miR-9 is considered a brain-specific miRNA important for neurogenesis (Krichevsky et al., 2003), but there is conflicting evidence for its expression in adult brain (Farh et al., 2005;Wu et al., 2006). We establish, using mature miR-9 specific primers (Figure S4), that miR-9 is expressed in alcohol-relevant brain regions, including SON and striatum (Figure 3C). Using the same primers, serial dilutions of synthetic miR-9 (Figure S4), and individually dispersed SON neurons (Figure 4A, B) and striatal neurons (Figure 4C), we determined that a single SON neuron expressed miR-9 at the level of ~300 mature miR-9 molecules per SON neuron (Figure 4D, E) and ~ 600 per striatal neuron (Figure 4F). Alcohol increased expression of miR-9 in both SON (Figure 4E) and striatal (Figure 4F) neurons on a time scale comparable to the downregulation of BK mRNA (Figure 2). These results suggest that miR-9 can mediate alcohol post-transcriptional regulation of neuronal BK mRNA.
