REGULATION OF NEURONAL SYNAPTIC COMPONENTS

控制突触形成和功能的分子机制 神经元尚不为人所知。为了深入了解这些机制， 拟议的研究将集中在尼古丁的一种特定成分上 胆碱能突触，神经元烟碱型乙酰胆碱受体(AChR)。 尤其是神经支配在调节AChR基因表达中的作用 和AChR函数、数量和分布的研究将在 雏鸡副交感睫状神经节神经元的发育 突触发生的过程。对于这些研究，唯一的来源是 突触前输入到副动眼神经核神经节，将 在突触之前的发育阶段进行手术消融 队形。为了确定神经支配是否诱导AChR基因表达， 将比较睫状神经节中前体和成熟AChR mRNA的水平 神经节前区存在和缺失时神经元的原位发育 应用核糖核酸酶保护分析和原位分析进行组织相互作用 杂交。神经支配对乙酰胆碱受体功能的影响 通过检查全细胞AChR反应和相对 不同AChR亚型在投入性剥夺和非投入性环境中的丰度和动力学 使用全细胞和单通道记录方法控制神经元。这个 神经元表面AChRs(包括沉默受体)的总数 将用放射性标记的抗AChR单抗建立。的作用 神经支配对神经元表面乙酰胆碱受体分布的调节 将通过间接免疫荧光标记与 抗AChR单抗。如果在原位发育的神经元上观察到AChR簇 当神经支配被外科手术阻止时，超微结构技术将 用于确定是否存在其他突触专门化，如 突触后密度，在这些部位组装和维持。至 检测神经源性因子调节神经元乙酰胆碱受体的能力 和AChR mRNA水平，睫状神经节神经元发育后 有无原位神经支配，将在培养中生长 适当的节前组织的存在，或有条件的介质 神经节前细胞。此外，特定的神经源性因子 调节肌肉中AChR和AChR mRNA水平，如AChR诱导 活性(ARIA)，降钙素基因相关肽和抗坏血酸 测试它们影响AChR和AChR转录水平的能力 神经元。这些研究将确定一些信号和分子 神经中控制突触组装和功能的机制 系统。关于控制形成的监管事件的知识 正常的功能连接将极大地增强我们识别、 可能还会逆转，发生在发育中的神经元中的缺陷 因疾病、伤害或发育障碍而导致的环境异常。

The molecular mechanisms that control synapse formation and function on neurons are not known. To gain insights into these mechanisms, the proposed studies will focus on a specific component of nicotinic cholinergic synapses, the neuronal nicotinic acetylcholine receptor (AChR). In particular, the role of innervation in regulating AChR gene expression and AChR function, number and distribution will be investigated in developing chick parasympathetic ciliary ganglion neurons during the process of synaptogenesis. For these studies, the sole source of presynaptic inputs to the ganglion, the accessory oculomotor nucleus, will be surgically ablated at a time in development which precedes synapse formation. To determine whether innervation induces AChR gene expression, precursor and mature AChR mRNA levels will be compared in ciliary ganglion neurons developing in situ in the presence and absence of preganglionic tissue interactions using RNase protection assays and in situ hybridization. The effects of innervation on AChR function will be determined by examining the whole-cell AChR response and the relative abundance and kinetics of the different AChR subtypes on input-deprived and control neurons using whole-cell and single-channel recording methods. The total number of surface AChRs (including silent receptors) on the neurons will be established using a radio-labeled anti-AChR mAb. The role of innervation in the regulation of AChR distribution on the neuron surface will be investigated by indirect immunofluorescence labeling with the anti-AChR mAb. If AChR clusters are observed on neurons developing in situ when innervation is surgically prevented, ultrastructural techniques will be used to determine whether other synaptic specializations, such as the postsynaptic density, are assembled and maintained at these sites. To examine the ability of neurally-derived factors to regulate neuronal AChR and AChR mRNA levels, ciliary ganglion neurons, after developing in the presence and absence of innervation in situ, will be grown in culture in the presence of the appropriate preganglionic tissue, or media conditioned by preganglionic cells. In addition, specific neurally-derived factors that regulate AChR and AChR mRNA levels in muscle, such as AChR-inducing activity (ARIA), calcitonin gene-related peptide and ascorbic acid, will be tested for their ability to influence AChR and AChR transcript levels in neurons. These studies will identify some of the signals and molecular mechanisms that control synapse assembly and function in the nervous system. Knowledge of the regulatory events that control the formation of normal functional connections will greatly enhance our ability to identify, and possibly reverse, the deficits that occur in neurons developing in an abnormal environment due to disease, injury or developmental disorders.