Using riboprobes covering the common primary transcript, we observed a marked enhancement of pri-miR-132/-212
expression following LTP induction (Fig. 5C, upper panel). This upregulation is transcription dependent as it was completely abolished by prior infusion of the RNA synthesis inhibitor ACD (Fig. 5C, lower panel). In situ hybridization using either colorimetric or fluorescence detection localized the changes in primary and mature miR-132 expression to granule cell somata of the upper and lower blades of the dentate gyrus, with no detectable changes in the dentate molecular layer (Fig. 5C and D). Thus, in Tanespimycin datasheet situ hybridization confirmed the RT-PCR analysis, and localized the enhancement in primary and mature miR-132 expression to granule cell somata.
Previous in vitro studies in primary hippocampal neuronal cultures have identified two common targets of miR-132 and -212: the Rac/Rho-family p250GAP and MeCP2 (Vo et al., 2005; Klein et al., 2007; Wayman et al., 2008). We performed Western blots for these proteins in homogenate samples from microdissected dentate gyrus collected 2 h post-HFS. There were no differences STA-9090 purchase in expression between HFS-treated and contralateral control dentate gyrus for p250GAP (1.8 ± 3.7%) or MeCP2 (1.4 ± 4.2%), whereas expression of activity-regulated cytoskeleton-associated protein (Arc) was strongly elevated (208 ± 20%). This study has uncovered novel features of miRNA regulation during LTP in the dentate gyrus of intact adult rats. Based on real-time PCR analysis of selected candidate miRNAs from a microarray screen, we demonstrated upregulation of miR-132 and -212, and downregulation of miR-219 expression during Cyclin-dependent kinase 3 LTP. It was anticipated that inhibition of LTP with an NMDAR antagonist would attenuate or eliminate these changes in mature miRNA levels. Although LTP was blocked, miR-132 and miR-219 both exhibited enhanced expression when HFS was applied in the presence of CPP, while the sign of miR-219 expression
switched from negative to positive. These results couple LTP to NMDAR-dependent downregulation of mature miR-132, -212 and -219. The regulation appears to be coordinate and specific insofar as expression of miR-124a and miR-134, both of which are expressed in granule cells, was unaffected by HFS in the presence or absence of NMDAR blockade. Furthermore, the regulation by NMDAR signaling appears to be specific to metabolism of these mature miRNAs, as NMDAR blockade had no effect on the expression of their primary and precursor transcripts. Seeking to explain the synaptic activity-dependent enhancement in miRNA expression, we turned to examine a possible role for mGluR signaling.