The Role of 5-HT6 Receptors in Primary Neuronal Cilia

描述（由申请人提供）：虽然近年来已经了解了大量关于成瘾的神经药理学，但我们仍然没有生物治疗方法可以预防或逆转大脑功能中的适应，这些适应“劫持”了正常动机行为中涉及的机制。纹状体等大脑区域是开发成瘾新疗法的关键靶点，但重要的是这些疗法不会破坏涉及常规动机行为的正常信息处理。这是多巴胺能药物的一个特殊问题，但其他神经递质，如血清素，也是成瘾生物学的核心。一个潜在的靶点是5-HT 6血清素受体，它在纹状体中大量表达，并影响与药物奖励和习惯形成有关的程序性学习。此外，5-HT 6受体是唯一定位于初级神经元纤毛的5-羟色胺受体，初级神经元纤毛是存在于大多数神经元上的细胞器，其在脑发育和正常认知功能中具有关键作用，但尚未完全表征。由于纤毛含有许多细胞信号系统，这些信号系统调节基因转录和可塑性而不直接干扰突触功能，这表明探索纤毛中5-HT 6定位的意义是关键的下一步。纤毛定位被认为是由GPCR的第三胞内环中的离散共有序列控制的，包括5-HT 6和一小组其他蛋白质。R21项目有两个目标。第一个目标将集中在5-HT 6受体中进行位点直接突变， 阻止它们定位于纤毛而不干扰它们的分子功能，然后我们将研究激活5-HT 6受体的信号传导后果，这些受体是否定位于纤毛中。我们将在5-HT 6基因敲除小鼠制备的原代培养纹状体神经元中进行大部分这些实验，为得出有关其功能的结论提供强有力的基础。我们将研究5-HT 6受体对纤毛和树突状形态，环AMP产生，DARPP 32磷酸化和基因表达的影响，使用PCR阵列专注于预测对5-HT 6和纤毛功能敏感的关键途径。在第二个目标中，我们将测试5-HT 6 R受体的纤毛定位是否对在背内侧纹状体中表达5-HT 6受体的已确立的行为效应至关重要，使用工具性学习作为关键行为结果。我们将在大鼠（其作用已得到充分证实）和野生型和5-HT 6敲除小鼠中进行这些实验，其中可以在存在和不存在内源性5-HT 6受体的情况下研究正常或突变型5-HT 6受体的作用。这些实验将创建一个平台，用于确定靶向纤毛中的细胞信号传导机制是否会为开发成瘾和其他强迫症的治疗方法提供新的机会。

DESCRIPTION (provided by applicant): While a great deal has been learned about the neuropharmacology of addiction in recent years, we still have no biological treatments that can prevent or reverse the adaptations in brain function that "highjack" the mechanisms involved in normal motivated behavior. Brain regions such as the striatum are key targets for developing novel treatments for addiction, but it is important that these treatments do not disrupt normal information processing involved in routine motivated behaviors. This has been a particular problem with dopaminergic drugs, but other neurotransmitters such as serotonin are also centrally involved in the biology of addiction. One potential target is the 5-HT6 serotonin receptor, which is heavily expressed in striatum and affects procedural learning that is involved in drug reward and habit formation. Additionally, the 5-HT6 receptor is the only serotonin receptor localized to the primary neuronal cilium, a cellular organelle present on most neurons that has a critical role in brain development and normal cognitive function that has not yet been thoroughly characterized. Since cilia contain a number of cellular signaling systems that modulate gene transcription and plasticity without interfering with synaptic function directly, thi suggests that exploring the significance of 5-HT6 localization in cilia is a crucial next step. Ciliary localization is thought to be governed by a discrete consensus sequence in the third intracellular loop of GPCRs including 5-HT6 and a small set of other proteins. This R21 project has two aims. The first aim will focus on making site direct mutations in 5-HT6 receptors that will prevent them from localizing to cilia without perturbing their molecular functions otherwise; we will then investigate the signaling consequences of activating 5-HT6 receptors that are or are not localized in cilia. We will perform most of these experiments in primary cultured striatal neurons prepared from 5-HT6 knockout mice, providing a strong basis for reaching conclusions about their function. We will examine the effects of 5-HT6 receptors on cilia and dendritic morphology, cyclic AMP production, DARPP32 hosphorylation, and gene expression using PCR arrays focusing on key pathways predicted to be sensitive to 5-HT6 and cilia function. In the second aim we will test whether ciliary localization of 5-HT6R receptors is critical to the well- established behavioral effects of expressing 5-HT6 receptors in dorsomedial striatum using instrumental learning as the key behavioral outcome. We will perform these experiments both in rats (where the effect is well established) and in wild-type and 5-HT6 knockout mice, where the effects of normal or mutant 5-HT6 receptors can be studied in the presence and absence of endogenous 5-HT6 receptors. These experiments will create a platform for determining whether targeting cell signaling mechanisms in the cilia will offer new opportunities for developing therapeutics for addiction and other compulsive disorders.