D4 Receptor-Mediated Effects of Methylphenidate in Mice

描述（由申请人提供）：注意缺陷多动障碍（ADHD）是一种复杂的行为状况，其部分特征是注意力分散、冲动、多动和异常的新奇行为。目前估计全国有250万儿童和成人患有多动症。ADHD的分子基础尚不清楚，但大量的临床数据支持这样的假设，即中枢神经系统多巴胺（DA）信号的失调在其病因学中起着重要作用。虽然大多数被诊断患有多动症的人都能从低剂量的精神兴奋剂利他林(r)（哌醋甲酯，MPD）中获益，但其确切的作用方式和长期暴露的后果尚不清楚。然而，由于MPD可以通过干扰DA转运体功能来提高细胞外DA水平，其临床益处可能涉及间接刺激DA受体（dar）。在五种已知的DAR亚型中，最近的家庭和双胞胎研究揭示了人类DA D4R基因（DRD4.7）的一个等位基因与ADHD之间的关联。从解剖学上讲，D4Rs在被认为与多动症相关的大脑区域表达。此外，早期遗传D4R-/-小鼠对精神兴奋剂可卡因和甲基苯丙胺表现出运动超敏感性，纹状体多巴胺含量升高，寻求新奇行为减少。综合这些观察结果，我们提出了这样的假设：DA D4Rs介导小鼠和人类MPD的影响。由于D4R在体内的选择性药理拮抗作用尚未得到令人信服的证明，我们选择在D4R缺陷小鼠中开始测试我们的假设，并发现同源（C57B1/6J上的N10） D4R-/-小鼠对MPD表现出剂量依赖性的运动超敏感性。在这些发现的鼓舞下，我们提出了以下实验过程：在具体目标1中，我们描述了旨在确定急性和/或慢性MPD暴露是否影响缺乏D4Rs的幼年和青春期小鼠的新奇寻求和冲动行为的研究。在目标2中，我们解决了这个问题：急性和慢性MPD暴露对野生型和d4r缺陷幼年和青春期小鼠adhd相关脑区基因表达的影响是什么？最后，在目标3中，我们建议产生三种新型敲入小鼠菌株，它们表达与ADHD相关的人类等位基因（DRD4.7）、“正常”的人类D4R等位基因（D4.4）或绿色荧光蛋白标记（GFP）小鼠D4R，以更可靠地定位小鼠脑组织中的受体蛋白。我们期望在成功完成这些目标后，能够更好地理解MPD暴露、D4R刺激、基因表达和与ADHD相关的啮齿动物行为之间的复杂关系

DESCRIPTION (provided by applicant): Attention Deficit Hyperactivity Disorder (ADHD) is a complex behavioral condition characterized, in part, by distractibility, impulsivity, hyperactivity, and abnormal novelty-seeking behavior. ADHD is currently estimated to affect 2.5 million children and adults nationwide. The molecular basis for ADHD is unknown but a wealth of clinical data supports the hypothesis that dysregulation of dopamine (DA) signaling in the central nervous system significantly contributes to its etiology. Although most individuals diagnosed with ADHD benefit from low doses of the psychostimulant Ritalin (r) (methylphenidate, MPD), its exact mode of action and long-term consequences of exposure are unclear. However, since MPD can elevate extracellular DA levels by interfering with DA transporter function, its cIinical benefit may involve an indirect stimulation of DA receptors (DARs). Of the five known DAR subtypes, recent family and twin studies have revealed an association between an allele of the human DA D4R gene (DRD4.7) and ADHD. Anatomically, D4Rs are expressed in brain regions thought to be relevant to ADHD. Furthermore, incipient congenic D4R-/-mice display locomotor supersensitivity to the psychostimulants cocaine and methamphetamine in addition to elevated striatal dopamine content and diminished novelty-seeking behavior. Taken together these observations suggested to us the hypothesis: DA D4Rs mediate MPD's effects in mice and humans. Since the selective pharmacological antagonism of D4Rs in vivo has yet to be convincingly demonstrated, we chose to begin testing our hypothesis in D4R-deficient mice and have found that congenic (N10 on C57B1/6J) D4R-/- mice display a dose-dependent locomotor supersensitivity to MPD. Encouraged by these findings we propose the following course of experimentation: In specific aim 1 we describe studies designed to establish whether acute and/or chronic MPD exposure influences novelty-seeking and impulsivity behaviors in juvenile and adolescent mice lacking D4Rs. In aim 2 we address the question: What are the effects of acute and chronic MPD exposure on gene expression in ADHD-relevant brain regions of wild-type and D4R-deficient juvenile and adolescent mice? Finally, in aim 3, we propose to generate three novel strains of knock-in mice that express either the human allele associated with ADHD (DRD4.7), a "normal" human D4R allele (D4.4), or Green Fluorescent Protein-tagged (GFP) murine D4Rs to more reliably localize the receptor protein in mouse brain tissue. It is our expectation that upon the successful completion of these aims, a better understanding of the complex relationship between MPD exposure, D4R stimulation, gene expression, and rodent behaviors relevant to ADHD will emerge