(A) Scheme of the adenoviral DNA constructs. A1: control vector driving the expression of GFP alone; A2: control vector driving the expression of GFP-IRES-Cre; A3: three-protein expression vector, which drives the expression of Munc18-1 (either wild-type or myc-tagged), Cre-recombinase, and GFP from an additional cassette in the viral genomic backbone (see ‘Materials and methods’). (B) Results from paired recordings from calyx synapses in Munc18-1lox/lox mice expressing either GFP alone (B1), GFP-IRES-Cre (B2), or M18WT-IRES-Cre and GFP (B3). Shown are the corresponding GFP-positive calyces (top), the presynaptic voltage-clamp protocols and presynaptic Ca2+ currents (middle), and the resulting postsynaptic EPSCs (bottom). Note the abolishment of release when Cre recombinase is expressed alone (B2 – note remaining quantal release in grey trace with enhanced scale), and the rescue of release when Cre-recombinase is expressed together with M18WT protein (B3). These measurements were done following virus injection at P1, which we found necessary for efficient elimination of endogenous Munc18-1 protein in Cre expressing calyces. (C and D) Summary of EPSC amplitudes recorded in response to 50 ms presynaptic depolarization (C; see B), for the following conditions: expression of GFP alone (left bar, black data points), expression of Cre-recombinase (middle; grey data points), and expression of Cre-recombinase together with M18WT (right; red data points and see Figure3—source data 1A). The presynaptic Ca2+ current amplitudes were unaffected by genetic removal of Munc18-1 (D and Figure3—source data 1B). (E) Adenovirus-mediated expression of myc-Munc18-1 in E2T packaging cells analyzed by SDS-PAGE and western blotting shows strong expression of recombinant protein (myc-Munc18-1, 67 kDa, actin, 43 kDa). (F) Immunohistochemistry of calyces of Held expressing the myc-tagged M18 construct in a P11 Munc18lox/lox mouse after injection at P1. Antibodies against GFP (left, green channel) and c-myc (middle, red channel) were used; the overlay image is shown on the right. Scale bar, 10 μm.