伏隔核CART通过钙离子调节CaMK-D3R负反馈调节可卡因奖赏效应

Cocaine- and amphetamine- regulated transcript (CART) peptides in nucleus accuments (NAcc) is as a key modulator in the rewarding and reinforcing effects of addiction drugs. The other studies demonstrated that the microinjection of CART 55-102 peptides into NAcc had no effect on locomotion， even attenuated the increase of locomotor activity produced by systemic cocaine and amphetamine administration. Our previous studies shown that CART 55-102 peptides dose-dependently inhibited the Ca2+ influx, and attenuated the cocaine-induced Ca2+ influx in cultured NAcc neurons. Moreover, microinjection of CART 55-102 peptides into NAcc blocked the cocaine-induced behavioral sensitization. Thus, CART in NAcc generate a negative regulation as they modulated the development of the rewarding and reinforcing effects of addiction drugs, but the associated regulated molecular mechanisms are not yet answered. The present studies will elucidate the activity alternation of the Ca2+/calmodulin/CaMKⅡand cAMP/PKA/pCREB signaling pathway; the protein-prtein interaction between CaMKⅡand D3R, and the functional alternation of D3R in NAcc after the microinjection of CART 55-102 peptides into NAcc using the series experiments of electrophysiology and immunobiochemicy, such as the tissue-attached configuration of the patch clamp, the Co-immunoprecipitation and autoradiography et al. Further, these studies will explore that effects of the over-expression of CaMKⅡ levels with microinjection of the herpes simplex virus (HSV) vector into NAcc on the sensitization and cAMP/PKA/pCREB signaling induced by cocaine will be inhibited by the microinjections of CART peptides into NAcc. The microinjection of CART peptides into NAcc in D3R-Knockout mice will have no effect on the cocaine-induced behavioral sensitiztion. Accordingly, CART peptides as the negative modulator in NAcc may eventually prove to be potential targets for the design of pharmacotherapies to treat addiction.

可卡因安非他明调节(CART)肽是成瘾药物奖赏的关键调控因子。研究报道证实定位注射至伏隔核的CART活性肽稀释成瘾药物产生的奖赏效应。前期实验证实，CART活性肽呈剂量依赖型抑制伏隔核神经细胞内钙离子浓度，并且稀释可卡因激活的钙离子内流。CART活性肽被定位注射至伏隔核可阻断可卡因行为敏感化的产生。因此推测，伏隔核存在的CART肽负反馈调控可卡因奖赏效应，但调控机制尚未清楚。本研究通过一系列实验观察定位注射至伏隔核区域内的CART肽调节可卡因增强的行为活动性和神经元内Ca2+/calmodulin/CaMKⅡ和cAMP-PKA信号通路活性变化，及观察CaMKⅡ调节D3R突变和功能的变化； 使用高表达CaMKⅡ病毒载体和D3R基因敲除小鼠，进一步证实CART肽负调控可卡因奖赏效应与伏隔核内CaMKⅡ调节D3R功能密切相关。

项目背景：定位注射到伏隔核区的CART 活性肽，稀释可卡因增加的行为活动性。本课题组前期研究报道，小鼠纹状体内CART基因和蛋白水平的变化趋势呈倒“U”型形成负反馈调节曲线。另外，相对于正常小鼠，D1R基因敲除小鼠稀释可卡因或者咖啡因增加的纹状体内CART肽水平，反之，D2R基因敲除小鼠增强这些刺激效应。 CART肽作为可卡因成瘾的关键负调节因子与多巴胺受体密切相关。主要研究内容：高表达 shRNA-CaMKⅡα的病毒载体和CART活性肽联合定位注射至大鼠伏隔核区域，评估可卡因产生的行为活动性和行为敏感性，该区域pCaMKⅡα-D3R连接蛋白水平； D3R磷酸化水平及下游效应信号分子cAMP，PKA和pCREB蛋白水平。关键数据和重要结果：定位注射至大鼠脑内伏隔核区的CART活性肽（1.0 or 2.5 μg, 0.5μl/side）稀释可卡因增强该区域内多巴胺受体D1和D2活性和D3磷酸化水平。可卡因激活伏隔核区域cAMP 水平，PKA 活性和 pERK 和pCREB 水平，但被定位注射至此区域的CART肽阻断。因此，CART肽主要通过降低DR功能抑制可卡因成瘾的奖赏效应。体外实验证实CART活性肽（0.1, 0.5 or 1 μM)）呈剂量依赖型地直接抑制钾离子极化激活的伏隔核神经元钙离子内流和CaMKIIa磷酸化水平，对pCaMKIIα–D3R连接蛋白的水平没有影响。但是，CART活性肽稀释可卡因增加的pCaMKIIα–D3R连接蛋白的水平。同样，定位注射伏隔核CART肽不影响大鼠的正常行为，但抑制可卡因激活大鼠产生行为敏感性。因此，伏隔核区域pCaMKIIα–D3R连接蛋白是CART肽调节成瘾药物产生奖赏效应的关键机制。伏隔核定位注射LV-shRNA-CaMKⅡα病毒，腹腔反复注射可卡因显著提高大鼠产生行为敏感性和伏隔核区域CaMKIIα磷酸化水平，pCaMKIIα–D3R连接蛋白水平，CREB磷酸化水平。但是，这些增加效应被定位注射至此区域的CART 活性肽阻断。因此，在伏隔核区CART肽可能通过抑制细胞内 Ca2+内流，阻断CaMKⅡα-D3R蛋白与蛋白的相互连接，从而增强D3R负调节的功能，抑制细胞内cAMP/PKA信号通路，最终稀释可卡因产生的行为敏感性。科学意义：以CART为靶点的新药研发，可能为将来临床戒毒治疗提供新策略。