| Summary: | 博士 === 長庚大學 === 基礎醫學研究所 === 96 === The endocannabinoids (eCBs), N-arachidonoylethanlamine (anandamine) and 2-arachidonoylglycerol (2-AG) belong to a family of endogenous lipids that could activate cannabinoid receptors. eCBs are released from cell in a stimulus-dependent manner by cleavage of membrane lipid precursors. Two cannabinoid receptors, CB1 and CB2 receptors have been identified so far. The CB1 receptors express most abundantly in the CNS, especially in the limbic forebrain area, such as cerebral cortex, hippocampus, and nucleus accumbens. The higher density of the cannabinoid receptors in the basal ganglia and cerebellum may correlate with its marked effects on spontaneous motor activity. Higher expression of cannabinoid receptors in limbic system may play an important role for memory and rewarding.
It is known that blockade of the cannabinoid CB1 receptor could suppress methamphetamine self-administration; however, the cellular mechanism remains unclear. In the first study, we intended to investigate the significance of brain CB1 receptors on the development of behavioral sensitization to methamphetamine. Male Sprague-Dawley rats treated with chronic methamphetamine (4 mg/kg, i.p.) for either 7 or 14 days developed behavioral sensitization to methamphetamine, evaluated by drug challenge (1 mg/kg) at withdrawal day 7. A progressive decrease in numbers of CB1 receptor (both Bmax and mRNA) but increase in binding affinity (Kd) was noticed during withdrawal days 3 to 7. Microinjection of CB1 antagonist SR 147778 into the nucleus accumbens at withdrawal day 7 significantly suppressed the behavioral sensitization to methamphetamine. In accumbal brain slices preparation, acute incubation with CB1 agonist CP 55940 dose-dependently enhanced cyclic AMP accumulation in sensitized rats; no change was noticed in control groups. Consequently, treatment of CP 55940 induced a dose-dependent (10 nM – 10 μM) phosphorylation on down-stream DARPP-32/Thr34 in sensitized rats, whiles only 10 μM CP 55940 was able to enhance the phosphoDARPP-32/T34 in control groups. Alternatively, both basal activity of PP-2B and CP 55940-induced changes in the amount of PP-2B in the nucleus accumbens were decreased in sensitized rats, but not in controls.
Chronic METH treatment decreased the expression of CB1R in the NAc at both protein and mRNA levels on withdrawal day 7. The decrease of CB1R mRNA indicates CB1R gene could be regulated by chronic treatment with METH- and dopamine-dependent addictive drug. We used 5』RACE approaches to isolate complete 5』UTR. The results of 5』RACE explored a 375 bp of 5』untranslated region of CB1 gene, also revealed that rat CB1R mRNA is encoded by two exons separated by a 17-kb intron. Using different primer pairs, we were able to obtain several 5』-deletion sequences and 3』-deletion sequences from PCR products. According to results from luciferase reporter assay conducted in HEK293 cells, we identified -114 to -130 bp of upstream of transcription start site (+1) could play a critical role for CB1R gene regulation, and a positive regulatory element existed at -1 to -66 bp. On the other hand, expressed in PC-12 and C6 cells, the best promoter activity located on -1 to -222, and the core promoter element might lie within -1 to -66, and with one or more negative regulatory elements lied within -223 to -522. Computer-assisted databank prediction suggests that there are ΔCREB, CRE-BP2, YY1, GATA-1 and Sp1 sites in the promoter region, which showed the possible transcription factors binding site of dopamine regulation in CB1R promoter.
Chronic METH treatment would change CB1R-mediated down-stream cAMP/PKA/DARP32/T34/PP2B signals in nucleus accumbal slices, which indicated that there exists a possible interaction with dopamine system and CB1R. Based on previous in vivo and in vitro studies, dopamine D2R and CB1R are co-localized in the nucleus accumbens and possible to form a heterodimer. Stimulatoin of either CB1R or D2R was evidenced to inhibit or evoke down-stream adenylyl clyclase and ERK1/2 signals, respectively. To investigate the possible signal crosstalk between CB1R and D2R, we used striatal primary culture to characterize if CB1R and dopamine D2R signaling would interact on ERK pathway. The results showed that chronic METH treatment would change the sensitivity of CB1R agonist CP55940-induced cAMP accumulation and ERK 1/2 activity. In primary striatal cultures, there are interactions between CB1 receptor and D2 receptor in regulation of ERK 1/2 signal. Activation of either CB1R or D2R in primary striatal cultures resulted an increase in ERK 1/2 phosphorylation. Co-stimulation of CB1R and D2R appeared to evoke a synergistic effect on phosphor-ERK1/2 signal. On the other hand, pertussis toxin pretreatment totally blocked the CB1R-, D2R- or CB1R/D2R-induced ERK 1/2 phosphorylation, indicates both receptors activated via the Gαi/o pathway. Pre-treatment with D2R antagonist could inhibit the CB1R-induced ERK 1/2 phosphorylation, suggests a possible heterodimer form of CB1R and D2R. Overall, through these in vivo and in vitro studies, we demonstrate that CB1R participates the progress of drug addiction through either its gene regulation or signaling interaction with dopamine D2 receptor. In the present studies, we conclude the involvement of endocannabinoid system, especially the CB1R, in drug addiction through a dopamine-dependent modulation, from behavioral to subcellular signals.
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