药物诱导的奖赏记忆在药物寻求、复吸中扮演着重要角色。伏隔核是成瘾药物作用的首要靶点(Nucleus accumbens, NAc。在伏隔核中，中等峭神经元根据受体表达分为表达多巴胺1型受体的神经元(Dopamine receptor 1,D1R-MSNs)和表达多巴胺2型受体的神经元(Dopamine receptor 2, D2R-MSNs。前人研究发现伏隔核中这两类神经元(D1R-MSNs和D2R-MSNs)分别介导奖赏、厌恶相关行为。吗啡、可卡因对两类神经元突触可塑调节方向相反，这由多巴胺一蛋白激酶A ( Protein kinase A, PKA)信号通路介导。该信号通路能够调节a-氨基-3-经基一5一甲基一4一异恶哩丙酸受体(AMPAR ) G1uR1 S 845位点磷酸化。AMPAG1uR15845位点磷酸化促进受体插膜，还能够放大受体电流、增加受体通道开放J陛，其结果是突触功能增强。然而，伏隔核G1uR1磷酸化是否以细胞特异性的方式调节药物引起的奖赏记忆还不清楚。
本研究以吗啡诱导的小鼠条件性位置偏好测量奖赏记忆。我们构建了AAV-DIO质粒运载G1uR1-845(E)基因，该病毒选择性地表达于含有Cre重组酶的细胞中。因此，通过在D1R-Cre, A2R-Cre转基因小鼠伏隔核内注射AAV-DIO-G1uR1-845(E)病毒，G1uR-845(E)基因能够特异性表达于伏隔核D 1 R-MSNs, D2R-MSNs上，模拟G1uR15845位点磷酸化。本研究首先通过WesternBlot检测了5天吗啡暴露后第1天、21天AMPA G1uR1和G1uR1 5845磷酸化受体蛋白表达。数据显示，不仅G1uR15845位点磷酸化蛋白表达下降，它与G1uR1蛋白的比率也显著下降。然而，吗啡暴露后21天未检测到任何变化。其次，为确定D2R-MSNs上G1uR15845位点磷酸化是否调节吗啡诱导的条件性位置偏好，我们分别在D 1 R-Cre、A2R-Cre小鼠伏隔核内注射AAV-DIO-G1uR-845(E)，并检测吗啡诱导的条件性位置偏好。吗啡诱导的小鼠条件性位置偏好至少维持21天，本研究数据显示，伏隔核内D2R-MSNs而不是D1R-MSNs上G1uR-845(E)表达，与对照组相比(注射AAV-DIO-EGFP，在吗啡诱导的条件性位置偏好获得后抑制第21天条件性位置偏好保持。随后，我们检测了伏隔核D1R-MSNs,D2R-MSNs表达G1uR1-845(E)对吗啡诱导的条件化位置偏好消退和重建的作用。结果显示，伏隔核D1R-MSNs G1uR1-845(E)表达特异性地阻断了吗啡诱导的条件化位置偏好重建并促进其消退。与此相反，G1uR1-845(E)表达对D 1 R-Cre小鼠、野生型小鼠均无作用。
综合上述结果，本研究揭示伏隔核D2R-MSNs上G1uR1-845(E)过表达，模拟AMPA G1uR1845位点磷酸化，不仅能够阻断吗啡诱导的条件性位置偏好保持，还抑制其重建并且促进其消退。这暗示，由AMPA G1uR1磷酸化介导的D1R-MSNs, D2R-MSNs活动的平衡，对调节吗啡相关的奖赏学习至关重要。
Drug-induced reward memory plays an important role in drug seeking and relapse.Nucleus accumbens (NAc) is the primary target of addictive drugs, In the NAc,medium spine neurons (MSNs) can be categorized into D1R-MSNs and D2R-MSNs based on the expression of dopamine receptor 1 and dopamine receptor 2 respectively.Previous studies revealed that the two types of neurons (D1R-MSNs and D2R-MSNs)in the NAc modulated reward and aversion-associated behaviors. Morphine and cocaine have opposite effects on the synaptic plasticity of D1R-MSNs and D2R-MSNs through dopamine-protein kinase A (PKA) signaling pathway. This signaling pathway can mediate a-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPA助G1uR1 phosphorylation at serine845 site. Besides facilitating AMPAR membrane trafficking, G1uR1一5845 phosphorylation augments AMPAR current and increases channel open probability, consequently, strengthening synaptic functions. However, it is not clear whether G1uR1-phsphorylation in the NAc can regulate drug-induced reward memory in a cell-specific manner.
In this thesis, morphine-induced mice conditioned place preference (CPP) model was utilized for testing reward memory. We generated the AAV-DIO vectors carrying G1uR1一845(E) gene, which could selectively express G1uR1一845(E) in the cells containing Cre recombinase. Hence, via injecting AAV-DIO-G1uR1一845(E) into the NAc of D1R-Cre and A2R-Cre transgenic mice, G1uR-845(E) gene could specifically express in the Nucleus accumbens D1R-MSNs or D2R-MSNs, mimicking G1uR1 phosphorylation at site serine845. In present study, we firstly assayed the expression of AMPA G1uRland G1uR1 5845 phosphorylation within NAc at day 1 and day day 21 after 5-day (lOmg/kg per day) exposure to morphine by western blot method. The results showed that not only phosphorylated protein expression of AMPA G1uR1 5845 decreased, but also the ratio of G1uR1S845 phosphorylation protein to total G1uR1 protein was significantly declined. However, no change was detected at day 21 after morphine exposure. Secondly, in order to clarify whether AMPA G1uR1一5845 phosphorylation in D2R-MSNs of NAc selectively modulates morphine-induced CPP maintenance, we respectively injected AAV DIO-G1uR-845(E) into the NAc of D1R-Cre and A2R-Cre mice and morphine CPP test was conducted. It turned out that morphine-induced CPP expression in mice could last 21 days at least. The present data revealed that G1uR1一845(E) expression in the NAc of D2R-MSNs rather than D1R-MSNs inhibited morphine-CPP retention when the test was performed 21 days after CPP acquisition, compared with control groups (AAV DIO-EGFP was injected).Subsequently, we tested the effects of G1uR1一845(E) expression in the NAc of D1R-MSNs and D2R-MSNs on the CPP extinction and reinstatement. Our results exhibited that the expression of G1uR1一845(E) in the NAc of D2R-MSNs specifically blocked the morphine-induced CPP reinstatement and facilitated CPP extinction.
Conversely, G1uR1一845(E) had no effect on D1R-Cre mice or wild-type mice.Taken together, our results revealed that over-expressing G1uR1一845(E) in the D2R-MSNs of the NAc, mimicking AMPA G1uR1 phosphorylation at site serine845,could block the maintenance of morphine-induced CPP, as well as inhibit the reinstatement of morphine-CPP and facilitated CPP extinction. These data indicate that the balance of the activities of D1R-MSNs and D2R-MSNs in the NAc modulated by AMPA G1uR1 phosphorylation is important for modulation of morphine-associated reward memory.