Determining the role of dopamine D2L versus D2S receptors in vivo

描述(由申请人提供)：多巴胺能系统是学习和记忆、奖赏和成瘾、运动和催乳素分泌等不同神经过程的关键调节器。因此，四个多巴胺能通路之间的失调导致了不可挽回的神经疾病和精神疾病，如精神分裂症、帕金森氏病和药物成瘾。具体地说，D2多巴胺受体被认为是抗精神病药物的主要靶点；然而，传统的治疗方法仍然显示出有限的疗效，并且通常会引起不良反应。大多数临床前和临床研究认为D2受体是一个单一实体，但D2基因的选择性剪接会产生两种亚型--长型(D2L)和短型(D2S)。了解D2受体信号在调节多巴胺的生理反应中的相关性，以及D2受体激动剂和拮抗剂目前在治疗中的使用情况，有必要探讨D2L和D2S在体内是否可以被认为具有同等的功能。我们实验室最近的研究表明，D2S和D2L亚型之间存在明显的功能差异，其中D2S和D2L分别具有突触前和突触后的作用。然而，如果没有异构体选择性配体，就很难准确区分这两种异构体在体内的功能。相应地，我们的实验室已经产生了D2亚型的特异性敲除，以比较生理反应和生化差异，以确定哪个受体亚型在突触前/突触后起作用，它们的激活在Sigal转导和对滥用药物的反应方面是如何不同的，最后，哪个受体亚型是特定抗精神病药物的靶点。在目标1中，我们将通过比较D2S-/-和D2L-/-与其各自的野生型(WT)对多巴胺能激动剂和拮抗剂的反应，来分析每种D2亚型在体内黑质纹状体通路中的作用。我们还将分析基础条件下和药理学挑战下的D2L和D2S特异信号。在目标2中，我们将分析每种异构体在中边缘通路中的作用，其中我们将检测可卡因的运动、奖赏和敏化反应，并剖析可卡因诱导的即刻早期基因和转导通路的细胞特异性诱导。这些研究的成功完成将为D2受体介导的多巴胺能系统的控制提供重要信息。从而扩大了我们对多巴胺介导的反应的科学知识，并为D2靶向药物的药理设计提供了关键信息。 公共卫生相关性：用于治疗精神分裂症和帕金森病的药物的疗效似乎部分与阻断或激活多巴胺D2受体有关。然而，这些疗法会产生运动障碍和其他次要影响。这个项目的目的是为了进一步了解D2受体的生理学，并确定多巴胺D2受体亚型在体内的作用。在翻译水平上，我们认为对这些异构体的作用和信号的全面了解 可能会在未来导致能够将副作用降至最低的替代疗法。为此，我们培育了选择性缺失受体长(D2L)或短(D2S)亚型的动物。在行为和分子水平上研究这些小鼠，将有助于确定每种异构体在体内的作用。我们将分析它们对抗精神病药物以及滥用药物(如可卡因)的药理反应，以确定每个异构体在对这些化合物的反应中的具体参与。我们的研究将以多学科的方式处理科学问题，范围从行为学到分子分析。这些研究将扩大D2受体介导的多巴胺能系统控制的知识。

DESCRIPTION (provided by applicant): The dopaminergic system is a key regulator of distinct neural processes such as learning and memory, reward and addiction, locomotion, and prolactin secretion. Consequently, a dysregulation among the four dopaminergic pathways contributes to the pathophysiology of irrevocable neurological disorders and psychiatric illnesses such as Schizophrenia, Parkinson's disease, and drug addiction. Specifically, D2 dopamine receptor is known to be a primary target of antipsychotic drugs; however, conventional therapeutics still exhibit limited efficacy and typically cause adverse side-effects. Most preclinical and clinical studies consider D2 receptor as a single entity, but alternative splicing of the D2 gene generates two isoforms-a long form (D2L) and a short form (D2S). Knowing the relevance of D2 receptor's signaling in the regulation of physiological responses to dopamine, and the current use of D2 receptors agonists and antagonists in therapy, it is essential to explore whether D2L and D2S can be considered as equally functioning in vivo. Recent studies from our lab have indicated a distinct functional difference between D2S and D2L isoforms, where D2S and D2L have a presynaptic and postsynaptic role, respectively. However, without isoform-selective ligands, it is difficult to accurately discriminate the in vivo function of the two isoforms. Correspondingly, our lab has generated D2 isoform specific knockouts to compare physiological responses and biochemical differences to determine which receptor isoform is acting pre-/post-synaptically, how their activation is distinct in terms of sigal transduction and in response to drugs of abuse, and finally, which receptor isoform is targeted by specific antipsychotics. The following specific aims are proposed to characterize the D2 isoforms: In aim 1, we will analyze the role of each D2 isoform in the nigrostriatal pathway in vivo, by comparing the locomotion of D2S-/- and D2L-/- with its respective wild-type (WT) in response to dopaminergic agonists and antagonists. We will also analyze D2L- and D2S-specific signaling under basal conditions and in response to pharmacological challenges. In aim 2, we will analyze the role of each isoform in the mesolimbic pathway, where we will examine the motor, rewarding and sensitization responses to cocaine and dissect cell-specific induction of immediate-early-genes and transduction pathways induced by cocaine. Successful completion of these studies will give important information on the D2 receptor-mediated control of the dopaminergic system. Thus expanding our scientific knowledge of dopamine-mediated responses and providing crucial information for pharmacological designing of D2-targeted drugs. PUBLIC HEALTH RELEVANCE: The terapeutical benefits of drugs used in the treatment of Schizophrenia and Parkinson's Disease appear partly linked to blockade or activation of the dopamine D2 receptor. However, these therapies generate movement disorders and other secondary effects. The purpose of this project is to further our understanding of the physiology of the D2 receptor and determine the role of dopamine D2 receptor isoforms in vivo. At the translational level, we believe that the full knowledge on the role and signaling of these isoforms might lead in the future to alternative therapies able to minimize secondary effects. For this purpose we have generated animals lacking selectively either the long (D2L) or the short (D2S) isoform of the receptor. Studying these mice at the behavioral and molecular level will allow determining the role of each isoform in vivo. We will analyze their pharmacological responses to antipsychotics, as well as to drugs of abuse, such as cocaine to define the specific involvement of each isoform in the response to these compounds. Our studies will approach the scientific questions in a multidisciplinary way, ranging from behavior to molecular analyses. These studies will expand the knowledge of the D2 receptor-mediated control of the dopaminergic system.