However, the usefulness of the calyx preparation has been limited by the fact that many conventional mouse knock out (KO) models are perinatally lethal. To overcome this limitation and to allow for a direct study of the

presynaptic roles of RIM proteins, we have devised a Cre-lox based conditional KO approach at the calyx of Held synapse, using recently generated RIM1/2 floxed mouse lines (Kaeser et al., 2011). We show that the conditional removal of RIM proteins leads to a strong decrease of Ca2+ currents in the nerve terminal, providing direct evidence that RIMs enrich Ca2+ channels at the presynaptic active zone. RIM1/2 removal also led to a marked reduction in compound screening assay the number of readily releasable vesicles, which was paralleled by a similar reduction in the number of docked vesicles.

Moreover, RIM proteins help to speed the rates of transmitter release both intrinsically and by increasing the coupling of readily releasable vesicles with Ca2+ channels. Thus, RIM proteins coordinate essential functions that ensure fast rates of transmitter release Selleck BMN 673 at synapses. We wished to investigate the presynaptic function of RIM proteins at the calyx of Held, a large CNS model synapse at which direct measurement of presynaptic Ca2+ currents can be made (see Introduction). However, constitutive RIM1α/RIM2α double KO mice die at birth (Schoch et al., 2006), which precludes their analysis at the calyx of Held, and deleting individual RIM isoforms might produce only a weak phenotype because of the functional redundancy among the isoforms (Schoch

et al., 2006 and Kaeser et al., 2008). We therefore aimed to remove RIM1/2 conditionally at the calyx of Held, using recently produced floxed mouse lines in which the expression of all RIM1 and RIM2 isoforms, including RIM1β and RIM2β, can be abolished by expression of Cre-recombinase (Kaeser et al., 2011). For this purpose, we searched for a Cre-driver mouse line that expresses Cre-recombinase specifically in the lower auditory brainstem where calyces of Held are located. We found that in Krox20+/Cre mice (Voiculescu et al., 2000), Cre-activity as revealed Thymidine kinase by a tandem-dimer red fluorescence protein (tdRFP) reporter mouse line (Luche et al., 2007) was present in the lower auditory brainstem as well as in the trigeminal nucleus and some adjacent areas, but importantly, most other areas of the brainstem and CNS were devoid of Cre activity (Figure S1A, available online). In the ventral cochlear nucleus, which harbors globular bushy cells that generate calyces of Held (see Cant and Benson, 2003, and references therein), a large number of tdRFP-positive neurons were found (Figure S1B and Figure 1A).