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受体被假定在精神分裂症、帕金森病、药物成瘾和抑郁症等神经系统疾病中发挥作用。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。体内信号转导通路的偶联更难确定，特别是D3多巴胺受体。缺乏能够选择性区分D3多巴胺受体与其他d2样多巴胺受体的激动剂，加上D3受体的有限表达谱，导致无法完全确定其体内信号转导途径和特性。以前的研究没有定论，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，我们测试了大脑中D3多巴胺受体与离子通道偶联的假设，表现出区域和细胞类型特异性的信号功能差异，这是由与其他多巴胺受体亚型的差异共表达决定的。我们将使用由NINDS基因表达神经系统图谱（GENSAT）项目生成的新型Drd3-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绿色荧光蛋白（EGFP），有助于体内D3受体的鉴定和功能表征。鉴于其新颖性，到目前为止，这种转基因模型还没有得到充分的表征；因此，在第一个特定目的中，我们将在Drd3-EGFP转基因小鼠的五个脑区单细胞水平上确定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。在第二个特定目标中，我们将比较从Drd3- EGFP转基因小鼠的伏隔核和黑质脑区分离的荧光细胞中D3受体与离子通道在单细胞水平上的功能偶联。这两个区域在先前的报道中表达相对较高的D3受体，并且也与神经精神和神经退行性疾病有关。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，本项目将验证Drd3-EGFP转基因小鼠模型，为未来D3受体在正常和疾病状态下功能的研究提供依据。我们的长期目标是使用Drd3-EGFP小鼠来研究D3受体和构成其信号通路的蛋白质对药理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，我们将在一种新型转基因小鼠模型中表征D3多巴胺受体在体内的表达和功能。这些转基因小鼠中D3受体的表征将有助于该模型在未来的行为和药理学研究中使用，以测试针对D3受体信号转导途径的新药。确定D3多巴胺受体的体内功能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.