A study of substance P in the dorsal horn : involvement in phasic and tonic nociception

Abstract

Animal laboratory research represents a major approach to investigate pain. While it is not clear whether animals feel pain, an animal's reaction to noxious stimuli is a useful means of inferring responses that we associate with the experience of pain. "Nociception" or "reaction to a noxious stimulus" has been used operationally to address the subject of pain for investigation. "Phasic" nociception is used to describe transient nociception. "Tonic" nociception is used to describe continuous nociception. The spinal cord has been a major focal point for studies involving nociception. The neuropeptide, substance P (SP), has received much attention as a putative mediator of nociception in the dorsal horn. A substantial amount of conflicting data has surfaced concerning the role of SP in transmitting different types of nociception in the spinal cord. Currently, it is not known whether SP is involved in phasic and/or tonic nociception. Based upon the literatur~ and some preliminary data generated in this laboratory, it was hypothesized that SP in the dorsal horn mediates phasic and not tonic nociception. This dissertation addresses this hypothesis by employing current tests which have been established ereviously as accepted methods for both types of nociception. A series of experiments were undertaken to determine whether immunohistochemicalchanges of SP levels in the dorsal horn were associated with phasic and/or tonic nociception. Following the treatment of rats with a hindpaw needle placement (sham) or injection with saline or formalin, nociception was assessed with the automated measurement of stereotypic behavior. SP immunohistochemical levels were assessed with the techniques of immunocytochemistry and microdensitometry. The results showed that stereotypic behavior correlated very well with licking which was considered to be nociceptive. A profile of formalin-induced stereotypic behavior over time revealed both a phasic and a tonic phase of nociception. Both phases were reduced by pretreatment with systemically administered morphine or peripheral application of lidocaine. However, immunohistochemical changes of formalin-induced SP-like immunohistochemistry in the dorsal horn following pretreatment with lidocaine were associated directly with the phasic and not the tonic behavioral response. A second series of experiments were undertaken to examine the effect of SP receptor desensitization ~n phasic and tonic nociception. Multiple injections of a high dose of SP were made onto the rat spinal cord, and changes in the number or affinity of SP receptors or in SP receptor Gprotein interactions were investigated. Radioligand binding assays revealed that multiple populations of 3H-SP binding sites are prese!lt in the rat dorsal spinal cord. The high-affinity binding of 1251-BH-SP to dorsal spinal membranes Wl\S inhibited by the presence of the guanine nucleotide, Gpp(NH)p . . Successive administration of a high dose of SP orito the spinal cord induced a similar reduction in highaffinity 1251-BH-SP binding. The number of high-affinity SP binding sites (Bmax) was reduced and the IC50 of this bindingcomponentwas increased suggesting a possible involvement of receptor-G protein complexes. As demonstrated with the tail-flick and paw pressure tests, phasic behavioral responses were attenuated following multiple injections of SP onto the spinal cord. An attenuation of behavioral responses was associated with an increase in the IC50 of the high-affinity SP binding component which was qualitatively and quantitatively similar to that produced by Gpp(NH)p. The method of SP receptor desensitization was employed in two models of tonic nociception, the formalin test and the monosodium urate (MSU) test. In the formalin test, SP receptor desensitization reduced the first phase but had no effect on the second phase of pain-related behavior. In the MSU test, SP receptor desensitization did not affect MSU-induced pain-related behavior. These data showed that SP levels were associated with the phasic behavioral changes following noxious stimulation in a single behavioral.model of nociception. In two established models of phasic and tonic nociception, alteration of SP receptor activity in the dorsal horn changed phasic nociceptive behavior but did not alter tonic nociceptive behavior. These results support the hypothesis that SP mediates phasic and not tonic nociception.