SPECIFICITY OF CORD-SYMPATHETIC INNERVATION IN VITRO

将进行体外研究来分析调节的机制

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.