REGULATION OF NEURONAL SYNAPTIC COMPONENTS

描述(改编自申请者的摘要)： 建议的研究是确定与形成相关的分子机制。 特殊突触和突触周围区域对雏鸡纤毛发育的影响 神经节(CG)神经元。CG神经元表达两类烟碱能神经元 乙酰胆碱受体：由a3、a5、b4和偶尔b2组成的nAChRs 集中在突触后膜的亚单位和BGT-nAChRs 它含有A7亚基，定位于突触周围。这个 研究人员最近的发现表明，外源、嵌合的BGT-nAChRs 被设计为包含A3亚单位的长胞浆环，但不包含 来自a5或b4亚基的相应环在 突触后膜。这些结果被解读为表明内源性 NAChR复合体也可能使用A3环进行适当的突触定位。 除AChRs外，CG神经元还表达抑制性甘氨酸受体(GlyRs)。 GlyRs和nAChRs聚集在单独但接近的突触后 成熟CG神经元上的膜区。这些不同的星团共存于 突触前终末下的神经元表面有一个帽状突触。计划中的研究 我将在逻辑上将研究结果扩展为4个目标：(1)确定本质 定位A3环路中的子域；(2)确定该子域是否 将内源性nAChRs定位于突触；(3)定义 突触后膜微异质性与突触后神经元的空间组织 分离的nAChR和GlyR簇；以及(4)识别相互作用的蛋白质 与A3亚区结合，并可能在将nAChRs靶向突触的过程中发挥作用。这个 研究将涉及分子和形态分析，包括逆转录病毒。 介导的基因原位转移、共聚焦和电子显微镜和酵母 双杂交分析。

DESCRIPTION (Adapted from the applicant's abstract): The major goal of the proposed studies is to identify molecular mechanisms relevant to the formation of specialized synaptic and perisynaptic regions on developing chick ciliary ganglion (CG) neurons. CG neurons express two classes of nicotinic acetylcholine receptors: nAChRs composed of a3, a5, b4 and occasionally b2 subunits which are concentrated in the postsynaptic membrane, and Bgt-nAChRs which contain a7 subunits, and are localized perisynaptically. The investigator's recent findings show that exogenous, chimeric Bgt-nAChRs engineered to contain the long cytoplasmic loop of the a3 subunit, but not the corresponding loop from the a5 or b4 subunit, become localized in the postsynaptic membrane. These results are interpreted to suggest that endogenous nAChR complexes may also use the a3 loop for appropriate synaptic localization. In addition to AChRs, CG neurons express inhibitory glycine receptors (GlyRs). GlyRs and nAChRs are clustered in separate, but proximate, postsynaptic membrane regions on mature CG neurons. These distinct clusters coexist on the neuron surface under one calyx-type presynaptic terminal. The planned studies will logically extend the findings in 4 aims: (1) identify the essential targeting subdomain in the a3 loop; (2) determine whether this subdomain localizes endogenous nAChRs to the synapse; (3) define the extent of postsynaptic membrane microheterogeneity and the spatial organization of the separate nAChR and GlyR clusters; and (4) identify the protein that interacts with the a3 subdomain and may function in targeting nAChRs to the synapse. The studies will involve molecular and morphological analyses, including retroviral mediated gene transfer in situ, confocal & electron microscopy, and yeast two-hybrid assays.