Development of dopamine D2 receptor-targeted DARTs

多巴胺神经传递的改变与各种神经精神和 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、帕金森病和物质使用障碍。时间和预期的扭曲也是症状 在许多这些疾病中，作为额外并发症的来源。人类的神经影像学研究，以及 广泛的动物研究表明，皮质-纹状体-丘脑回路的多巴胺能调节 D2和D1受体信号传导对于准确和精确的时间控制都很重要。 理解药物在体内作用的复杂性的主要障碍涉及药物在体内作用的复杂性和多样性。 在整个大脑中定位相同的受体。例如，在纹状体中，多巴胺D2受体 （D2 Rs），位于间接通路中棘神经元（iMSN），胆碱能中间神经元，终末 多巴胺能投射从中脑，以及对多巴胺能皮质纹状体终端。鉴于这种 复杂性，可能无法推断多巴胺在特定D2 R的精确作用，或其后果。 通过拮抗剂阻断这种作用，因为相同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中时，或者甚至在同一神经元内的不同位置中时，电路上的功能。当地 颅内注射药物已被用作区分药物的局部作用与其 全身效应，但在一个复杂的区域，如纹状体，这是不足以区分的行动， D2 R在不同的神经元亚型上表达。而依赖于Cre的D2 R敲除可以开始解决 在这些问题上，这种方法的局限性包括发展影响，补偿，以及D2 R 纹状体外的神经元也被删除，这使得解释效果变得复杂。 药物急性限制性拴系（DARTs）是一种新的方法，允许靶向内源性 受体与细胞类型特异性使用直接药理学治疗。这一战略已被用于 成功地靶向体内特定神经元群体中的离子型谷氨酸受体， 首次在体外用于毒蕈碱乙酰胆碱GPCR，但尚未开发用于研究GPCR， vivo.重要的是，DART方法的优点是，未修饰的天然表达的 受体是可以控制的。利用化学、蛋白质工程、基因工程和生物技术领域的专业知识 小鼠工程和行为分析，我们建议使用这种方法来询问D2 R功能， 体内，最初的重点是确定纹状体中的神经元细胞类型，其中D2 Rs调节时序。 我们已经在开发D2 R靶向DART方面取得了初步成功，我们已经在体外和体内验证了这一点。 目的1：优化DARTs用于细胞类型特异性多巴胺D2 受体拮抗作用。目的2：使用DARTs识别神经元群体 在背侧纹状体多巴胺D2受体调节时间。

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.