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.