Introduction: Morphine is a potent painkiller but has different side effects such as analgesic tolerance and dependence. The precise underlying mechanisms of morphine tolerance have remained elusive. Voltage-gated calcium channels play key roles in neurotransmitter release and neuronal adaptations. It has been shown that blockade of L-type voltage-gated calcium channel abolishes the development of opioid-induced analgesic tolerance. A growing body of evidence also shows that genetic and epigenetic regulations may underlie neuronal changes resulted from repeated use of morphine. In particular, non-coding RNAs such as microRNAs (miRNAs) and circular RNAs have crucial roles at gene expression level and post-transcriptional alterations of different signaling molecules in morphine tolerance. We aimed to examine and compare changes in the gene expression of Cav2.2 subtype of calcium channels, circular RNA circ-Cacna1b, miR-383-3p, mir-182 and mir-133b-3p in the cerebellar cortex after tolerance and washout of morphine tolerance in rat. Method: Male Wistar rats were used. Morphine tolerance was induced after ten days repeated subcutaneous (s.c.) injections of morphine (10 mg/kg) twice per day. A control group received saline (1 ml/kg) injections twice daily instead of morphine for 10 consecutive days. Two other groups of saline- or morphine-treated rats were subjected to 30 days of a withdrawal period after 10 days of the repeated injections. Biologically relevant non-coding RNAs to Cav2.2 mRNA were chosen based on bioinformatics tools such as miRTarget and Target Scan. For gene expression study, rats were sacrificed, each rat’s whole brain was removed (tolerance groups on day 10 and withdrawal groups on day 30 of drug-free period), and the 50 mg of the bilateral cerebellar cortex was separated in each rat. The gene expression was examined using a quantitative RT-PCR method. The real-time PCR data was analyzed using the 2-ΔΔCT (Livak) method. Then, an independent t-test was used for pairwise comparisons. P<0.05 was set as a statistically significant level. Result: The results of the RT-PCR in the cerebellar cortex revealed significant decrease in the Cav2.2 expression but significant increases circ-Cacna1b, miR-383-3p, miR-182, and miR-133b-3p levels in the morphine-tolerated group compared to the saline-treated control group (P<0.001). On day 30 of withdrawal, the result revealed no significant change in the gene expression of Cav2.2, but on contrary to the tolerance, the expression of circ-Cacna1b, miR-383-3p, miR-182, miR-133b-3p miRNA significantly decrease (P<0.001) in the cerebellar cortex of morphine-withdrawn rats compared with the respective saline-treated control group. Conclusion: It can be concluded that repeated morphine treatment differently affects the expression of Cav2.2 voltage-dependent calcium channel and related miRNAs and circular RNA in the cerebellar cortex after morphine tolerance and withdrawal. The result showed the gene expression of Cav2.2 returns to the normal situation after morphine withdrawal but the examined miRNAs and circular RNA gene expression showed opposite results after withdrawal compared to the gene expression results in the morphine-tolerated group. It can be proposed that changes at molecular levels in the cerebellar cortex may also be involved, at least partly, in morphine-induced analgesic tolerance, dependence, and withdrawal signs. The present results may propose some more cognitive functions beyond being a site of movement control and balance for the cerebellum, which desire further investigation in the future.
