ORGANIZATION OF NOCICIPTION DRIVEN NEURONS

本申请中概述的研究是用 具体目的是阐明有关 涉及多肽的功能解剖回路 脊髓伤害性信息处理中的递质/调节器 感官信息。为了这项研究的继续，以及 基于之前的数据。我们将使用合适的标记来 含P物质的初级感觉神经纤维与 这些是背角固有的，并将分析它们的突触 到功能识别的细胞内填充的背角上 神经元。只要有可能。我们将确定是否 功能表征的神经元是一个投射或局部 电路神经元。我们会进行这些研究，以针对 假设： L在生理上有一个可识别的区别 以P物质为优势受体的伤害性神经元 初级感觉起源的纤维与那些接受 P物质从内源性来源输入的优势 背角；2-脑啡肽免疫反应伤害性神经元 突触主要来自初级P物质纤维 感觉起源：3--P物质输入与 树突的尖状部分是一个一致的发现，这种输入 起源于初级感觉纤维。要检验以上几点， 假设，我们为这些实验设计的所有详细说明 将直接功能、神经化学 以及严格条件下的超微结构信息。为 这，所有的实验都将在成年猫身上进行， 阿尔法-氯醛糖麻醉。延髓背角的神经元 腰椎脊髓将在功能和功能上得到充分的描述 然后细胞内注射辣根过氧化物酶(HRP)。 动物将通过血管灌流和相关的 脊髓的节段在可控震颤器中切开并处理 用于展示功能和免疫信号。这个 含有细胞内注射细胞的切片要小心 在光学和电子显微镜水平上进行了分析。在 超微结构水平，免疫信号将通过 基于杂交瘤研究进展的新技术应用 技术，如内部放射性标记和双特定技术 用于展示抗原部位的单抗，在 与免疫金技术相关联。我们的目标是 几个信号的同时演示 超微结构水平与形态和突触的关系 按职能确定的单位的组织。的重要意义。 量化数据将使用非参数进行评估 统计数据。这种方法应该会提供对 参与脊髓加工的功能性突触回路 感官信息，尤指伤害性信息。这些观察结果 从这些研究中衍生出的概念是中期和长期的 范围与疼痛的临床方面相关。

The research outlined in this application was devised with the specific aim of elucidating some key issues regarding the functional-anatomical circuits involving peptide transmitters/modulators in the spinal processing of nociceptive sensory information. For the continuation of this research, and based on previous data. we will use suitable markers to differentiate substance P-containing primary sensory fibres from those intrinsic to the dorsal horn, and will analyze their synapses onto functionally identified, intracellularly filled dorsal horn neurons. Whenever possible. we will establish whether the functionally characterized neuron is a projection or a local circuit neuron. These studies will be carried out to address to the hypotheses that: l there is a physiologically identifiable difference between nociceptive neurons which receive a predominance of substance P fibres of primary sensory origin vs those that receive a predominance of substance P input from sources intrinsic to the dorsal horn; 2 - enkephalin immunoreactive nociceptive neurons receive synapses predominantly from substance P fibres of primary sensory origin: 3 - the association of substance P input with aspiny parts of dendrites is a consistent finding and this input originates from primary sensory fibers. To test the above hypotheses, we have devised for these experiments all elaborate combined protocol which renders direct functional, neurochemical and ultrastructural information under stringent conditions. For this, all the experiments will be carried out in adult cats under alpha-chloralose anesthesia. Neurons of the dorsal horn of the lumbar spinal cord will be fully characterized functionally and then injected intracellularly with horseradish peroxidase (HRP). The animals will be fixed by vascular perfusion and the relevant segments of the spinal cord sectioned in a Vibratome and processed for the demonstration of functional and immunological signals. The slices containing intracellularly injected cells will be carefully analyzed at the light and electron microscopic levels. At the ultrastructural level, immunological signals will be revealed by applying novel procedures based on advances of hybridoma technology, such as internally radiolabelled and bi-specific monoclonal antibodies for the demonstration of antigenic sites, in association with immunogold techniques. Our purpose is the simultaneous demonstration of several signals at the ultrastructural level in relation to the morphology and synaptic organization of functionally identified units. The significance of the quantitative data will be assessed using non-parametric statistics. This approach should provide new insights into the functional synaptic circuits involved in the spinal processing of sensory information, particularly nociception. These observations and the concepts derived from these studies are of medium and long range relevance to clinical aspects of pain.