SPECIFICITY OF CORD-SYMPATHETIC INNERVATION IN VITRO

将进行体外研究以分析调节 胚胎椎旁神经突触神经支配特异性 交感神经节（SG）的神经元从胚胎脊髓节。 提供神经节功能性神经支配的最佳培养条件 将通过匹配13天大胎儿的横截面外植体来定义 小鼠胸腰段脊髓与适当的目标神经节从17- 18天龄的胎鼠，例如，上胸髓（T2）与上级 第五胸脊髓中段颈神经节（T5） 神经节，上腰髓（L2）与第二腰神经节。 将通过以下方法评价脊髓和神经节外植体之间的神经支配： 脊髓诱发复合神经节动作电位的细胞外记录 或来自索神经节纤维束。 细胞内记录， 去鞘神经节的标准神经元群体将提供关于 自发或诱发的突触电位（大小和频率）或动作 潜力 胆碱乙酰转移酶（ChAT）和酪氨酸羟化酶 脊髓-SG外植体的放射化学测定将与 生理数据 ChAT免疫细胞化学将尝试在 神经支配的SG外植体。 脊髓神经元的神经解剖学定位 将通过选择性标记来建立神经节的神经支配， 辣根过氧化物酶的微离子电渗注射或荧光 将路西法黄染成黄色。 将使用几种特异性范式：1）不适当的区域 胸髓（即背侧或腹侧）内的细胞将与 神经节，并与适当的 中间-外侧区域; 2）不适当的颈椎和 将骶髓与神经节共培养; 3） 胸髓和腰髓将在位置不匹配的情况下共培养， 各自的神经节。 如果表型或位置索错配， 事实上，导致神经节的异常神经支配， 将在适当的脐带区域进行实验; 4） 修改共培养条件以增强cord-SG的表达 特异性将通过引入SG的靶组织或通过 向培养基中加入高K+或地塞米松。 将尝试选择性地干扰SG的神经支配 使用针对PNS神经元特异性部分的抗体的细胞 膜和其他特定的封闭剂。

In vitro studies will be undertaken to analyze the mechanisms regulating the specificity of synaptic innervation of embryonic paravertebral sympathetic ganglia (SG) by neurons from embryonic spinal cord segment. Optimal culture conditions to provide functional innervation of the ganglia will be defined by matching cross-sectional explants of 13-day-old fetal mice thoraco-lumbar cord with appropriate target ganglia from 17- to 18-day-old fetal mice, e.g., upper thoracic cord (T2) with the superior cervical ganglion, middle thoracic cord (T5) with the 5th thoracic ganglion, upper lumbar cord (L2) with the 2nd lumbar ganglion. Innervation between cord and ganglion explants will be evaluated by extracellular recordings of cord-evoked compound ganglion action potentials or from the cord-ganglion fiber tract. Intracellular recordings from a standard neuron population of desheathed ganglia will provide data on spontaneous or elicited synaptic potentials (size and frequency) or action potentials. Choline acetyltransferase (ChAT) and tyrosine hydroxylase radiochemical assays of the cord-SG explants will be correlated with the physiological data. ChAT immunocytochemistry will be attempted in the innervated SG explants. Neuroanatomical localization of the cord neurons innervating the ganglia will be established by selective labeling with microiontophoretic injections of horseradish peroxidase or the fluorescent dye Lucifer Yellow. Several specificity paradigms will be used: 1) inappropriate regions within the thoracic cord (i.e. dorsal or ventral) will be co-cultured with the ganglia and compared to co-cultures with the appropriate intermedio-lateral region; 2) inappropriate cross-sections of cervical and sacral cord will be co-cultured with the ganglia; 3) cross-sections of thoracic and lumbar cord will be co-cultured in positional mismatch with the respective ganglia. If phenotypic or positional cord mismatches do, in fact, result in aberrant innervation of the ganglia, competition experiments with the appropriate cord regions will be carried out; 4) modifications of the co-culture conditions to enhance expression of cord-SG specificity will be brought by introducing target tissues of the SG or by adding high K+ or dexamethasone to the culture medium. Attempts will be made to selectively interfere with innervation of the SG cells using antibodies raised against specific moieties of PNS neuronal membranes and other specific blocking agents.