As noted above, expression of miR-212 is regulated by CREB. In many cases, miRNAs have been shown to influence signaling cascades that increase their expression and further modify the activity those cascades through positive or negative feedback mechanisms (Tsang et al., 2007). As CREB overexpression in ventral striatum is known to diminish the motivational properties of cocaine (Carlezon et al., 1998), we tested the hypothesis that miR-212 may regulate cocaine intake in extended access rats by amplifying striatal CREB activity through positive feedback mechanisms. Consistent with a profound stimulatory effect of miR-212 on CREB in cultured cells in vitro, we found that levels of CREB that was phosphorylated at serine 133 (i.e., activated CREB) was significantly increased (Hollander et al., 2010). Furthermore, forskolin-stimulated expression of the CREB-responsive gene fos was also increased by miR-212, as was the activity of a luciferase-based CREB reporter construct (CRE-containing element from promoter of EVX-1). Dominant negative or phosphorylation-deficient mutant forms of CREB attenuated these stimulatory effects of miR-212 on CREB signaling. More importantly, we also found that miR-212 amplified CREB signaling in the striatum
