Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

Abstract

Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its expression in mouse brain was monitored by quantitative RT-PCR (RT-qPCR). Pilocarpine-induced seizures led to a robust, rapid, and transient increase in the primary transcript of miR-132 (pri-miR-132) followed by a subsequent rise in mature microRNA (miR-132). Activation of neurons in the hippocampus, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h of stimulation. Expression levels of primary and mature-miR-132 increased significantly between postnatal Days 10 and 24. We conclude that miR-132 is an activity-dependent microRNA in vivo, and may contribute to the long-lasting proteomic changes required for experience-dependent neuronal plasticity.

Figure 1

Genomic location of miR-132 and Levels of hippocampal primary- and mature-miR-132 following acute pilocarpine treatment. (A), Genomic location of primers used to recognize the primary sequence of miR-132. In mice, miR-132 is located on chromosome 11. The start of pri-miR-132 resides in an exon and ranges from bp 74986063–74986107. This is followed by an intronic sequence, which separates the remaining pri-miR-132 exon ranging from bp 74987260–74987519. The sense primer binds to bp 74987362–74987383 and the antisense primer binds to 74987430–74987451. All experiments in this study use total RNA that is DNase treated to prevent amplification of genomic sequence during RT-qPCR. (B, C), Mice were injected with saline or pilocarpine (300 mg/kg), sacrificed at indicated times, and RNA levels were determined by RT-qPCR. (B), Relative pri-mir-132 normalized to ARBP transcript levels from same samples (saline: n=4 mice, pilocarpine: n=3 mice, analyzed in triplicate). Two tailed t-test p =0.0182. (C), Both groups received diazepam 60 min after the initial injection. Relative mature-mir-132 normalized to snoRNA-202 (saline: n=10 mice, pilocarpine: n=8 mice, analyzed in triplicate). Two tailed t-test p = .0132.

Figure 2

Acute cocaine treatment significantly increases striatal primary-miR-132 levels while. Mice were injected with saline or cocaine (20 mg/kg), sacrificed at indicated times, and RNA levels were determined by RT-qPCR. Relative pri-mir-132 normalized to ARBP. Pri-mir-132 significantly increased 45 min post-cocaine injection compared to saline treatment and returned to baseline by 90 min (n=4–5 mice/group, analyzed in triplicate). One-way ANOVA *p < .05 (Tukey’s post-hoc test).

Figure 3

Analysis of primary-miR-132 in the olfactory bulb following odorant-exposure, and in the hippocampus following contextual fear conditioning (CFC). (A), Mice were exposed to a cotton swab soaked in water or citralva (5 μM), sacrificed at indicated times, and RNA levels were determined by RT-qPCR. Odor exposure significantly increases pri-mir-132 (n=4–5 mice/group, analyzed in triplicate). One-way ANOVA *p < .01 (Tukey’s post-hoc test). (B–D), Relative pri-miRNA levels in hippocampus following CFC, compared to naive mice. (B), Significant increase in primary-miR-132 following CFC (All groups n=5–6; except naive, n=12; paired-30, n=11, analyzed in triplicate). One-way ANOVA *p < .05, **p < .001 (Tukey’s post-hoc test). (C), Expression levels were analyzed under naive, unpaired, context alone, or paired (p30) conditions. Mice were sacrificed 30 minutes after exposure to each condition and reported as relative levels. (naive, n=12; unpaired, n=6; context, n=5; p30, n=11) One-way ANOVA, **p < .001 (Tukey’s post-hoc test). (D) Relative levels of primary-miR-132 expressed in hippocampus following CFC across a full time course (All groups n=5–6; except naive, n=12; paired-30, n=11, analyzed in triplicate). One-way ANOVA *p < .05, **p < .001 (Tukey’s post-hoc test).

Figure 4

Transcripts of primary and mature-miR-132 progressively increase during post-natal development in various brain regions. (A) Typical qPCR amplification plot of hippocampal mature-miR-132 levels expressed during post-natal days 10, 17 and 24. (B–D) Relative levels of primary (left y axis) and mature mir-132 (right y axis) expression levels in the hippocampus, olfactory bulb, and striatum are depicted for the indicated developmental time points. n=5–6 mice/group, analyzed in triplicate. One-way ANOVA **p < .001 (Tukey’s post-hoc test).