We found that one published Doc2A shRNA, but not another similarly effective published Doc2A shRNA and two different Doc2B shRNAs, altered the intrinsic properties of neurons and impaired neurotransmitter release (Figure S1

available online). Gemcitabine cost Viewed together, the expression and KD results thus argue against a selective role for Doc2A in Ca2+-triggered asynchronous release in hippocampal neurons, consistent with previous studies (Groffen et al., 2010 and Pang et al., 2011a). As a consequence, we focused on Syt7 as the most abundant candidate Ca2+ sensor that is universally expressed in all neurons and developed multiple shRNAs to knock down Syt7 mRNAs. We identified four shRNAs that suppress Syt7 expression by >80% (Figure 2A). These shRNAs, as well as the Syt7 knockouts (KOs) discussed below, interfered with expression of all Syt7 splice variants. In wild-type (WT) neurons, the Syt7 shRNAs had no effect on release as measured by inhibitory postsynaptic currents (IPSCs) elicited by a 10 Hz, 1 s stimulus train (Figure 2B). In Syt1 KO neurons, however, all four Syt7 shRNAs BMN-673 similarly suppressed the remaining asynchronous release

by ∼70% (Figures 2C and 2D). The shRNAs had no effect on intrinsic neuronal properties (Figure S2). The selective effect of the Syt7 shRNAs on release in Syt1 KO neurons but not in WT neurons could potentially be associated with changes in the expression of Syt7 or other candidate Ca2+ sensors in Syt1 KO neurons. However, quantification of all Doc2 and synaptotagmin mRNAs in WT and Syt1 KO neurons failed to uncover a specific change (Figure S3). KD experiments always give rise to concerns about specificity (Figure S1). Such specificity can be assessed by three tests. First, the same KD phenotype should be obtained with multiple independent shRNAs. Second, the KD phenotype should be rescued by expression

of shRNA-resistant WT mRNA, although this experiment is only valid if the WT rescue—which necessarily involves overexpression of the rescue mRNA—does not in itself produce a phenotype and does not impair others the shRNA-mediated KD by “sponging” up shRNAs. Third, the KD phenotype should be phenocopied by a genetic KO, although there may be genuine differences between the KD and KO phenotypes that could arise from incompleteness of shRNA-mediated KDs or from compensation effects of constitutive KOs. The first criterion for the Syt7 KD phenotype specificity was met in Figures 2C and 2D, where the lack of an effect of the Syt7 KD in WT neurons (Figures 2A and 2B) serves as an additional negative control.