DIRECT APPROACH TO SYNAPTIC ORGANIZATION OF NOCICEPTION

本申请中概述的研究是用 明确的目的是阐明脊椎调节是否 由肽能感觉传递的伤害性感觉信息 纤维，出现在突触前或突触后。我们的假设是， 根据现有证据，P物质- 含有感觉纤维和脑能脊髓神经元的活动 肾小球或非肾小球树突的突触后分布 非伤害性刺激驱动的背角神经元。为此目的，一个 超微结构免疫细胞化学与免疫组化的结合 伤害性感觉驱动的腔内记录和注射 将使用神经元。这样的方法是唯一可以 为上述问题提供了直接答案，并已 以前从未以系统的方式使用过。所有的实验 将在阿尔法-氯醛糖下对成年猫进行 麻醉。脊髓腔内伤害性感觉神经元 会被细胞内注射辣根过氧化物酶 (HRP)。动物将通过血管灌流和 脊髓的相关节段在振动器中切开并 为证明过氧化物酶的存在而加工。薄片 含有细胞内注射的细胞将被进一步处理 用于显示P物质或脑不朽活动 在电子显微镜水平上。要使用的抗体是 P4C1(抗P物质/抗HRP双特异性抗体)和 NOc1(抗脑磷脂单抗)。的感官起源 在某些动物中，P物质纤维可通过 背根神经节注射氚氨基酸， 并将放射自显影与免疫细胞化学相结合 电生理学。在第二阶段，三重标签包括 两者的细胞内注射和同时免疫染色 将尝试相同制剂中的多肽。为此， 基于二氨基联苯胺的免疫细胞化学也将结合 使用放射免疫细胞化学(使用内部标记的版本 NOC1抗体或免疫金。病毒的检测 GABA、强啡肽、5-羟色胺、胆碱的免疫反应 乙酰转移酶和生长抑素联合应用 还将进行电生理学检查。这项研究概述了 以上内容预计将带来有关 脊髓中疼痛的调制。

The research outlined in this application was devised with the specific aim of clarifying whether the spinal modulation of the nociceptive sensory information, transmitted by peptidergic sensory fibers, occurs at a pre- or postsynaptic site. Our hypothesis, based on the available evidence, is that both substance P- containing sensory fibers and encephalonergic spinal neurons act postsynaptically on glomerular or non-glomerular dendrite's of noniception driven dorsal horn neurons. For that purpose, a combination of ultrastructural immunocytochemistry with intracullular recordings and injections of nociception driven neurons will be used. Such an approach is the only one that can provides a direct answer to the question outlined above, and has never been used before in a systematic way. All the experiments will be carried out on adult cats under alpha-chloralose anesthesia. Nociception driven neurons of the lumour spinal cord will be injected intracellularly with horseradish peroxidense (HRP). The animals will bs fixed by vascular perfusion and the relevant segments of the spinal cord sectioned in a vibratome and processed for the demonstration of peroxidase. The slices containing intracellularly injected cells will be further processed for the demonstration of substance P or encephalon immoractivities at the electron microscoPic level. The antibodies to be used are the P4C1 (anti-substance P/anti-HRP bi-specific antibody) and the NOC1 (antienkeplalin monoclonal antibody). The sensory origin of the substance P fibers will be astablished in some animals by injections of tritiated amino acids in the dorsal root ganglia, and combining radioautography with immunocytochemistry and electrophysiology. At a second stage, a triple labeling involving intracellular injection and simultaneous immunostaining for both peptides in the same preparation will be attempted. For that, diaminobenzidine-based immunocytochemistry will be combined either with radioimmuocytochemistry (using the internally labeled version of the NOC1 antibodys or immunogold. The detection of immunoreactivities for GABA, dynorphin, serotonin, choline acetyltransferase and somatostatin in combination with electrophysiology will be also carried out. The research outlined above is expected to bring important new information on the modulation of pain in the spinal cord.