Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine

Abstract

Background: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine.

Methods: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35 mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity.

Results: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated cAMP-response element-binding protein in the medial prefrontal cortex of impoverished condition but not enriched condition rats.

Conclusion: These findings suggest that environmental enrichment, via upregulation of prefrontal microRNA-221 expression, suppresses the nicotine-induced activation of extracellular signal-regulated kinase and cAMP-response element-binding protein, which provides a potential mechanism underlying enhanced locomotor sensitivity to nicotine.

Keywords: ERK; Environmental enrichment; medial prefrontal cortex; microRNA; nicotine.

Figure 1.

Environmental enrichment increases expression of specific miRs in the PFC of rats in response to repeated administration of nicotine. (a) Expression analysis using Agilent GeneSpring showed that miRs levels were significantly enhanced in the prefrontal cortex (PFC) of enriched condition (EC) rats treated repeatedly with nicotine (Nic; 0.35mg/kg, s.c., 15 days). Data are expressed as mean ± SEM of log 2 normalized intensity values. Columns (left to right): 1–3, EC rats with nicotine treatment (EC-Nic, n=3); 4–7, impoverished condition (IC) rats with nicotine treatment (IC-Nic, n=4); 8–11, standard condition (SC) rats with nicotine treatment (SC-Nic, n=4). Rows (left top to bottom) display a list of 6 microRNAs (miRs) that were found to be significant among groups with over 2-fold expression. Quantitative real-time PCR (qPCR) validation of miR-221 (b) and miR-483 (c) from the PFC of EC, IC, and SC rats with repeated saline or nicotine injections. 2-ΔΔCt values were calculated by subtracting the ΔCt value of the control sample (miR-423-3p) from the ΔCt of miR-221 or miR-483. Two-way ANOVA on ΔΔCt value of miR-221 revealed significant main effects of housing condition (F_{(2, 18)} = 6.4, P<.01) and treatment (F_{(1, 18)} = 5.8, P<.05), and a housing × treatment interaction (F_{(2, 18)} = 4.2, P<.05). Two-way ANOVA on ΔΔCt value of miR-483 revealed a significant housing condition × treatment interaction (F_{(2, 18)} = 3.7, P<.05). There was a trend for main effects of housing condition (F_{(2, 18)} = 3.3, P=.059) and treatment (F_{(1, 18)} = 3.8, P= .068). Data are expressed as mean ± SEM. * P<.05, denotes difference between the nicotine- and saline-treated groups. #P<.05, denotes difference within nicotine-treated groups among EC, IC, and SC rats. n=4 rats/group. Total horizontal activity counts were presented as mean values of each rat within 60-minute session on day 15. Correlation of the expression levels (ΔΔCt) of miR-221 (d) and miR-483 (e) in the PFC with total horizontal activity in EC, IC, and SC rats following a 15-day nicotine administration. Dashed lines represent the 95% confidence interval of the liner regression fit (solid line).

Figure 2.

Ingenuity pathway analysis of miR-221 and miR-483 target genes. (a) -log(P values) for significantly overrepresented canonical pathways (mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase [ERK] and neurotrophin-3 (NT3)/Trk signaling) and biological functions (learning and locomotion) among miR-221 target genes. (b) Combined MAPK/ERK signaling and neurotrophin/Trk signaling pathways. Yellow symbols are targets of miR-221. (c) miR-221 target genes implicated in learning. (d) miR-221 target genes implicated in locomotion.

Figure 3.

Overexpression of miR-221 diminishes nicotine-induced increases in phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) levels. (a) Confirmation of lentiviral (LV)-miR-221 expression in PC12 cells: Image (40×) of phase contrast (left panels) and GFP expression (right panels) in PC12 cells transfected with LV-miR-1 or LV-miR-221, respectively. (b) Quantitative real-time PCR (qPCR) verification of increased miR-221 levels in PC12 cells transfected with LV-miR-1 or LV-miR-221. PC12 cells with no transfection were used as a negative control. Histobars represent means ± SEM of 3 independent experiments. 2-ΔΔCt values were calculated by subtracting the ΔCt values of the control sample (miR-423-3p) from the ΔCt of the experimental sample (miR-221). *P<.001, compared with PC12 cells with no transfection or LV-miR-1. Transfected cells were first exposed to nicotine (1 µM or 100 µM) or vehicle (Veh) for 10 minutes and then pERK1/2 levels were determined. (c) Representative immunoblots of pERK1/2 and ERK1/2 in LV-miR-1 or LV-miR-221 transfected cells following exposure to nicotine or Vehicle (Veh). Total ERK1/2 and pERK1/2 were run at the same time with the same amount of proteins. (d) The ratio of pERK1/2 to total ERK1/2 in corresponding PC12 cell groups. Data are presented as the percentage of pERK1/2 to total ERK1/2 densitometry values of immunoreactivity. Histobars represent means ± SEM of 4 independent experiments. * P<.05, compared to the respective vehicle controls. #P<.05, compared to the respective LV-miR-1 group within same concentration of nicotine.

Figure 4.

Overexpression of miR-221 in the medial prefrontal cortex (mPFC) increases nicotine-mediated locomotor activity in impoverished condition (IC) rats. At the age of 54 days, enriched condition (EC) and IC rats received bilateral injections of either LV-miR-1 or LV-miR-221 into the mPFC. (a) Representative placement mapping for coronal sections showing LV injection sites within the mPFC, where green circles represent injection sites. Cg 1, cingulate; PrL, prelimbic, IL, infralimbic cortices. (b) Coronal section (10× magnification) of lentiviral (LV) injections tagged with GFP expression into the mPFC. (c) Quantitative real-time PCR (qPCR) validation of miR-221 overexpression from the mPFC of rats injected bilaterally with either LV-miR-1 or LV-miR-221. n =7 rats/groups. *P<.05, compared with LV-miR-1. One week after lentiviral injection surgery, all rats were habituated to the locomotor activity chambers for two 60-minute sessions, once per day with no injection (d-e). Twenty-four hours after the second habituation session, all rats were injected subcutaneously with saline and placed into the activity chambers for 60 minutes to measure baseline activity (f). #P<.001, compared with EC group (n = 5–10 rats/group). Twenty-four hours after saline baseline measurement, all rats were administered nicotine (Nic, 0.35mg/kg, s.c.) or saline (Sal) on days 1 to 15. Total horizontal activity during the 30-minute preinjection habituation period was recorded in EC (g) and IC (h) rats across sessions. Total horizontal activity in EC (i) and IC (j) rats during the 60-minute postinjection period following repeated saline (Sal) or nicotine (Nic) injections.

Figure 5.

Overexpression of miR-221 in the medial prefrontal cortex (mPFC) attenuates nicotine-mediated phosphorylated extracellular signal-regulated kinase1/2 (pERK1) levels in impoverished condition (IC) rats. After locomotor sensitization paradigm, levels of pERK1/2 and ERK1/2 in the PFC of enriched condition (EC) and IC rats that received either lentiviral (LV)-miR-1 or LV-miR-221 were determined. Top panels show representative immunoblots of pERK1/2 and ERK1/2 immunoreactivity in the PFC of EC (a) and IC (b) rats after the last injection of nicotine (Nic; 0.35mg/kg, s.c.) or saline (Sal). Bottom panels show the ratio of pERK1/2 levels to total ERK1/2 levels in the PFC of corresponding EC (c) and IC (d) rats. Data are presented as the ratio of pERK1/2 to total ERK1/2 densitometry values of immunoreactivity. n =4–6 rats/group. *P<.05, compared with LV-miR-1 Sal group. #P<.05, compared with LV-miR-1 Nic group.

Figure 6.

LV-miR-221 overexpression reduces nicotine-mediated phosphorylated cAMP-response element-binding protein (pCREB) levels in impoverished condition (IC) rats. Protein expression levels of pCREB were determined after nicotine sensitization paradigm in the prefrontal cortex (PFC) of enriched condition (EC) and IC rats that received either lentiviral (LV)-miR-1 or LV-miR-221. Top panels show representative immunoblots of pCREB and CREB immunoreactivity in the PFC of EC (a) and IC (b) rats after the last injection of nicotine (Nic; 0.35mg/kg, s.c.) or saline (Sal). Bottom figures show the ratio of pCREB levels to total CREB levels in the PFC of EC (c) and IC (d) rats. Data are presented as the ratio of pCREB to total CREB densitometry values of immunoreactivity. n =4–6 rats/group. *P<.05, compared with LV-miR-1 Sal group. #P<.05, compared with LV-miR-1 Nic group.