Introduction: Repeated administration of morphine induces dependence and analgesic tolerance, which limits the use of the drug in chronic pain management. Elucidating the underlying mechanisms of morphine dependence and tolerance are of great interest but they still have remained unclear. The involvement of non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) as epigenetic regulators of many physiological and pathological conditions have been revealed during the last two decades. In particular, the involvement of circRNAs in some neurodegenerative diseases has been investigated. However, little is known about the role of circRNAs in the induction of morphine tolerance. We aimed to examine the expression of circOprm which is a circRNA transcribed from the mu-opioid receptor gene (Oprm1), mir-124, and mir-339 in the striatum after repeated administration of morphine in rats and also after the drug withdrawal. Method: Sixteen male Wistar rats (n = 8 in each group) were used. A rat model of morphine tolerance was induced by repeated injections of morphine (10 mg/kg) twice daily for 10 consecutive days. A control group received saline (1 ml/kg) instead of morphine during 10 days of the repeated injection. A hotplate test of analgesia was used to assess induction of analgesic tolerance after 10 days of the repeated injection compared to day 1 of the injections. Besides, two groups of rats after receiving 10 days of saline or morphine treatments were subjected to additional 30 days of morphine withdrawal. On day 10, two hours after the hotplate test of analgesia each rat was anesthetized, decapitated, and the striatum was immediately dissected on an ice-chilled surface. In rat model of withdrawal groups, the striatum was extracted on day 30 of the drug-free period. The dissected tissues were immediately frozen in liquid nitrogen and stored in a freezer until further evaluation. Changes in the gene expression of the circRNA were assessed with real-time PCR. The real time-PCR raw data was converted to 2-ΔΔCT values and then analyzed with an independent t-test. A statistically significant level was set at P<0.05. Results: The present results revealed that morphine (10 mg/kg) induced complete analgesia on day 1 of the injection (P < 0.001) compared to the saline-treated control group. However, the analgesic effect of morphine significantly decreased on day 10 compared to day 1 of the injections (P < 0.001), which revealed the induction of morphine analgesic tolerance. The results of gene expression revealed that the Oprm1 gene expression significantly increased in the striatum between the experimental groups after induction of tolerance and also after 30 days of the drug withdrawal. The results also revealed that the expression of circOprm, mir-124, and mir-339 significantly decreased in the striatum in morphine-tolerant rats compared to the saline-treated group. Besides, the results revealed that after 30 days of withdrawal the decrease in the expression of circOprm was still significant, mir-124 expression more severely decreased but mir-339 expression returned to its basal level compared to the control group. Conclusion: It can be concluded that the expression of circOprm and some miRNAs especially mir-124 and mir-339 involved in the alterations in the expression of mu-opioid receptors in the striatum in the induction of morphine tolerance and withdrawal. These results reveal that circOprm and the examined miRNAs are differently involved in molecular mechanisms of morphine tolerance and withdrawal
