Development of dopamine D2 receptor-targeted DARTs

多巴胺神经传递的改变与各种神经精神疾病和 神经退行性疾病，包括精神分裂症、抑郁症、注意力缺陷多动障碍、帕金森氏症和药物使用障碍。在时间和预期上的扭曲也是症状 作为其他并发症的来源，在许多这些疾病中。人类的神经成像研究，以及 广泛的动物研究表明，多巴胺能调制皮质-纹状体-丘脑回路 D2和D1R信号对于精确和精确的时间控制是重要的。 理解体内药物作用的错综复杂的一个主要障碍与复杂和多样的 同一受体在整个大脑中的定位。例如，在纹状体中，多巴胺D2受体 (D2Rs)，位于间接通路中的棘神经元(IMSN)、胆碱能中间神经元、终末 中脑以及谷氨酸能皮质纹状体终末的多巴胺能投射。鉴于此， 复杂性，可能不可能推断多巴胺在特定D2Rs上的准确作用或其后果 拮抗剂阻断这一作用，因为同一类型的受体可以具有互补或相反的作用 当存在于不同的神经元，甚至在同一神经元内的不同位置时，对电路功能的影响。本地 颅内注射药物已被用作区分药物的局部作用和其作用的一种策略 全身性影响，但在纹状体等复杂区域，这不足以区分 D2Rs表达于不同的神经元亚型。虽然依赖于Cre的D2R基因敲除可以开始解决 这种方法的这些问题和限制包括发育影响、补偿和D2Rs 纹状体之外的部分也被删除，使得解释效果变得复杂。 药物被拴系限制(DARTS)是一种允许内源性靶向的新方法 使用直接药物治疗的具有细胞类型特异性的受体。这一策略已经被使用 在体内成功地靶向特定神经元群体中的离子型谷氨酸受体，并已被 在体外首次亮相，但还没有被开发用于研究GPCRs。 活着。重要的是，DART方法具有这样的优势，即未经修饰的、原生表达的活性 受体是可以控制的。利用化学、蛋白质工程、基因工程方面的协作专业知识 小鼠的工程学和行为分析，我们建议使用这种方法来询问D2R功能在 体内，最初的重点是识别纹状体中D2Rs调节时序的神经细胞类型。 我们已经在开发D2R靶向DART方面取得了初步成功，我们已经在体外和 并因此提出了以下目标：目标1：优化细胞类型特异性多巴胺D2的DART 清醒行为动物的受体拮抗作用。目标2：使用DART识别神经元群体 在背侧纹状体，多巴胺D2受体调节计时。

Alterations in dopamine neurotransmission have been implicated in various neuropsychiatric and neurodegenerative disorders, including schizophrenia, depression, attention-deficit hyperactivity disorder, Parkinson’s disease, and substance use disorder. Distortions in timing and anticipation are symptoms, as well as a source of additional complications, in many of these disorders. Neuroimaging studies in humans, as well as a wide range of animal studies, have shown that dopaminergic modulation o f cortico-striatal-thalamic circuits alters timing and that both D2 and D1 receptor signaling are important for accurate and precise temporal control. A major barrier to understanding the intricacies of drug action in vivo relates to the complex and manifold localization of the same receptor throughout the brain. For example, in the striatum, dopamine D2 receptors (D2Rs), are located on indirect pathway medium spiny neurons (iMSNs), cholinergic interneurons, the terminals of dopaminergic projections from the midbrain, as well as on glutamatergic corticostriatal terminals. Given this complexity, it can be impossible to infer the precise action of dopamine at specific D2Rs, or the consequences of blocking this action by antagonists, as the same type of receptor can have complementary or opposing effects on circuit function when present in different neurons, or even in different locations within the same neuron. Local intracranial injections of drugs have been used as a strategy to differentiate the local actio ns of drugs from their systemic effects, but in a complex region such as the striatum, this is not adequate to differentiate actions of D2Rs expressed on different neuronal subtypes. While Cre-dependent knockout of D2R can begin to address these issues, limitations of this approach include developmental effects, compensation, and the fact that D2Rs outside of the striatum are also deleted, making the effects complex to interpret. Drugs Acutely Restricted by Tethering (DARTs) is a new approach that allows targeting of endogenous receptors with cell-type specificity using direct pharmacological treatment. This strategy has been used successfully to target ionotropic glutamate receptors in specific neuronal populations in vivo and has also been debuted in vitro for a muscarinic acetylcholine GPCR but has not yet been developed for studying GPCRs in vivo. Importantly, the DART approach has the advantage that the activity of unmodified, natively expressed receptors can be controlled. Leveraging collaborative expertise in chemistry, protein engineering, genetic engineering of mice, and behavioral analysis, we propose to use this method to interrogate D2R function in vivo, with an initial focus on identifying the neuronal cell types in the striatum in which D2Rs regulate timing. We have had initial success in developing D2R-targeted DARTs, which we have validated both in vitro and in vivo and thus propose the following aims: Aim 1: Optimize DARTs for cell-type-specific dopamine D2 receptor antagonism in awake behaving animals. Aim 2: Use DARTs to identify the neuronal population in the dorsal striatum in which dopamine D2 receptors modulate timing.