D4 Receptor-Mediated Effects of Methylphenidate in Mice

描述（由申请人提供）：注意力缺陷多动障碍（ADHD）是一种复杂的行为状况，部分特征是注意力分散、冲动、多动和异常的新奇寻求行为。ADHD目前估计影响全国250万儿童和成人。ADHD的分子基础尚不清楚，但大量的临床数据支持中枢神经系统中多巴胺（DA）信号失调对其病因学有重要贡献的假设。虽然大多数被诊断患有ADHD的人受益于低剂量的精神兴奋剂利他林（r）（哌甲酯，MPD），但其确切的作用模式和暴露的长期后果尚不清楚。然而，由于MPD可以通过干扰DA转运蛋白功能来升高细胞外DA水平，因此其临床益处可能涉及DA受体（DAR）的间接刺激。在五种已知的DAR亚型中，最近的家族和双胞胎研究揭示了人类DA D4 R基因（DRD4.7）的等位基因与ADHD之间的关联。在解剖学上，D4 R在被认为与ADHD相关的大脑区域中表达。此外，早期同系D4 R-/-小鼠除了纹状体多巴胺含量升高和寻求新奇行为减少外，还表现出对精神兴奋剂可卡因和甲基苯丙胺的运动超敏性。总之，这些观察结果向我们提出了以下假设：DA D4 R介导MPD在小鼠和人类中的作用。由于D4受体在体内的选择性药理学拮抗作用尚未得到令人信服的证明，我们选择开始在D4受体缺陷小鼠中测试我们的假设，并发现同类（C57 B1/6 J上的N10）D4受体-/-小鼠显示出对MPD的剂量依赖性运动超敏性。受这些发现的鼓舞，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D4 Rs的青少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D4 R缺陷的幼年和青春期小鼠ADHD相关脑区基因表达的影响是什么？最后，在目标3中，我们提出产生三种新的敲入小鼠品系，其表达与ADHD相关的人类等位基因（DRD 4.7）、“正常”人类D4 R等位基因（D4.4）或绿色荧光蛋白标记（GFP）的小鼠D4 R，以更可靠地将受体蛋白定位在小鼠脑组织中。我们期望，在成功完成这些目标后，将出现对MPD暴露、D4 R刺激、基因表达和与ADHD相关的啮齿动物行为之间复杂关系的更好理解

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