A lowered threshold for pain occurs in and around the injured area following tissue damage. The decrease in threshold for perception of a stimulus as noxious is referred to as hyperalgesia. When tissue injury and inflammation occur in the periphery, dorsal horn neurons exhibit increased excitability in parallel with the hyperalgesia. A current hypothesis is that prolonged increases in membrane excitability or increased synaptic efficiency of nociceptive neurons in the dorsal horn contributes to hyperalgesia. We have determined that hyperalgesia created by peripheral inflammation is accompanied by increased binding of SP in the spinal cord. The studies proposed in this application will characterize the binding further and explore whether increased activation of NK-1 receptors in the spinal cord by SP may contribute to hyperalgesia. The studies address the following questions: Question 1: Does the increase in [125I]BH-SP binding in the spinal cord with inflammation reflect an increase in affinity or an increase in number of NK-1 receptors? Does a change in the level of mRNA for the NK-1 receptor in dorsal horn neurons parallel the change in receptor number? Scatchard analysis of data acquired with receptor autoradiography will determine whether the increase in SP binding is due to a decrease in Kd and/or an increase in Bmax of [125I]BH-SP binding to the NK-1 receptor. In situ hybridization using oligonucleotide probes for the mRNA of the NK-1 receptor will be used to determine whether the increase in SP binding is associated with an increase in the synthesis of mRNA for the NK-1 receptor by dorsal horn neurons and spinothalamic tract (STT) cells. Question 2: Do NK-1 receptors contribute to the hyperalgesia observed in inflammation? The ability of a non-peptide NK-1 receptor antagonist to alter the threshold of an inflamed paw for a thermal stimulus will be determined in a behavioral assay. Additional studies will address whether NK-1 receptors can be up-regulated by chronic, intrathecal administration of the NK-1 receptor antagonist, and whether hyperalgesia accompanies this biochemical change. Question 3: How is the NK-1 receptor regulated by SP as well as other peptides and amino acids with which SP is co-released from primary afferent neurons? These experiments will be conducted on primary cultures of neonatal rat spinal neurons. The binding characteristics of radiolabeled SP will be analyzed in primary cultures of neonatal rat spinal neurons following treatment of the cultures with SP, calcitonin gene-related peptide, or a selective agonist for the NMDA receptor. Question 4: Does SP alter the response of spinal neurons to glutamate? Is intracellular Ca++ involved in mediating the effect? The ability of SP to enhance neuronal responses to activation of NMDA receptors will be explored using neonatal rat dorsal horn neurons in culture. We will measure activation of NMDA receptors by quantifying changes in intracellular Ca++ with the fluorescent dye Indo-1. By depleting the intracellular pool of Ca++ released by IP3 and blocking the activity of protein kinase C, which is activated by IP3/Ca++, we will explore the pathway by which SP may enhance the response to NMDA receptor activation.
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