SPECIFICITY OF CORD-SYMPATHETIC INNERVATION IN VITRO

将进行体外研究，以分析调节机制。 胚胎椎旁突触神经支配的特异性 胚胎脊髓节段神经元的交感神经节。 为神经节提供功能性神经支配的最佳培养条件 将通过匹配13天大的胎儿的横切面外植体来定义 小鼠胸腰段脊髓具有适当的靶点神经节-17- 18日龄胎鼠，如上胸髓(T2)与上胸 颈神经节，中胸髓(T5)与第5胸椎 上腰髓神经节(L2)和第二腰神经节。 脊髓和神经节外植体之间的神经支配将通过 脊髓诱发复合神经节动作电位的细胞外记录 或来自脊髓神经节纤维束。小区内录制来自 脱鞘神经节的标准神经元种群将提供关于 自发或诱发的突触电位(大小和频率)或动作 潜力。胆碱乙酰转移酶和酪氨酸羟基酶 脐带-SG外植体的放射化学分析将与 生理数据。ChAT免疫细胞化学将在 神经支配SG外植体。脊髓神经元的神经解剖学定位 神经节的神经支配将通过选择性标记 微离子导入注射辣根过氧化物酶或荧光素 染成路西法黄色。 将使用几个具体的范例：1)不适当的区域 在胸髓内(即背侧或腹侧)将与 并与共培养的神经节和适当的 中外侧区；2)颈椎和颈椎的不适当横断面 将骶髓与神经节共同培养；3)横断面 胸腰髓将在位置不匹配的情况下进行共培养 各自的神经节。如果表型或位置线不匹配，则在 事实上，导致神经节的异常神经支配，竞争 将对合适的脐带区域进行实验；4) 改良共培养条件以提高CORD-SG的表达 通过引入SG的目标组织或通过以下方式带来特异性 在培养基中加入高K+或地塞米松。 将尝试选择性地干扰SG的神经支配 使用针对三叉神经节神经元特异性部分产生的抗体的细胞 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.