DIRECT APPROACH TO SYNAPTIC ORGANIZATION OF NOCICEPTION

伤害感受突触组织的直接方法

• 批准号: 3412246
• 负责人: A CLAUDIO CUELLO
• 金额: $ 11.59万
• 依托单位: MCGILL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3412246
• 关键词: afferent nerve; autoradiography; cats; choline acetyltransferase; dynorphins; electron microscopy; electrophysiology; enkephalins; gamma aminobutyrate; immunocytochemistry; monoclonal antibody; neural information processing; neuroanatomy; neurons; pain; serotonin; somatostatin; spinal cord; substance P; synapses

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.

本申请中概述的研究是根据 明确的具体目的是， 肽能感觉传递的伤害性感觉信息 纤维，发生在突触前或突触后部位。 我们的假设， 根据现有的证据，P物质- 含有感觉纤维和脑能脊髓神经元的神经元 突触后的肾小球或非肾小球树突的 背角神经元的非感受性。 为此，A 超微结构免疫细胞化学与 伤害感受驱动的细胞内记录和注射 将使用神经元。 这种方法是唯一可以 直接回答了上述问题，并 从未被系统地使用过。 所有实验 将在成年猫身上进行， 麻醉 脊髓肿瘤的伤害感受驱动神经元 将在细胞内注射辣根过氧化物酶 （HRP）。 动物将通过血管灌注固定， 在振动刀中切割脊髓的相关节段， 用于过氧化物酶的演示。 切片 含有细胞内注射的细胞将被进一步处理 为了证明P物质或脑免疫 在电子显微镜水平上。 使用的抗体是 P4 C1（抗P物质/抗HRP双特异性抗体）和 NOC 1（抗脑啡肽单克隆抗体）。 感官起源 P物质纤维将在某些动物中建立， 在背根神经节中注射氚化氨基酸， 结合放射自显影和免疫细胞化学， 电生理学 在第二阶段，三重标记涉及 细胞内注射和同时免疫染色 将尝试在相同的制备中使用肽。 为此， 将结合基于二氨基联苯胺的免疫细胞化学， 用放射免疫细胞化学（使用内部标记的版本 NOC 1抗体或免疫金。 的检测 GABA、强啡肽、5-羟色胺、胆碱的免疫反应性 乙酰转移酶和生长抑素联合 还将进行电生理学检查。 该研究概述了 预计以上将带来重要的新信息 脊髓疼痛的调节。