Gene Expression Profile of Calcium/Calmodulin-Dependent Protein Kinase IIα in Rat's Hippocampus during Morphine Withdrawal

Abstract

Introduction: Calcium/calmodulin-dependent protein kinase II (CaMKII) which is highly expressed in the hippocampus is known to play a pivotal role in reward-related memories and morphine dependence.

Methods: In the present study, repeated morphine injections once daily for 7 days was done to induce morphine tolerance in male Wistar rats, after which gene expression profile of α-isoform of CaMKII (CaMKIIα) in the hippocampus was evaluated upon discontinuation of morphine injection over 21 days of morphine withdrawal. Control groups received saline for 7 consecutive days. For gene expression study, rats' brains were removed and the hippocampus was dissected in separate groups on days 1, 3, 7, 14, and 21 since discontinuation of of morphine injection. A semi-quantitative RT-PCR method was used to evaluate the gene expression profile.

Results: Tolerance to morphine was verified by a significant decrease in morphine analgesia in a hotplate test on day 8 (one day after the final repeated morphine injections). Results showed that gene expression of CaMKIIα at mRNA level on day 1, 3, 7, 14 and 21 of morphine withdrawal was significantly altered as compared to the saline control group. Post hoc Tukey's test revealed a significantly enhanced CaMKIIα gene expression on day 14.

Discussion: It can be concluded that CaMKIIα gene expression during repeated injections of morphine is increased and this increase continues up to 14 days of withdrawal then settles at a new set point. Therefore, the strong morphine reward-related memory in morphine abstinent animals may, at least partly be attributed to, the up-regulation of CaMKIIα in the hippocampus over 14 days of morphine withdrawal.

Keywords: Gene Expression; Hippocampus; Morphine withdrawal; Semi-Quantitative RT-PCR.

Figure 1

Repeated injections of morphine induced analgesic tolerance to the drug. Eight groups of animals were used. Four groups of them received 7 days repeated administrations of saline, and 1 day after the final injection (day 8) they received saline (1 ml/kg) or one dose of morphine (5, 7.5 and 10 mg/kg) 30 min before the hotplate test. The other four groups of the animals received 7 days repeated administrations of morphine (10 mg/kg), and 1 day after the final injection (day 8) they received saline (1 ml/kg) or one dose of morphine (5, 7.5 and 10 mg/kg) 30 min before the hotplate test. Each bar represents mean±S.E.M. of pain reaction latency related to eight rats per group. Two-way ANOVA revealed significant effects of repeated injections of morphine (factor A) and groups (factor B), and their interaction [for factor A, F (1, 56) = 17.59, P < 0.001; for factor B, [F (3, 56) = 14.16, P < 0.001; for factor A * factor B, [F (3, 56) = 10.83, P < 0.001]. ***P < 0.001 compared to control group that received saline on the test day. + + +P < 0.001 compared to respective group of saline pretreated that received morphine 10 mg/kg on the test day.

Figure 2

Profiling the ratio of CamKII/β-actin mRNA expression in the hippocampus during 21 days of morphine withdrawal. Six groups of rats were used. One group of rats (n = 6) as control group received seven days repeated injections of saline (1 ml/kg), and their brain were excised 1 day after the final injection to examine gene expression in the hippocampus. The other five groups of rats received seven days repeated injections of morphine (10 mg/kg), and their brains were excised on days 1, 3, 7, 14 and 21 after the final injection of morphine to examine gene expression profile in the hippocampus. Each bar represents mean±S.E.M. related to the ratio of CamKII/ β-actin band density of 5 rats per group. *P < 0.05 compared to the saline treated control group.