levels (see Figure S1 available online). To confirm that the W464R point mutation interferes with the Ca2+-CaM-Munc13-1 interaction in vivo, we immunoprecipitated Munc13-1 from Munc13-1W464R and WT brains and tested for coprecipitated interaction partners. While Syntaxin-1

was coprecipitated with WT Munc13-1 and Munc13-1W464R, we did not detect any Ca2+-CaM binding to Munc13-1W464R, although the levels of CaM in WT and Munc13-1W464R brains were indistinguishable (Figure 1G). These data show that the KI mutation has a specific and selective effect restricted to the interaction of Munc13-1 with Ca2+-CaM, does not interfere with

gene transcription, mRNA translation, protein stability and turnover, or other protein-protein interactions of Munc13-1, and does not have overt effects on brain structure. In preparation for functional analyses, we studied the expression and localization of Munc13-1 already at the calyx of Held of WT and Munc13-1W464R mice (Figure 2). Munc13-1 was previously reported to localize to AZs of most glutamatergic and GABAergic synapses in the brain (Augustin et al., 1999a; Varoqueaux et al., 2005). Correspondingly, immunostaining for Munc13-1 in sections containing the medial nucleus of the trapezoid body (MNTB) from WT mice at P9–P11 (before hearing onset) or P15–P17 (after hearing onset) revealed punctate immunopositive structures that surrounded postsynaptic cell bodies. Colabeling with antibodies to the AZ marker Bassoon (Dondzillo et al., 2010) indicated that Munc13-1 is localized at AZs of calyx of Held synapses (Figures 2A and 2C). The same staining pattern for Munc13-1 was observed in calyces of Munc13-1W464R mice (Figures 2B and 2D).