Role of Spinal g-Aminobutyric AcidA Receptors in Formalin-Induced Nociception in the Rat

This study investigated the role of g-aminobutyric acid (GABA) and GABAA receptors in the spinal cord in the expression of pain behaviors evoked by injection of formalin in concentrations ranging from 0.25 to 2.5% in the hindpaw of the rat. Two approaches were used. The first approach compared the effect of drug treatment to saline at each concentration of formalin. The second approach examined the effect of drug treatment on the concentration-response functions of formalin, i.e., its EC50. Intrathecal (i.t.) pretreatment with 0.03 to 0.3 mg of bicuculline, a GABAA receptor antagonist, dose-dependently increased the number of flinches and weighted pain scores in the interphase and phase 2, but did not alter responses in phase 1. In the interphase, the EC50 values of formalin for number of flinches or weighted pain score in bicuculline-pretreated rats were decreased to one-third or one-fourth, respectively, of their values in saline-pretreated rats. In phase 2, the EC50 values of formalin for number of flinches or weighted pain score in bicucullinepretreated rats were similarly decreased to one-half of their value in saline-pretreated rats. These results suggest that formalin was a significantly more noxious stimulus in the presence of bicuculline. Pretreatment with the GABAA receptor agonists, muscimol (0.3 mg) or isoguvacine (10 or 30 mg i.t.), significantly decreased the number of flinches in phase 1 and phase 2, but produced only a marginal decrease in the weighted pain score at the highest doses. These findings suggest that there is little tonic activation of GABAA receptors by GABA in the spinal cord before or immediately after the injection of formalin. However, approximately 10 min after the induction of injury by formalin, there is a release of GABA and activation of GABAA receptors in the spinal cord that 1) contributes to the period of quiescence between phase 1 and phase 2 and 2) coincidentally diminishes the magnitude of pain behaviors in phase 2, possibly by limiting the development of central sensitization in the spinal cord. Persistent nociceptive input to the spinal cord induces prolonged alterations in the response properties, neurochemistry and phenotype of dorsal horn neurons (Dubner and Ruda, 1992) and primary afferent fibers (Neumann et al., 1996; Woolf, 1996a). One such change is the induction of central sensitization in dorsal horn neurons after electrical stimulation of C-fiber afferents or the peripheral application of noxious substances such as mustard oil, carrageenan or formalin (Woolf and Wall, 1986; Neugebauer and Schaible, 1990; Woolf et al., 1994; Xu et al., 1995). The enhanced excitability of dorsal horn neurons is thought to mediate the hyperalgesia and allodynia that develop after tissue injury. Studies of the mechanisms responsible for the induction and maintenance of central sensitization and the behavioral sequelae to tissue injury have predominantly emphasized the role of excitatory neurotransmitters such as glutamate and substance P, as well as intracellular messengers (Coderre et al., 1993). Despite substantial evidence that GABA and its receptors are appropriately situated to modulate nociceptive transmission in the dorsal horn of the spinal cord (Hammond, 1997), comparatively little attention has been paid to the role of GABA in central sensitization in the spinal cord (Sivilotti and Woolf, 1994) or in the behavioral sequelae to tissue injury (Hao et al., 1991; Yamamoto and Yaksh, 1991; Smith et al., 1994; Dirig and Yaksh, 1995). Most of these studies examined the effects of GABA receptor agonists, and only a very few used GABA receptor antagonists to assess the role of endogenous GABA in central sensitization and the behavioral sequelae to tissue injury (Yamamoto and Yaksh, 1993). Yet, small-diameter primary afferent neurons are known to make synaptic contacts on the dendrites of GABAergic neurons in the dorsal horn (Carlton and Hayes, 1990; Hayes and Carlton, 1992), and stimulation of afferent inputs to slices of the spinal cord evokes GABAA receptormediated inhibitory postsynaptic potentials in dorsal horn neurons (Yoshimura and Nishi, 1995). Activation of nociceptive afferents by injection of formalin, carrageenan or the topical application of mustard oil is therefore likely to evoke a release of GABA, as well as glutamate, substance P and Received for publication January 21, 1997. 1 This work was supported by U.S.P.H.S. grant DE11423 from the National Institute for Dental Research (to D.L.H.). ABBREVIATIONS: GABA, g-aminobutyric acid; NMDA, N-methyl-D-aspartate; i.t., intrathecal; LTP, long-term potentiation; EC50, effective concentration; CL, 95% confidence limit. 0022-3565/97/2822-0928$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:928–938, 1997 928 at A PE T Jornals on Sptem er 9, 2016 jpet.asjournals.org D ow nladed from calcitonin gene-related peptide, in the spinal cord. It is reasonable to expect that the behavioral sequelae to tissue injury reflect the summation of inhibitory processes mediated by GABAA and GABAB receptors, and excitatory processes mediated by NMDA and neurokinin receptors. The recent report that i.t. administration of GABA receptor antagonists does not alter formalin-evoked pain behaviors (Dirig and Yaksh, 1995) is contrary to this expectation. However, the use of a single, high concentration of formalin (5%) and the existence of a “ceiling effect” for the number of flinches may have precluded identification of an increase in formalin-induced pain behaviors by GABA receptor antagonists. This observation led us to reexamine the role of GABA and GABAA receptors in the development and maintenance of persistent pain behaviors as modeled by the formalin test with the important distinction that the effects of the GABAA receptor ligands were examined at concentrations of formalin ranging from 0.25% to 2.5%. Subsequent comparison of the concentration-effect curves for formalin in the presence of increasing doses of the GABAA receptor antagonist bicuculline or the GABAA receptor agonists, muscimol and isoguvacine, permitted a quantitative estimate of the extent to which antagonism or mimicry of the actions of GABA at the GABAA receptor, respectively, enhanced or suppressed nociception. It was hypothesized that i.t. administration of low doses of bicuculline would enhance formalin-induced pain behaviors. Moreover, a preferential enhancement of pain behaviors in phase 2 of the formalin test, which are mediated by activation of NMDA receptors (Coderre and Melzack, 1992; Yamamoto and Yaksh, 1992), was expected as removal of an inhibitory GABAergic input to dorsal horn neurons should facilitate the activation of NMDA receptors by glutamate. Intrathecal administration of isoguvacine or muscimol was expected to suppress pain behaviors in both phase 1 and phase 2. A preliminary report of some of these data has appeared (Kaneko and Hammond, 1997). Materials and Methods Animal preparation. This study was approved by the Institutional Animal Care and Use Committee of the University of Chicago. Male Sprague-Dawley rats (Sasco, Madison, WI; 300–350 g) were housed in groups of three and maintained on a 12-hr light/dark cycle with free access to food and water. The rats were anesthetized with halothane, and a polyethylene catheter (PE-10) was inserted through an incision in the atlanto-occipital membrane. One end of the catheter was advanced caudally to the rostral edge of the lumbar enlargement, and the other end was tunneled subcutaneously and externalized on top of the head (Yaksh and Rudy, 1976; Hammond, 1988). The rats were housed individually after surgery and allowed at least 7 days to recover before testing. Formalin test. Animals were placed individually in Plexiglas testing chambers (30.5 3 30.5 3 30.5 cm) and allowed to acclimate for at least 60 min. A mirror was situated behind the chamber and another was situated at a 45° angle below the floor of the chamber to allow an unobstructed view of the rat’s paws. After acclimation, 100 ml of formalin (0.25–2.5%) was injected s.c. into the plantar surface of the left hind paw, and the rat was returned to the testing chamber. Its behavior was observed for the next 60 min. The time spent in each of four mutually exclusive categories of behavior was determined by use of a BASIC computer program generously provided by Dr. K.B.J. Franklin (Department of Psychology, McGill University, Montreal, Canada). The behaviors were those originally described by Dubuisson and Dennis (1977) and reiterated by Abbott et al. (1995) as “0 5 normal weight bearing on the injected paw, 1 5 limping during locomotion or resting the paw lightly on the floor, 2 5 elevation of the injected paw so that at most the nails touch the floor, and 3 5 licking, biting” or shaking the injected paw. A weighted pain score was calculated by multiplying the amount of time spent in each category by its assigned category weight, summing these products and then dividing by the total time in each 5-min block of time. In addition, the number of flinches that occurred was counted (Wheeler-Aceto and Cowan, 1991). Proper placement of the i.t. catheter was verified at the conclusion of the formalin test by the occurrence of hindlimb paralysis after an i.t. injection of 10 ml of 2% tetracaine hydrochloride or, in rats sacrificed by CO2 inhalation, by direct visualization of the catheter tip after laminectomy and injection of India ink. Experimental design. Animals were used only once in this study and received only one dose of drug and one concentration of formalin. The first series of experiments was designed to determine the time course and dose dependence of the effect of the GABAA receptor antagonist bicuculline methiodide on nociceptive behaviors induced by injection of formalin in the hindpaw. In the pretreatment study, rats received an i.t. injection of either saline or 0.03, 0.1 or 0.3 mg of bicuculline 10 min before the injection of a concentration of formalin ranging from 0.25% to 2.5%. In the post-treatment study, either saline or 0.3 mg of bicuculline was injected i.t. 7 to 8 min after the injection of a concentration of formalin ranging from 0.25% to 1.25%. Doses of bicuculline greater than 0.3 mg were not tested because 1) higher doses produce spontaneous vocalization, allodynia and caudally directed biting and scratching behavior (Yaksh, 1989; McGowan and Hammond, 1993) and 2) 0.3 mg of bicuculline effectively antagonizes the increase in tail-flick latency produced by i.t. administration of the GABAA receptor agonist isoguvacine (McGowan and Hammond, 1993). The second series of experiments examined the time course and dose dependence of the effect of i.t. administration of a GABAA receptor agonist on formalin-evoked nociceptive behaviors. In these experiments, either 10 or 30 mg of isoguvacine or 0.3 mg of muscimol was injected i.t. 10 min before the injection of a concentration of formalin ranging from 0.25% to 2.5%. Statistical analysis. The number of flinches and weighted pain scores were determined for each 5-min interval after the injection of formalin, and the data were expressed as the mean 6 S.E.M. for that 5-min interval. Two approaches were used to assess the effect of drug treatment on formalin-induced pain behaviors. The first approach compared the effect of drug treatment with that of saline at each concentration of formalin. This analysis was performed by a two-way analysis of variance for repeated measures in which drug treatment was one factor and time was the second (repeated) factor. Post hoc comparisons of individual mean values were made by the NewmanKeuls test. The second approach examined the effect of drug treatment on the stimulus-response functions of formalin. For this analysis, concentration-effect curves for formalin were constructed in drugand saline-treated rats for phase 1, phase 2 and interphase behaviors. For this purpose, phase 1 was defined as the first 5 min, interphase was defined as the period 10 to 15 min after injection of formalin and phase 2 was defined as the period 20 to 50 min after injection of formalin. The analysis of phase 1 behaviors used the total number of flinches and the weighted pain score for that 5-min interval. The analysis of interphase behaviors used the average number of flinches and the average of the weighted pain scores determined 10 and 15 min after the injection of formalin. The analysis of phase 2 behaviors used the average number of flinches and the average of the weighted pain scores determined between 20 and 50 min after the injection of formalin, respectively. Least-squares linear regression of the individual data was used to determine the concentration of formalin (EC50) that produced one-half the maximal number of flinches or increase in weighted pain score. These criteria corresponded to a weighted pain score of 1.1 (maximum pain score was 2.2) and 50 flinches for phases 1 and 2 and to a weighted pain score of 0.6 (maximum pain score was 1.2) and 25 flinches for the inter1997 Spinal GABAA Receptors 929 at A PE T Jornals on Sptem er 9, 2016 jpet.asjournals.org D ow nladed from phase. Fieller’s theorem was used to determine CL (Finney, 1964). The significance of differences in the EC50 values of formalin in drugand saline-treated rats was determined by analysis of covariance (Zar, 1984). P # .05 was considered significant. Drugs and injections. All drugs were injected i.t. in a volume of 10 ml followed by 10 ml of saline to flush the catheter. The drugs were freshly prepared, adjusted to pH 6.8 to 7.1 and filtered before administration. Bicuculline methiodide, muscimol and tetracaine hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Isoguvacine hydrochloride was purchased from Research Biochemicals Inc. (Natick, MA).