Repeated intrathecal (i.t.) injections of SP in mice leads to a """"""""desensitization"""""""" or decreased behavioral response to SP. Our research suggests that desensitization to the behavioral effects of SP is dependent on an accumulation of N-terminal metabolic fragments of SP and excitatory amino acid (EAA) activity. The experiments proposed will further test the hypothesis that N-terminal metabolites of SP regulate pain perception by inhibiting the release as well as the effect of nociceptive transmitters in the spinal cord, in part, by a direct interaction with mu-type opioid receptors. Objective 1: We will raise monoclonal antibodies to N-terminal fragments of SP and use them in RIAs and as potential pharmacologic antagonists in vivo to examine the role of SP fragments in the spinal cord. Objective 2: We will assess the ability of SP fragments to regulate the release of SP in the spinal cork by using in vivo microdialysis in the conscious, freely-moving rat to monitor the effects of N- and C-terminal fragments of SP on KCI- or nociception-induced changes in the concentrations of SP and EAAs. We will establish the role of opiate receptors in this regulation using selective opiate antagonists. Objective 3: We will continue to characterize the binding site(s) on N-terminal SP fragment in the CNS by determining whether SP metabolites interact exclusively with mu1-type opioid binding sites or whether they also interact with distinct site(s), unique from that of either mu1 or SP. Binding will then be examined in mice after an intraplantar injection of Freund's adjuvant to determine whether pain result from a change in SP N- terminal binding. Objective 4: We will examine N-terminal SP binding using autoradiographic techniques to see if binding is differentially localized within the brain and spinal cord and to quantify the density of binding sites, especially in areas thought to be involved in pain-transmission and areas known to contain SP(1-11)-like immunoreactivity. Objective 5: We will determine whether desensitization to the behavioral effects of i.t. SP or N-methyl-D-aspartate (NMDA) or sensitization to kainate or quisqualate alters nociception using the hot plate and writhing assays. Opiate antagonists will be used to determine whether opioids of their receptor are involved in any changes detected.
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