Taken together, our study shows that RIM proteins coordinately regulate Ca2+ channel targeting, vesicle docking and priming, and Ca2+ channel-vesicle colocalization at the presynaptic active zone. For the generation of calyx-specific conditional KO mice, we have made use of recently generated floxed mouse lines for RIM1αβ (Kaeser et al., 2008) and RIM2αβγ (Kaeser et al., 2011) and of a previously available Cre knockin mouse line in the Krox20 locus (Voiculescu et al., 2000). The presynaptic neuron pool that generates the large calyx of Held nerve terminals onto MNTB neurons is located in the contralateral ventral cochlear nucleus (VCN) and is largely represented

by globular bushy cells (Cant and Benson, Anti-diabetic Compound Library price www.selleckchem.com/screening/epigenetics-compound-library.html 2003). Krox20 is a transcription factor that is highly specifically active in rombomeres 3 and 5 of the developing hindbrain (Voiculescu et al., 2000), which give rise to a majority of neurons in the VCN (Farago et al., 2006 and Maricich et al., 2009). We found homogeneous phenotypes among the recorded

calyx of Held synapses, which indicates, together with morphological analysis (Figure 1B; L. Xiao, N. Michalski, and R.S., unpublished observations; Renier et al., 2010), that the entire population of calyx of Held-generating neurons stems from Krox20Cre-positive neurons. Thus, the Krox20Cre mouse line enables the conditional removal of floxed alleles at the calyx of Held synapse, which will make it a useful tool to advance our understanding also of other proteins of the presynaptic vesicle cycle. Using direct presynaptic recordings at the nerve terminal, we demonstrated that RIM proteins are essential for localizing Ca2+ channels to the active zone, as shown by the clear decrease in the presynaptic Ca2+ current density in RIM1/2

Adenosine triphosphate cDKO calyces. These direct recordings at the nerve terminal show that RIMs determine presynaptic Ca2+ channel density without changing major biophysical parameters of Ca2+ channel gating, although we cannot exclude that RIMs influence Ca2+ channel inactivation with prolonged pulses (>50 ms, Figure 2; Kiyonaka et al., 2007); this, however, will be relevant only for prolonged presynaptic AP trains. Analyzing RIM-dependent presynaptic Ca2+ channel targeting has been limited because previously no KO mice deleting all RIM1/2 isoforms were available (Schoch et al., 2002 and Calakos et al., 2004) and because previously investigated synapses, including the C. elegans neuromuscular synapse ( Koushika et al., 2001), did not allow for measurements of presynaptic Ca2+ currents. Using cultured RIM1/2 cDKO neurons, Kaeser et al. (2011) have observed an ∼2-fold reduction in Ca2+ transients in presynaptic boutons during an AP. This, together with our finding that presynaptic APs are unchanged ( Figure 2), is evidence for a reduced Ca2+ channel density also at small bouton-like synapses.