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

项目总结 阿片类药物在美国的流行急剧上升。尽管对公共卫生造成了巨大的影响，但基本的 阿片类药物使用障碍(OUD)的发病机制尚不清楚。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。伏隔核(NAC)内区域 腹侧纹状体是通过多巴胺受体信号强化阿片类药物的关键节点。 此外，NAC的内侧壳(MNAcSh)越来越多地与阿片类药物的强化有关 小鼠自我管理模型中的行为。阿片类药物促进中脑DA神经元释放DA，其 NAC内的投射与谷氨酸能输入汇聚。NAC神经元群体的95%由 GABA能中棘神经元(MSN)在很大程度上被分为两个群体，根据其表达情况 D_1(D_1R)与D_2(D_2R)受体。实际上，D1R+和D2R+MSN投射到不同的产出结构中 对运动控制有相反作用的基底节环路：D1R+MSN对 运动通过直接途径，而D2R+MSN通过间接途径对运动实施抑制控制 路径。此外，D1R+和D2R+MSN表达的DA受体与不同的G蛋白偶联 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D1R+的作用不同 对比D2R+NAC MSN。具体地说：1)吗啡能增强大鼠脑内谷氨酸能突触强度 NAC D1R+MSN，而D2R+MSN表现出相反的作用；2)吗啡暴露优先产生 与D1R+MSN相比，NAC D2R+MSN中的无声突触。解剖学和功能证据表明 纹状体D1R+和D2R+MSN群体的组成比之前认为的更不均匀。直到 最近，由于不能选择性地靶向D1/2R共表达的MSN，因此很难剖析 考虑到这些共表达的MSN在纹状体中的密切联系，它们各自的贡献。结果, 大多数研究都是在体外进行的，依赖于异位受体的过度表达，导致了相互矛盾的发现和 体内相关性尚不清楚。此外，D1/2R共表达的MSN在阿片类药物滥用中的具体作用尚未得到证实 接受检查。为了解决这个问题，我们开发了工具来剖析大脑的功能和解剖特性 同一个大脑中不同的MSN亚群。这包括使用交叉遗传 INTRSECT2.0系统，它允许我们标记和操作仅表达D1R或D2R的MSN的子集 D1R和D2R共表达具有很强的时间和空间特异性。有了这些工具，我们将 确定D1/2R共表达的MSN是否代表一个不同的MSN群体，不同于仅 快递D1R或D2R。我们假设D1/2R共表达的NAC MSN具有独特的解剖学、生理学、 和行为功能对阿片类药物的作用至关重要。为了解决这个问题，我们将确定生理特性 针对D1/2R-在阿片类药物作用中共表达NAC MSN(目标1)，并确定D1R和D2R的贡献 在特定的NAC MSN亚群中发出阿片诱导行为的信号(目标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).