D4 Receptor-Mediated Effects of Methylphenidate in Mice

描述(申请人提供)：注意缺陷多动障碍(ADHD)是一种复杂的行为状况，部分特征是注意力不集中、冲动、多动和异常的新奇寻求行为。据估计，目前全国有250万儿童和成人患有ADHD。ADHD的分子基础尚不清楚，但大量的临床数据支持这一假说，即中枢神经系统中多巴胺(DA)信号的失调在其病因中起重要作用。虽然大多数被诊断为ADHD的人受益于低剂量的精神刺激剂利他林(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可通过干扰DA转运体功能而升高细胞外DA水平，其临床益处可能涉及间接刺激DA受体(DAR)。在已知的五种DAR亚型中，最近的家族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ADHD相关的大脑区域表达。此外，早期同源基因D4R-/-小鼠除了纹状体多巴胺含量升高和寻求新奇行为减少外，还对精神刺激剂可卡因和甲基苯丙胺表现出运动超敏感。综上所述，这些观察向我们提出了假设：DA D4Rs介导了MPD对小鼠和人类的影响。由于D4Rs在体内的选择性药理拮抗作用尚未得到令人信服的证明，我们选择开始在D4R缺陷小鼠中测试我们的假设，并发现同源基因(C57B1/6J上的N10)D4R-/-小鼠对MPD表现出剂量依赖的运动超敏。受到这些发现的鼓舞，我们建议进行以下实验：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的少年和青少年小鼠的寻求新奇和冲动行为的研究。在目标2中，我们解决的问题是：急性和慢性MPD暴露对野生型和D4R缺陷的青少年和青少年小鼠ADHD相关脑区域的基因表达有什么影响？最后，在目标3中，我们建议产生三个新的敲入小鼠品系，它们要么表达与ADHD相关的人类等位基因(DRD4.7)，要么表达“正常”人类D4R等位基因(D4.4)，或者表达绿色荧光蛋白标记(GFP)的小鼠D4Rs，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，将更好地理解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