Regulation of MiR-124, Let-7d, and MiR-181a in the accumbens affects the expression, extinction, and reinstatement of cocaine-induced conditioned place preference

Abstract

Molecular adaptations underlying drug seeking and relapse remain largely unknown. Studies highlight post-transcriptional modifications mediated by microRNAs (miRNAs) in addiction and other neurological disorders. We have previously shown that chronic cocaine suppresses miR-124 and let-7d and induces the expression of miR-181a in mesolimbic pathway. To further address the role and target gene regulation network of these miRNAs in vivo in cocaine addiction, we developed lentiviral vector (LV)-expressing miRNAs and their corresponding silencers for stable and regulatable miRNA expression. We tested in vivo miRNA gain and loss of function on cocaine-induced conditioned place preference (CPP) by localized LV-miRNA regulation in the nucleus accumbens (NAc). LV-miR-124 and let-7d expression in the NAc attenuates cocaine CPP, whereas LV-miR-181a enhances it. Silencing miRNAs by corresponding LV-miRNA silencers expressing perfect miRNA target sequences inversed this effect on cocaine CPP. Doxycycline treatment for switching off silencer expression abolished the observed behavioral changes. Behavioral changes mediated by LV-miRNA regulation resulted in dynamic alterations in transcription factors, receptors, and other effector genes involved in cocaine-induced plasticity. Our results describe a complex regulatory pathway mediated by miRNAs in cocaine-mediated neuronal adaptations.

Figure 1

Localized microRNA (miRNA) regulation in the nucleus accumbens (NAc) affects cocaine-induced conditioned place preference (CPP). (a) Schematic representation of behavioral training and testing schedule used. Ten groups of animals (n=12) were treated with lentiviruses expressing either green fluorescent protein (GFP) control or miRNAs (non-regulatable lentiviral vector (LV)-miR-124, miR-181a, or let-7d) or miRNA-Silencers (Sil) (regulatable LV-miR-124-Sil, miR-181a-Sil, or let-7d-Sil, either with or without doxycycline), and subjected to cocaine-induced CPP. All groups (n=12) were further subdivided into two sets cocaine (n=6) and saline (n=6) (control). After 3 days pre-conditioning recording, during the conditioning phase (days 4–9), animals received either saline or cocaine (20 mg/kg i.p.) in their corresponding cue-paired chamber (see Methods), followed by recording on day 10. After extinction over 12 days with no injection, saline priming was performed, followed by 24 h of cocaine priming and the reinstatement was recorded. (b) Expression of miR-124 in the NAc attenuates cocaine-induced CPP, whereas local knockdown of miR-124 promotes cocaine-induced CPP. (c) Conversely, miR-181a expression enhances cocaine-induced CPP, whereas local knockdown of miR-181a attenuates cocaine-induced CPP. (d) Similar to miR-124, expression of let-7d in the NAc attenuates cocaine-induced CPP, whereas local knockdown enhances cocaine-induced CPP. In all cases, doxycycline-mediated inhibition of corresponding miRNA-Sil expression brings the CPP score back to the control group levels. Data show the mean (±SEM). ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from cocaine-paired LV-GFP group; ⁺P<0.05, ⁺⁺P<0.01, and ⁺⁺⁺P<0.001 represents values significantly different from cocaine-paired corresponding LV-miRNA-expressing group; two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.

Figure 2

Regulation of microRNA (miRNA) expression in the nucleus accumbens (NAc) affects the rate of extinction in cocaine-induced conditioned place preference (CPP). Post-training extinction data of cocaine-paired animals from the different lentiviral vector (LV) groups are shown. Ten groups of animals (n=12/group) were pre-tested, trained, and tested for CPP as displayed in Figure 1. After the CPP recording on the 10th day, rats were subjected over 12 days to CPP extinction in 20 min daily sessions with full chamber access but no injections (see Methods). LV-mediated miR-124 (a), miR-181a (b), or let-7d (c) regulation in the NAc modulates the extinction rate of cocaine-induced CPP compared with the control LV-green fluorescent protein (GFP) levels, likely due to the high starting point in extinction. Values represent mean±SEM.

Figure 3

Effect of microRNA (miRNA) regulation in the nucleus accumbens (NAc) on cocaine reinstatment. After the cocaine-induced conditioned place preference (CPP) recording and establishment of extinction (by means of prolonged drug withdrawal over 12 days), animals were subjected to priming injections of 0.9% saline (1 ml/kg i.p.), followed 24 h later by a priming dose of either saline or cocaine (2 mg/kg i.p.) and place preference was recorded for 20 min (see Methods). Lentiviral vector (LV)-miRNA regulation in the NAc affects reinstating properties of cocaine after single priming with low-dose cocaine (2 mg/kg). LV-miR-124 (a) and LV-let-7d (c) expression results in an attenuated reinstatement of CPP, whereas silencing miR-124 and let-7d leads to an enhanced reinstatement of cocaine-induced CPP compared with the control LV-green fluorescent protein (GFP) group. Conversely, silencing of miR-181a (b) shows an attenuated reinstatement for cocaine-induced CPP compared with the control LV-GFP group. Values represent mean±SEM. ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from LV-GFP group; ⁺P<0.05, ⁺⁺P<0.01, and ⁺⁺⁺P<0.001 represents values significantly different from cocaine-paired corresponding LV-miRNA expressing group, by two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.

Figure 4

Effect of lentiviral vector (LV)-miR-124 regulation on the reinstatement of cocaine-induced conditioned place preference (CPP). Four groups of animals (n=12) were subjected to cocaine CPP. Immediately after the establishment of CPP, three groups were stereotaxically injected with either LV-miR-124 or miR-124-Silencers (Sil) or control green fluorescent protein (GFP), a fourth retained as naive un-operated animals. LV-miR-124 expression in the NAc after previous cocaine conditioning failed to attenuate the reinstating properties of cocaine. Silencing miR-124 leads to an enhanced reinstatement of cocaine CPP compared with LV-GFP group, whether injected either previously or after cocaine CPP, whereas LV-miR-124 expression after previous cocaine conditioning results in reinstatement of CPP comparable to that of the control GFP and shows a significantly enhanced reinstatement compared with the similar group injected before cocaine conditioning. Values represent mean±SEM. ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from the same cocaine paired corresponding LV-miRNA group injected either previously or after cocaine conditioning, by two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.

Figure 5

Quantitative real-time-polymerase chain reaction (qRT-PCR) shows specific expression or silencing of mature microRNAs (miRNAs) after lentiviral vector (LV)-miRNA regulation in the nucleus accumbens (NAc). Samples from rats bilaterally injected in the NAc with corresponding lentiviruses were analyzed at the end of the behavioral studies and used for quantification of mature miRNA levels (see Methods). (a) Quantification of mature miR-124 levels after LV-miR-124 and LV-miR-124-Silencers (Sil) expression in the presence (no expression) or absence (expression) of doxycycline. (b) Quantification of mature miR-181a levels after LV-miR-181a and LV-miR-181a-Sil expression in the presence or absence of doxycycline. (c) Quantification of mature let-7d levels after LV-let-7d and LV-let-7d-Sil expression in the presence or absence of doxycycline. Expression levels of mature miRNAs were calculated relative to U6 small nuclear RNA (snRNA) levels (see Methods) and represented as fold change compared with the control LV-GFP group. ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from LV-GFP group, by two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.

Figure 6

Differential expression of miR-124, let-7d, and miR-181a in the nucleus accumbens (NAc) alters mRNA expression of target genes. Reverse transcription-polymerase chain reaction (RT-PCR) quantification of cocaine-induced and plasticity-related microRNA (miRNA) target genes, after lentiviral-mediated regulation of miR-124 (a), miR-181a (b), and let-7d (c) transcript levels. Rats (n=12/group) were bilaterally injected into the NAc with either lentiviral vector (LV)-miRNA or the corresponding LV-miRNA-Silencer or LV-green fluorescent protein (GFP) (see Methods). Expression levels were calculated relative to cyclophilin and represented as fold change compared with the LV-GFP control group. ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from LV-GFP-injected rats, by two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.

Figure 7

miR-124, let-7d, and miR-181a cause changes at protein levels of miRNA targets genes after cocaine treatment. Immunoblots (left panels) and corresponding protein quantification graphs (right panels) display widespread changes in protein levels of several cocaine- and plasticity-related genes after lentiviral vector (LV)-mediated regulation of miR-124 (a), miR-181a (b), and let-7d (c) upon cocaine treatment. Protein levels were quantified from rats bilaterally injected into the nucleus accumbens (NAc) with either LV-miRNA or the corresponding LV-miRNA-Silencer or LV-green fluorescent protein (GFP) (see Methods). Expression levels were calculated relative to β-actin as control and represented as fold change compared with the LV-GFP group. Graphs on the right show densitometric quantification of the corresponding band intensities relative to the loading control. ^*P<0.05, ^**P<0.01, and ^***P<0.001 represents values significantly different from LV-GFP-injected rats, by two-way analysis of variance (ANOVA), followed by Bonferroni post hoc tests.