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. 2014 Mar 26;34(13):4581-8.
doi: 10.1523/JNEUROSCI.0445-14.2014.

microRNA-206 in rat medial prefrontal cortex regulates BDNF expression and alcohol drinking

Jenica D Tapocik  1 , Estelle Barbier, Meghan Flanigan, Matthew Solomon, Alexandra Pincus, Andrew Pilling, Hui Sun, Jesse R Schank, Courtney King, Markus Heilig
Affiliations

microRNA-206 in rat medial prefrontal cortex regulates BDNF expression and alcohol drinking

Jenica D Tapocik et al. J Neurosci. .

Abstract

Escalation of voluntary alcohol consumption is a hallmark of alcoholism, but its neural substrates remain unknown. In rats, escalation occurs following prolonged exposure to cycles of alcohol intoxication, and is associated with persistent, wide-ranging changes in gene expression within the medial prefrontal cortex (mPFC). Here, we examined whether induction of microRNA (miR) 206 in mPFC contributes to escalated alcohol consumption. Following up on a microarray screen, quantitative real-time reverse transcription PCR (qPCR) confirmed that a history of dependence results in persistent (>3weeks) up-regulation of miR-206 expression in the mPFC, but not in the ventral tegmental area, amygdala, or nucleus accumbens. Viral-mediated overexpression of miR-206 in the mPFC of nondependent rats reproduced the escalation of alcohol self-administration seen following a history of dependence and significantly inhibited BDNF expression. Bioinformatic analysis identified three conserved target sites for miR-206 in the 3'-UTR of the rat BDNF transcript. Accordingly, BDNF was downregulated in post-dependent rats on microarray analysis, and this was confirmed by qPCR. In vitro, BDNF expression was repressed by miR-206 but not miR-9 in a 3'-UTR reporter assay, confirming BDNF as a functional target of miR-206. Mutation analysis showed that repression was dependent on the presence of all three miR-206 target sites in the BDNF 3'-UTR. Inhibition of miR-206 expression in differentiated rat cortical primary neurons significantly increased secreted levels of BDNF. In conclusion, recruitment of miR-206 in the mPFC contributes to escalated alcohol consumption following a history of dependence, with BDNF as a possible mediator of its action.

Keywords: BDNF; addiction; alcohol dependence; medial prefrontal cortex; microRNA; self-administration.

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Figures

Figure 1.
Figure 1.
Blood alcohol content (BAC) data for alcohol-dependent rats over the course of 7 weeks. The BAC range averaged 200–300, a considerably high level of intoxication.
Figure 2.
Figure 2.
Increased cortical miR-206 expression after 3 weeks' protracted withdrawal in alcohol vapor-exposed rats. A, Left, Representative miR-206 FISH image within the mPFC (10× magnification). Right, The area in which miR-206 in situ hybridization was measured (+2.0 to +3.0 bregma, including both the infralimbic and prelimbic regions of the mPFC). B, In situ hybridization identified that miR-206 levels were increased in the mPFC after alcohol vapor 3 weeks after protracted abstinence (***p < 0.01, compared with control, n = 8 per group).
Figure 3.
Figure 3.
Effects of cortical miR-206 overexpression on alcohol self-administration. A, Representative mapping of viral injection sites within the mPFC denoted by an X (between +2.2 and +3.7 bregma). B, miR-206 overexpression significantly increased rewards during operant self-administration. The self-administration data represent the average of the last 10 d of self-administration. Data are presented as mean ± SEM, ***p < 0.01, significant difference; n = 8 per group. C, Injection site of the miR-206 overexpression virus (tagged with GFP, in green) and colabeled with BDNF (in red) at 10× magnification. D, A zoomed-in picture of the white panel in C (63× magnification) of the miR-206 overexpression AAV-infected cells (top, green; white arrows denote two infected cells) and BDNF expression (bottom, red; white arrows denote reduced BDNF expression). E, Colocalization of miR-206 overexpression AAV-infected cells and BDNF. F, Quantification of BDNF in infected cells, presented as percentage of the average density of BDNF expression per infected cell compared with scrambled, *p = 0.028.
Figure 4.
Figure 4.
miR-206 regulates the expression of BDNF via three seed matches on its 3′-UTR. A, B, Nanostring verification of BDNF (A) and its receptor, NTRK2, expression (B). C, Target scan identified 3 putative binding targets of miR-206 on the 3′-UTR of BDNF. D, Luciferase activity measured in HEK293T cells cotransfected with either miR-206 mimic, miR-9 mimic, or scrambled mimic, and a luciferase reporter plasmid carrying the 3′-UTR of BDNF. Renilla luciferase activity was normalized by firefly luciferase expression levels and is presented as percentage of activity achieved by the 3′-UTR of BDNF in the presence of either mimic (miR-206, miR-9, scrambled). E, Luciferase activity measured in HEK293T cells with miR-206 or scrambled mimic and a luciferase reporter plasmid with mutations at site 1 (214–220), site 2 (392–398), site 3 (1294–1300), combinations with these sites, or all sites mutated. **p < 0.01, *p < 0.05, #p = 0.05–0.1 (trend significance).
Figure 5.
Figure 5.
PCR validation of BDNF and BDNF isoform IV in the NAc, AMG, and VTA. BDNF and BNDF IV were significantly upregulated in the NAc, but not in the AMG or VTA. *p < 0.05.
Figure 6.
Figure 6.
miR-206 inhibition increases secreted BDNF in primary cortical rat neurons. A, miR-206 knockdown lentivirus (KD LV) significantly increased secreted BDNF in rat primary cortical neuron cultures with respect to scrambled lentivirus-infected controls. There was a main effect of treatment, F(1,9) = 5.67, p = 0.041, and a main effect of day, F(2,18) = 13.03 p = 0.0003, but no treatment vs day interaction, F(2,18) = 0.8221, p = 0.45. B, Representative bright-field images of rat primary cortical neurons after 3 weeks in culture at 20× magnification. C, Sample of two neurons that were infected with the miR-206 KD LV containing an mCherry reporter 2 weeks postinfection and 3 weeks in culture at 40× zoom magnification.
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