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.
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