Dissecting the functional relevance of unique subpopulations of striatal dopamine receptors in opioid use disorder

项目摘要 阿片类药物的流行在美国急剧上升。尽管公共卫生影响巨大， 阿片类药物使用障碍（OUD）的发展机制尚不清楚。纹状体多巴胺（DA） 神经传递对阿片类药物奖赏相关行为至关重要。内核内的核小体（NAc）区域 腹侧纹状体作为通过多巴胺受体信号传导的阿片条件强化的关键节点。 此外，NAc的内侧壳（mNAcSh）越来越多地涉及阿片样物质强化 小鼠自我给药模型中的行为。阿片类药物增强中脑DA神经元的DA释放， 投影与NAc中的超声波输入会聚。95%的NAc神经元群体由以下组成： GABA能中型多刺神经元（MSN）主要分为两个群体，基于表达 DA D1（D1 R）与D2（D2 R）受体。实际上，D1 R+和D2 R + MSN投射到不同的输出结构， 对运动控制具有相反作用的基底神经节回路：D1 R + MSNs对 D2 R + MSN通过直接途径对运动施加抑制控制，而D2 R + MSN通过间接途径对运动施加抑制控制。 通路此外，由D1 R+与D2 R + MSN表达的DA受体偶联至不同的G蛋白 它们通过不同的细胞内途径发出信号。在阿片类药物的背景下，吗啡对D1 R + 与D2 R + NAc MSN相比。具体地说：1）吗啡给药增加海马神经元突触强度， NAc D1 R + MSNs，而D2 R + MSNs显示相反的作用;和2）吗啡暴露优先产生 与D1 R + MSN相比，NAc D2 R + MSN中的沉默突触。解剖学和功能学证据表明， 纹状体D1 R+和D2 R + MSN群体的组成比以前认为的更异质。直到 最近，由于不能选择性靶向D1/2 R共表达的MSN， 考虑到这些共表达MSN在纹状体中的紧密接近，它们各自的贡献。因此，在本发明中， 大多数研究都是在体外进行的，并依赖于异位受体的过度表达，导致了相互矛盾的发现， 体内相关性不明确。此外，D1/2 R共表达MSN在阿片类药物滥用中的具体作用还没有得到证实。 接受检查为了解决这一问题，我们开发了工具来剖析功能和解剖特性， 不同的MSN亚群在同一个大脑中。这包括使用交叉遗传 INTRSECT2.0系统，该系统允许我们标记和操作单独表达D1 R或D2 R的MSN子集 与同时表达D1 R和D2 R的那些相比，具有很大的时间和空间特异性。有了这些工具，我们将 确定D1/2 R共表达MSN是否代表不同的MSN群体，与仅表达D1/2 R的神经元不同。 表达D1 R或D2 R。我们假设D1/2 R共表达NAc MSN具有独特的解剖学，生理学， 以及对阿片类药物作用至关重要的行为功能。为了解决这个问题，我们将确定 特异于D1/2 R-共表达NAc MSN在阿片样物质作用中的作用（目的1），并确定D1 R和D2 R的贡献 特定NAc MSN亚群中阿片样物质诱导行为的信号传导（Aim 2）。

PROJECT SUMMARY The opioid epidemic has skyrocketed in the United States. Despite the enormous public health impact, the basic mechanisms underlying the development of opioid use disorder (OUD) remain unclear. Striatal dopamine (DA) neurotransmission is critical for opioid reward-related behaviors. The nucleus accumbens (NAc) region within ventral striatum serves as a crucial node for opioid-conditioned reinforcement via dopamine receptor signaling. Furthermore, the medial shell of the NAc (mNAcSh) has increasingly been implicated in opioid reinforcement behavior in a mouse self-administration model. Opioids enhance DA release from midbrain DA neurons, whose projections converge with glutamatergic input in the NAc. 95% of the NAc neuronal population consists of GABAergic medium spiny neurons (MSNs) are largely segregated into two populations based on expression of DA D1 (D1R) versus D2 (D2R) receptors. Indeed, D1R+ and D2R+ MSNs project to different output structures in the basal ganglia circuitry which have opposite effects on motor control: D1R+ MSNs exert stimulatory effects on movement via the direct pathway whereas D2R+ MSNs exert inhibitory control over movement via the indirect pathway. Moreover, the DA receptors expressed by D1R+ versus D2R+ MSNs are coupled to different G proteins which signal via different intracellular pathways. In the context of opioids, morphine differentially acts on D1R+ versus D2R+ NAc MSNs. Specifically: 1) morphine administration increases glutamatergic synaptic strength in NAc D1R+ MSNs, while D2R+ MSNs show opposite effects; and 2) morphine exposure preferentially generates silent synapses in NAc D2R+ MSNs compared to D1R+ MSNs. Anatomic and functional evidence suggests that the composition of striatal D1R+ and D2R+ MSN populations is more heterogenous than previously thought. Until recently, the inability to selectively target D1/2R-co-expressing MSNs has made it very difficult to dissect the respective contributions of these co-expressing MSNs given their close proximity in the striatum. As a result, most studies have been in vitro and relied on ectopic receptor overexpression, leading to conflicting findings and unclear relevance in vivo. Moreover, the specific roles of D1/2R-co-expressing MSNs in opioid abuse have yet to be examined. To address this, we developed tools to dissect the functional and anatomical properties of different MSN subpopulations within the same brain. This includes use of the intersectional genetic INTRSECT2.0 system, which allows us to label and manipulate subsets of MSNs expressing D1R or D2R alone versus those co-expressing D1R and D2R with great temporal and spatial specificity. With these tools, we will determine if D1/2R-co-expressing MSNs represent a distinct MSN population, different from neurons that only express D1R or D2R. We hypothesize that D1/2R-co-expressing NAc MSNs have unique anatomic, physiologic, and behavioral functions critical for opioid actions. To address this, we will determine the physiological properties specific to D1/2R-co-expressing NAc MSNs in opioid actions (Aim 1), and identify contributions of D1R and D2R signaling in specific NAc MSN subpopulations to opioid-induced behaviors (Aim 2).