ANTISENSE KNOCKOUT OF CNS D2 DOPAMINE RECEPTORS

尽管多巴胺能受体激动剂和拮抗剂是已知的， 许多精神和神经疾病的标准疗法， 多巴胺受体的神经生物学研究受到一种 无法选择性地操纵个别多巴胺受体亚型。 药理学工具仅允许两个受体“家族”（D1和D2） 最近的克隆研究表明， 至少5种多巴胺受体亚型本实验 利用这些最新的分子进展， 选择性阻断多巴胺产生的寡脱氧核苷酸 体内受体。反义基因敲除可以高度特异性地抑制 mRNA翻译成功能性受体蛋白，提供了第一个 时间，选择性装置，用于获取关于 单独的多巴胺受体亚型。初步数据包括 显示D2反义寡核苷酸CNS输注产生特异性D2多巴胺 受体敲除沿着多巴胺介导的 行为和自身感受器功能。我们将使用受体放射自显影术 和匀浆结合以影响D2多巴胺的参数分析 受体敲除在时间过程、剂量相关性和受体 的特异性局部脑内应用反义寡核苷酸将 允许选择性敲除突触后与突触前D2受体。 D2多巴胺受体敲除的功能方面将通过以下方法确定： 自发性和药物诱导的运动活动的分析。 体内 微透析将用于评估特异性D2敲除对 纹状体乙酰胆碱释放和对释放的自动调节， 纹状体多巴胺的合成。 体内电生理实验 将被用来确定多巴胺受体亚型负责 自身受体介导的多巴胺终末兴奋性的变化， 监测反义处理对多巴胺神经元自发性的任何影响 活动最后，我们建议应用体内反义技术， 对其他多巴胺受体亚型的研究，从D3开始， 提供试点数据的接收器。关于我们 特定多巴胺受体亚型在中枢作用中的作用 将从这些研究中获得多巴胺。这些数据与 了解多巴胺在正常和异常行为中的作用， 发展更具体的方法来影响这些过程。

Despite the fact that dopaminergic receptor agonists and antagonists are standard therapy for a number of psychiatric and neurological disorders, study of the neurobiology of dopamine receptors is severely hampered by an inability to selectively manipulate individual dopamine receptor subtypes. Pharmacological tools permit only two receptor "families" (D1 and D2) to be distinguished whereas recent cloning studies demonstrate the existence of at least 5 dopamine receptor subtypes. The present experiments capitalize on these recent molecular advances by utilizing antisense oligodeoxynucleotides to selectively block the production of dopamine receptors in vivo. Antisense knockout allows the highly specific arrest of mRNA translation into functional receptor protein providing, for the first time, a selective means for acquiring functional information regarding individual dopamine receptor subtypes. Preliminary data are included to show that CNS infusion of D2 antisense oligo produces specific D2 dopamine receptor knockout in rat brain along with decreases in dopamine-mediated behaviors and autoreceptor function. We will use receptor autoradiography and homogenate binding to effect a parametric analysis of D2 dopamine receptor knockout in terms of time course, dose-relatedness, and receptor specificity. Local, intracerebral application of the antisense oligo will allow for selective knockout of post-versus pre-synaptic D2 receptors. Functional aspects of D2 dopamine receptor knockout will be determined by analysis of spontaneous and drug-induced motor activity. In vivo microdialysis will be used to assess the impact of specific D2 knockout on striatal acetylcholine release and on autoregulation of the release and synthesis of striatal dopamine. In vivo electrophysiological experiments will be used to determine the dopamine receptor subtype responsible for autoreceptor-mediated changes in dopamine terminal excitability, and to monitor any effects of antisense treatment on dopamine neuron spontaneous activity. Finally, we propose to apply the in vivo antisense technique to the study of other dopamine receptor subtypes, beginning with the D3 receptor for which pilot data is presented. Unique information on the role(s) of specific dopamine receptor subtypes in the central actions of dopamine will be obtained from these studies. The data are relevant to understanding the role of dopamine in normal and abnormal behavior and to the development of more specific ways to influence these processes.