Thus, Cre activity in Krox20+/Cre mice is present in a neuron population that includes calyx of Held-generating neurons in the cochlear nucleus, which explains the presence of tdRFP-positive large nerve endings in the MNTB. We crossed the Krox20Cre/+ mice with floxed RIM1/2 mice (Kaeser et al., 2011) to generate conditional RIM1/2 double KO mice specific for the auditory brainstem. Because of germline recombination in the Krox20Cre line (Voiculescu et al., 2000), we obtained Cre-positive RIM1/2 lox/Δ

mice (see Experimental Procedures). These mice were viable and fertile, which allowed us ZD1839 mouse to investigate synaptic transmission in brain slice preparations. We will refer to synapses recorded in Cre-positive RIM1/2 lox/Δ mice as RIM1/2 cDKO synapses (for conditional double KO). As a control group, we used Cre-negative, BKM120 datasheet RIM1/2 lox/Δ littermate mice (see Experimental Procedures). Excitatory postsynaptic currents (EPSCs) evoked by afferent fiber stimulation at calyx of Held synapses in RIM1/2 cDKO mice had amplitudes of only 1.86 ± 1.73

nA (n = 12 cells), significantly smaller than in Cre-negative littermate control mice (9.8 ± 4.2 nA; n = 15 cells; Figures 1C and 1D). The evoked EPSCs also had slightly, but significantly slower rise times (Figures 1E and 1F; p = 0.0038), reflecting slowed release kinetics that will be analyzed in more detail below (see Figure 4 and Figure 5). The amplitude of spontaneous, miniature EPSCs (mEPSCs) was unchanged (Figure 1G), consistent with a presynaptic transmitter release deficit. Synaptic phenotypes were consistently observed in all synapses studied in RIM1/2 cDKO mice tuclazepam (n = 73). This indicates that Cre-mediated removal of the RIM proteins was effective, without detectable inhomogeneity across the population of the studied neurons. Having established the conditional removal of all long RIM isoforms at the calyx of Held, we are now in a position to directly study the presynaptic

function of RIM1/2 proteins. In current clamp recordings from calyces of Held, presynaptic APs elicited by afferent fiber stimulation were unchanged in RIM1/2 cDKO calyces (Figure 2), showing that changes in the AP waveform do not underlie the reduced transmitter release. We next investigated presynaptic Ca2+ currents in voltage-clamp recordings of the calyx of Held. Surprisingly, these recordings revealed a strong reduction of the amplitude of Ca2+ currents in RIM1/2 cDKO calyces (Figure 2C). In both genotypes, presynaptic Ca2+ currents started to activate at around −20 mV and the maximal Ca2+ current was observed at ∼0 mV (Figures 2C and 2D); however, the maximal Ca2+ current was only 500 ± 310 pA (n = 19 cells) in RIM1/2 cDKO calyces, whereas it was 1040 ± 250 pA (n = 9) in calyces of control mice (Figure 2E; p < 0.001).