, 1999 and Lou et al., 2008), but the isoform responsible for this enhancement was not known. A broad-spectrum PKC inhibitor, though with questionable selectivity (Lee et al., 2008), reduced enhancement by PDBu to ∼40% of control, and inclusion of a more selective PKC blocking peptide in the

presynaptic terminal reduced the enhancement to less than 20% of that observed in control conditions (Hori et al., 1999). Previous studies suggested that the calcium-insensitive isoform PKCɛ mediates this enhancement because it is present at the calyx of Held and is activated by phorbol esters (Saitoh et al., 2001). However, our observation that phorbol ester-induced potentiation of evoked EPSCs is reduced by ∼70% in PKCα/β double knockouts compared to controls indicate that these two isoforms account for the bulk of the contribution of PKCs to EPSC enhancement by phorbol esters. check details Moreover, our results are consistent with the observation that ∼50% of phorbol ester-induced potentiation in the hippocampus is impaired in PKCβ knockout mice (Weeber et al., 2000). The component of phorbol ester-induced

enhancement that is not mediated by PKCs is likely mediated by the synaptic protein Munc13, either as a result of phorbol esters directly activating Munc13, or as a result of phorbol ester binding to the N-terminal domain of Doc2α, thereby allowing it to interact with Munc13 (Hori et al., 1999 and Lou et al., 2008). Phorbol esters enhance mEPSC frequency ∼6-fold in wild-type animals Decitabine molecular weight (Figure 8). In the absence of PKCα

and PKCβ, this enhancement is reduced by ∼50% (compare black and purple traces in Figure 8I). This result agrees with previous observations using pharmacology (Lou et al., 2008 and Oleskevich and Walmsley, 2000) and suggests that PKC plays a less important role in potentiating spontaneous release compared to evoked release. In double knockout animals, the impairment of the phorbol ester-induced increase in mEPSC frequency (Figure 8I), although moderate, contrasts with the lack of effect on tuclazepam tetanus-induced increase in mEPSC frequency (Figure 9C). Further studies are needed to understand this potential difference in the regulation of spontaneous activity. PKCα (in 129S2 genetic background) and PKCβ (in C57BL/6J genetic background) single knockout animals, generated by M. Leitges (Leitges et al., 1996 and Leitges et al., 2002), were bred together to obtain offspring heterozygous for both genes (het-het animals). Crosses of het-het animals generated α+/+ β+/+ (WT), α−/− β+/+ (αKO), α+/+ β−/− (βKO), and α−/− β−/− (double knockout) animals with a frequency of 1:16 each. All animal handling and procedures abided by the guidelines of the Harvard Medical Area Standing Committee on Animals.