Fig 5.

Basal synaptic transmission and STP characteristics of Munc13-1/2 DKO neurons rescued by bMunc13-2, Munc13-3, or their CaM-insensitive variants. Wild-type bMunc13-2 and Munc13-3 (bMunc13-2^WT and Munc13-3^WT, respectively) and their CaM-insensitive variants (bMunc13-2^CaM* and Munc13-3^CaM*, respectively) were expressed in glutamatergic, autaptic hippocampal neurons from Munc13-1/2 DKO mice, and synaptic transmission was monitored using the whole-cell voltage clamp technique. Example traces (A) and averaged amplitudes (C) of evoked excitatory postsynaptic currents (EPSCs) in neurons expressing either bMunc13-2^WT or bMunc13-2^CaM* are shown. Example traces (B) and averaged RRP size (D) released upon application of hypertonic sucrose solution (0.5 M) for 6 s and plot of the average P_vr (E) are also shown. (F) Phorbol dibutyrate (PDBu; 1 μM) was applied for a period of 60 s, and the potentiation of evoked EPSCs, normalized to the average EPSC amplitude before application, was plotted. Shown are example traces of evoked EPSC (G) and sucrose application (H) from neurons that were matched according to their initial EPSC amplitudes and averaged RRP size (I) and P_vr (J) resulting from this data set. (K and L) Example traces of EPSCs and responses to application of sucrose recorded in neurons expressing Munc13-3^WT or Munc13-3^CaM*. Average EPSC amplitude (M), RRP size (N), P_vr (O), and potentiation in response to a 2-min application of 1 μM PDBu (P) in Munc13-3^WT- and Munc13-3^CaM*-expressing neurons, as described in panels A to F, are shown. (Q) STP in neurons expressing bMunc13-2^WT (n = 74) or bMunc13-2^CaM* (n = 73). A 5-s stimulation at 10-Hz frequency (gray horizontal bar) was applied during a period of stimulation at 0.2 Hz. All data points are normalized to the first EPSC of the 10-Hz train. The different STP phases observed are indicated by arrows. Inset: magnification of the 10-Hz stimulation period. (R) STP characteristics in amplitude-matched neurons (bMunc13-2^WT, n = 25; bMunc13-2^CaM*, n = 24) as described for panel Q. (S) STP in neurons expressing Munc13-3^WT (n = 37) or Munc13-3^CaM* (n = 31) as described for panel Q. In all bar graphs, n values are indicated at the bottom of the bar. ***, P < 0.001; *, P < 0.05; ns, not significant.