Introduction: MicroRNAs (miRNAs) are small non-coding RNAs that are involved in brain physiology and pathology. This type of small regulatory RNAs control gene expression via either degrading mRNAs after binding to their 3′ untranslated region (3′UTR) or inhibition of translation. In recent years, a growing body of research have focused on the role of miRNAs in morphine tolerance and addiction. According to many published pharmacological studies, morphine tolerance and addiction is associated with not only changes in the mu-opioid receptor signaling but also in the N-methyl-D-Aspartate (NMDA) receptor downstream signaling pathway. Striatum as a target of the mesocorticolimbic pathway is involved in neural disturbances related to morphine tolerance and addiction. However, there is no report about role of miRNAs on gene expression of NMDA receptor and downstream signaling molecules in the striatum of morphine tolerant subjects. In this study, we aim to examine changes in the expression of miR133b and miR365b and their association with the GluN1 as the main subunit of NMDA receptors, calcium/calmodulin-dependent kinase II α (CamKIIα), nitric oxide synthase (NOS) and nuclear factor kappa B (NFκB) in the striatum of morphine-tolerant rats. Method: Two groups of male Wistar rats were used. Morphine tolerance was induced by injections of morphine (10 mg/kg) twice daily for 8 days. A control group also received saline (1 ml/kg) instead of morphine during the schedule. Induction of morphine tolerance was assessed using a hotplate test of analgesia on day 8. Two hours after the last injections on day 8, each rat was anesthetized, decapitated and the striatum was dissected on an ice-chilled surface. The expression of miR133b and miR365b as well as the gene expression of the GluN1, CamKIIα, NOS and NFκB was assessed using a real-time PCR method. A two-way repeated measure ANOVA was used to analyze the hotplate data. The real time-PCR data was analyzed using the 2-ΔΔCT method and an independent t-test was used for the pairwise comparisons. P<0.05 was considered as statistically significant level. Result: The results of the hotplate test of analgesia confirmed induction of morphine analgesic tolerance after the 8 days treatments (P<0.001). The results of the real-time PCR indicated that the expressions of both miR133b (P<0.01) and miR365b (P<0.01) significantly increased in the striatum of morphine tolerant rats. The results also revealed significant decreases in the mRNA level of the GluN1 (P<0.01), CamKIIα (P<0.01), NOS (P<0.001) and NFκB (P<0.01) after morphine tolerance compared to the control group. Conclusion: It can be concluded that morphine tolerance increases expression of miR133b and miR365b in the striatum that in turn may cause significant decreases in the gene expression of the NMDA receptor and downstream signaling pathway. Therefore, miR133b and miR365b should be considered as key regulators of the gene expression associated with morphine tolerance in the striatum.
