Functional characterization of D3 dopamine receptor in Drd3-EGFP transgenic mice

描述（由申请人提供）：神经递质多巴胺激活两大类多巴胺受体。这些包括D1样（D1和D5）和D2样多巴胺受体（D2，D3和D4）。基于其在大脑中的表达模式，以及使用D3偏好激动剂的研究，D3受体被假定在神经系统疾病如精神分裂症、帕金森病、药物成瘾和抑郁症中发挥作用。在异源表达系统中，D2样多巴胺受体偶联并调节腺苷酸环化酶、离子通道和蛋白激酶。在体内耦合到信号转导途径已更难以确定，特别是对于D3多巴胺受体。缺乏可以选择性区分D3多巴胺受体与其他D2样多巴胺受体的激动剂，加上D3受体有限的表达谱，导致无法完全确定其体内信号转导途径和特性。以前的研究尚未得出结论，或者在不同的细胞群中显示出不同的功能效应。迄今为止的结果表明，体内D3受体在大脑中的功能尚不清楚，可能表现出区域和细胞类型特异性差异。在这个R21项目中，我们测试的假设，在大脑中的D3多巴胺受体耦合到离子通道，表现出区域和细胞类型特异性的信号功能的差异，这是由与其他多巴胺受体亚型的差异共表达。我们将使用NINDS基因表达神经系统图谱（GENSAT）项目产生的新型Drd 3-EGFP转基因小鼠模型来验证这一假设。这些转基因小鼠在天然表达野生型D3多巴胺受体的细胞中表达荧光增强的绿色荧光蛋白（EGFP），促进了体内D3受体的鉴定和功能表征。鉴于其新奇，迄今为止，这种转基因模型尚未得到充分表征;因此，在第一个具体目标中，我们将在Drd 3-EGFP转基因小鼠的五个脑区域中，在单细胞水平上确定D3受体和其他多巴胺受体亚型在突触前和突触后细胞类型中的mRNA表达谱。在第二个具体的目标，我们将比较D3受体的功能耦合离子通道在单细胞水平上的荧光细胞分离的Drd 3- EGFP转基因小鼠的黑质和黑核脑区。这两个区域先前已被报道以相对高的水平表达D3受体，并且还涉及神经精神和神经退行性疾病。这个为期两年的详细R21项目将首次确定大脑中D3受体信号功能的区域和细胞类型特异性差异。此外，该项目将验证Drd 3-EGFP转基因小鼠模型，用于未来正常和疾病状态下D3受体功能的研究。我们的长期目标是使用Drd 3-EGFP小鼠研究对D3受体及其信号通路蛋白的药理学和遗传操作的行为和分子反应。公共卫生相关性：在这个R21项目中，我们将在一个新的转基因小鼠模型中描述D3多巴胺受体的表达和功能。这些转基因小鼠中D3受体的表征将促进该模型在行为和药理学研究中的未来使用，这些研究测试靶向D3受体信号转导通路的新型药物。D3多巴胺受体的体内功能测定将有助于了解其在精神分裂症、帕金森病、药物成瘾和抑郁症等疾病中的作用。

DESCRIPTION (provided by the applicant): The neurotransmitter dopamine activates two major classes of dopamine receptors. These include the D1-like (D1 and D5) and the D2-like dopamine receptors (D2, D3 and D4). Based on its expression pattern in the brain, and on studies using D3-preferring agonists, the D3 receptor is postulated to play a role in neurological disorders such as schizophrenia, Parkinson's disease, drug addiction, and depression. In heterologous expression systems, the D2-like dopamine receptors couple to and modulate adenylyl cyclases, ion channels and protein kinases. The in vivo coupling to signal transduction pathways has been more difficult to determine, particularly for the D3 dopamine receptor. The lack of agonists that can selectively distinguish the D3 dopamine receptor from other D2-like dopamine receptors, coupled with the limited expression profile of the D3 receptor has contributed to the inability to fully determine its in vivo signal transduction pathways and properties. Previous studies have been inconclusive or have shown different functional effects in different cell populations. The results to date suggest that in vivo D3 receptor function in the brain is not clear and likely exhibits region- and cell type-specific differences. In this R21 project we test the hypothesis that the D3 dopamine receptors in the brain couple to ion channels, exhibiting regional and cell type specific differences in signaling function that is determined by differential co expression with other dopamine receptor subtypes. We will test this hypothesis using the novel Drd3-EGFP transgenic mouse model generated by the NINDS Gene Expression Nervous System Atlas (GENSAT) Project. These transgenic mice express the fluorescent enhanced green fluorescent protein (EGFP) in cells natively expressing wild type D3 dopamine receptors, facilitating the identification and functional characterization of D3 receptors in vivo. Given its novelty, to date, this transgenic model has not been fully characterized; therefore in the first specific aim, we will determine the mRNA expression profile of D3 receptor and other dopamine receptor subtypes in pre- and post-synaptic cell types at the single cell level in five brain regions of the Drd3-EGFP transgenic mice. In the second specific aim we will compare the functional coupling of D3 receptors to ion channels at the single cell level in fluorescent cells isolated from the nucleus accumbens and substantia nigra brain regions of the Drd3- EGFP transgenic mice. These two regions have been previously reported to express D3 receptors at relatively high levels, and have also been implicated in neuropsychiatric and neurodegenerative disorders. This two year detailed R21 project will determine for the first time, the regional- and cell type- specific differences in D3 receptor signaling function in the brain. In addition, this project will validate the Drd3-EGFP transgenic mouse model for future studies of D3 receptor function in normal and disease states. Our long term goal is to use the Drd3-EGFP mice to study behavioral and molecular responses to pharmacological and genetic manipulation of D3 receptor and the proteins that constitutes its signaling pathways. PUBLIC HEALTH RELEVANCE: In this R21 project we will characterize the expression and function of the D3 dopamine receptors in vivo in a novel transgenic mice model. Characterization of D3 receptors in these transgenic mice will facilitate the future use of this model in behavioral and pharmacological studies that test novel drugs targeting the D3 receptor signal transduction pathways. Determination of in vivo function of D3 dopamine receptors will help understand its role in diseases such as schizophrenia, Parkinson's disease, drug addiction and depression.