Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Neutral endopeptidase 24.11 and aminopeptidase N are the two enzymes involved in the degradation of enkephalins in vivo. Several inhibitors of these zinc-metallopeptidases, selective for one enzyme or with dual selectivity, have been designed, protecting enkephalins from catabolism both in vitro and in vivo. These compounds have various pharmacological properties deriving from this protection, such as antinociceptive effects (for review, see Roques et al., 1993). Among the designed mixed inhibitors of enkephalin catabolism, RB101 has been shown to be able to cross the blood-brain barrier (Fournié-Zaluski et al., 1992), eliciting antinociceptive effects in mice and rats after systemic administration (Fournié-Zaluski et al., 1992; Maldonado et al., 1994; Noble and Roques, 1995; Noble et al., 1992).

Considerable evidence has accumulated for a role of CCK during nociceptive transmission (see references in Baber et al., 1989; Stanfa et al., 1994). CCK, which is discretely distributed in the central nervous system (Gall et al., 1987; Vanderhaegen et al., 1975), has been suggested to play a counteracting role to endogenous opioids (Faris et al., 1983). CCK antagonists has been shown to potentiate the effects of opioids (for review, see Baber et al., 1989). The interactions between CCK receptor antagonists and opioids for the control of nociception are mainly mediated via CCK_B receptors (Chapman et al., 1995; Dourish et al., 1988, 1990; Maldonado et al., 1993; Valverde et al., 1994; Wiesenfeld-Hallin et al., 1990), which are found predominantly in the central nervous system (Hill and Woodruff, 1990; Kopin et al., 1992; Moran et al., 1986; Pelaprat et al., 1987; Wank et al., 1992). Furthermore, Vanderah et al. (1994) showed that i.c.v. treatment with a CCK_B receptor antisense oligonucleotide in mice resulted in an enhancement of the antinociceptive effects of i.c.v. morphine. CCK_B receptors are highly concentrated in laminae I and II of the rat dorsal horn of the spinal cord, and studies with neonatal capsaicin have illustrated that CCK_B receptors are located on C primary afferent fibers and post-synaptically on dorsal horn neurons (Ghilardi et al., 1992). Thus, both the laminar and synaptic locations of CCK receptors seem to be similar to those of the opioid receptors (see references in Besse et al., 1991; Lombard et al., 1995).

c-Fos expression, at the spinal level, is one of the long-term intracellular events (for review, see Morgan, 1991; Morgan and Curran, 1995) that could be used as an indirect marker of nociceptive processes (see references in Abbadie et al., 1994a; Zieglgänsberger and Tölle, 1993). Noxious (A and C fiber intensity) but not innocuous (A/ fiber intensity) electrical stimulation of the sciatic nerve has been shown to induce c-Fos expression in superficial and deeper lumbar dorsal horn neurons (Herdegen et al., 1991). The laminar distribution of c-Fos is in good keeping with the findings of electrophysiological studies, which have shown that a high proportion of superficial and deep laminae dorsal horn neurons are driven by noxious inputs (see references in Besson and Chaouch, 1987). Furthermore, in keeping with the evidence that spinal c-Fos is predominantly evoked by noxious stimuli, several studies showed that preadministered morphine significantly reduced spinal c-Fos expression induced by various types of peripheral nociceptive stimulation, such as intraplantar formalin injection (Gogas et al., 1991; Jasmin et al., 1994; Presley et al., 1990), intraplantar carrageenin injection (Honoré et al., 1995b), i.p. acetic acid injection (Hammond et al., 1992), noxious heat (Abbadie et al., 1994c; Tölle et al., 1990, 1991, 1994a,b), noxious cold (Abbadie et al., 1994b), noxious mechanical stimulation (Abbadie and Besson, 1993) and chemical stimulation of the meninges (Nozaki et al., 1992). In agreement with a physiological role of endogenous enkephalins in nociception control, two previous studies demonstrated that enkephalin-catabolizing enzyme inhibitors such as kelatorphan (Tölle et al., 1994b) and RB101 (Abbadie et al., 1994c) significantly decreased noxiously evoked spinal c-Fos expression.

We previously showed that the effects of low doses of i.v. morphine on noxiously induced spinal c-Fos expression could be revealed by coadministration of a CCK_B receptor antagonist (Chapman et al., 1995). Because CCK_B receptor antagonists, such as CI988 (Hughes et al., 1990), have also been shown to potentiate the antinociceptive effects of RB101 (Maldonado et al., 1993; Noble et al., 1993; Valverde et al., 1994), it was interesting to investigate whether this positive interaction could be reflected by modifications of the spinal expression of the immediate-early gene protein product c-Fos. For this purpose, the effects of various doses of i.v. RB101 on carrageenin-induced spinal c-Fos expression have been investigated. In the second part of our study, we have evaluated the effect of an ineffective dose of i.v. RB101, administered with an ineffective dose of i.p. CI988, on carrageenin-induced spinal c-Fos expression. To further improve our results, we have studied the effects of coadministered RB101 and devazepide, a CCK_A receptor antagonist. In these studies, we have used the intraplantar injection of carrageenin (Winter et al., 1962) in rats, which induces increased induction of c-fos mRNA and expression of c-Fos protein in the dorsal horn of the spinal cord (Draisci and Iadarola, 1989; Honoré et al., 1995a,b; Noguchi et al., 1991, 1992) and which produces acute restricted inflammation associated with thermal and mechanical hyperalgesia (Hargreaves et al., 1988; Iadarola et al., 1988; Joris et al., 1990; Kayser and Guilbaud, 1987). Part of this study has been previously presented as an abstract (Honoré et al., 1996).

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Experimental animals. Experiments were performed on 90 adult, male, albino, Sprague-Dawley rats (Charles River, France), weighing 225 to 250 g. Guidelines on ethical standards for investigations of experimental pain in conscious animals were followed (Zimmermann, 1983). Rats were kept in an animal room at a constant temperature of 22°C, with a 12-hr alternating light/dark cycle.

Chemicals. RB101 [N-[(RS)-2-benzyl-3[(S)-(2-amino-4-methylthio)butyldithio]-1-oxopropyl]-L-phenylalanine benzyl ester] and CI988 [PD 134,308 or 4-{[2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3,3,1,1³⁷]dec-2-yloxycarbonyl]amino]propyl]amino]-1-phenylethyl]amin}-4-oxo-[R-(R*,R*)]butanoate-N-methyl-D-glucamine] were synthesized as previously described (Fournié-Zaluski et al., 1992; Hughes et al., 1990). Naloxone was purchased from DuPont de Nemours France (naloxone hydrochloride, injectable solution, 0.4 mg/ml). Devazepide (L364,718) was prepared in Tween 80 (0.1%; Sigma). RB101 was dissolved in ethanol (10%), Cremofor EL (10%) and distilled water. CI988 was prepared in an aqueous suspension with carboxymethylcellulose (0.5%, sodium salt; Sigma).

Experimental protocol. Intraplantar carrageenin (6 mg, in 150 µl of saline) was injected in the hind limb and rats were perfused 90 min after this injection, a delay which has been shown to evoke c-Fos expression in numerous neurons in the dorsal horn of the spinal cord (Honoré et al., 1995b). In this study, nonstimulated control animals and control animals receiving an intraplantar injection of saline were not included, because we previously showed that in these two groups almost no c-Fos labeling (<5 Fos-LI neurons/section) was observed at the level of the spinal cord (Honoré et al., 1995a). The delay of 90 min was also chosen according to the duration of action of RB101 (Noble et al., 1992). In that study, Noble et al. (1992) investigated the effects of RB101 in numerous analgesic tests, such as the hot-plate and phenylbenzoquinone-induced abdominal constriction tests in mice and the tail-flick and electric stimulation tests in rats. They showed that the duration of action of RB101 is approximately 1 hr. In our study, we have chosen to observe spinal c-Fos expression 90 min after carrageenin injection, which, due to the delay in protein expression, reflects the situation at least 30 min earlier.

Immunohistochemistry. At 90 min after intraplantar carrageenin injection, the animals were deeply anaesthetized with pentobarbital (55 mg/kg i.p.; Sanofi) and perfused intracardially with 200 ml of PBS, followed by 500 ml of 4% paraformaldehyde in 0.1 M PB. The spinal cord was then removed, postfixed for 4 hr in the same fixative and cryoprotected overnight in 30% sucrose in PB. Frontal frozen sections (40 µm thick) were cut and collected in PB to be processed immunohistochemically as free-floating sections.

Counting of c-Fos-labeled neurons. Tissue sections were first examined using dark-field microscopy to determine the segmental level, according to the method of Molander et al. (1984), as well as the gray matter landmarks. The sections were then examined under light-field microscopy at ×10 magnification to localize c-Fos-positive cells. Labeled nuclei were counted using a camera lucida attachment. To study the laminar distribution, four regions were defined, as follows: superficial dorsal horn (laminae I and II; superficial), nucleus proprius (laminae III and IV; nucleus proprius), neck of the dorsal horn (laminae V and VI; neck) and the ventral gray (laminae VII-X; ventral).

Evaluation of inflammation. To assess the level of the peripheral inflammation, we considered two indicators of peripheral edema, i.e., paw diameter (the measure of the edema of the site of inflammatory stimulation with intraplantar injection of carrageenin) and ankle diameter (the measure of the extension of the edema). For each rat one measure of both paw and ankle diameters was performed with a caliper square immediately before perfusion. Carrageenin-enhanced paw and ankle diameters of the control group of rats (P_c and A_c, respectively) and the drug-treated rats (P_t and A_t, respectively) were measured. For comparison, paw and ankle diameters of nonstimulated rats (P_n and A_n, respectively) were measured. The carrageenin-induced paw and ankle diameters were determined as the difference between the paw and ankle diameters of carrageenin-stimulated and nonstimulated rats. The effects of drugs were determined as percent changes of the carrageenin-induced paw and ankle diameters of drug-treated rats (P_t  P_n and A_t  A_n, respectively), compared with the paw and ankle diameters of control carrageenin-treated rats (P_c  P_n and A_c  A_n, respectively); the following formulae were used were used: for the paw diameter, [(P_t  P_n)/(P_c  P_n)] × 100; for the ankle diameter, [(A_t  A_n)/(A_c  A_n)] × 100. Studies of carrageenin-evoked spinal c-Fos-LI neurons and peripheral edema were performed in the same rats; therefore, possible correlations between the parameters were determined.

Statistical tests. Statistical analysis was performed to compare the total number of Fos-LI neurons, using one-way analysis of variance for the different groups of animals and two-way analysis of variance for the different groups of animals and the laminar region. To compare the ankle or paw diameters, we used one-way analysis of variance for the different groups of animals. For multiple comparisons, the Fisher's protected least-significant difference test was used. Linear regression was used for dose-dependent effects of RB101 on a single parameter. The investigator responsible for plotting and counting the Fos-LI neurons was blind to the experimental conditions for each animal.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

In nonstimulated rats receiving i.v. RB101 (40 mg/kg), extremely low numbers of Fos-LI neurons were observed in the dorsal horn of the lumbar spinal cord (1-3 Fos-LI neurons/L4 and L5 40-µm section). Ninety minutes after intraplantar carrageenin, Fos-LI nuclei, which were stained to a variable degree, were located in the ipsilateral dorsal horn of the spinal cord. All Fos-LI nuclei were analyzed without considering the intensity of the staining. In agreement with previous studies on intraplantar carrageenin-evoked spinal c-Fos expression (Honoré et al., 1995a), Fos-LI neurons were observed in lumbar segments L2 to L6, with maximal labeling in segments L4 and L5.

Effects of RB101 on Carrageenin-induced c-Fos Expression and Peripheral Edema

c-Fos expression. In the first experimental series, the total number of Fos-LI neurons observed 90 min after carrageenin was 59 ± 3 Fos-LI neurons/40-µm section, in segments L4 and L5. The Fos-LI neurons were preferentially located in the superficial laminae (I and II) of the dorsal horn (86 ± 3%), whereas fewer neurons were observed in the nucleus proprius (1 ± 1%), the deep laminae (V and VI; 8 ± 2%) and the ventral horn (4 ± 1%) of the spinal cord.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Effects of RB101 (10, 20 or 40 mg/kg i.v.) and coadministration of RB101 (40 mg/kg i.v.) and naloxone (1 plus 1 mg/kg i.v.) on carrageenin-induced spinal c-Fos expression. A, each schema includes all labeled neurons in one 40-µm section; each dot represents one labeled neuron. The boundaries of the superficial laminae and the reticular part of the neck of the dorsal horn are outlined. B, results are expressed as the mean ± S.E.M. of the number of Fos-LI neurons for the total number of Fos-LI neurons per section in L4 and L5 segments (Total) and per laminar distribution. Significance is expressed compared with the control carrageenin group, by analysis of variance and protected least-significant difference Fisher's test. Significant differences between pharmacological groups are indicated by arrows (**P < .01, ***P < .001).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Regression lines for the effects of RB101 (5, 10, 20 or 40 mg/kg i.v.) on the total number of Fos-LI neurons/section (A) and on the number of Fos-LI neurons in superficial laminae (B) of the dorsal horn of the rat lumbar spinal cord.

Peripheral edema. We observed the development of unilateral peripheral edema associated with the intraplantar injection of carrageenin. Both the paw and ankle diameters of the injected hind paw were increased (181 ± 5% and 118 ± 2% of nonstimulated rat paw and ankle diameters, respectively), whereas the contralateral hind paw was unaffected (105 ± 0% and 98 ± 1% of nonstimulated rat paw and ankle diameters, respectively). Neither RB101, naloxone nor coadministered RB101 and naloxone influenced the carrageenin-induced peripheral edema.

Effects of the Coadministration of RB101 and CI988, a CCK_B Receptor Antagonist, on Carrageenin-induced c-Fos Expression and Peripheral Edema

c-Fos expression. In the second experimental series, the total number of Fos-LI neurons observed 90 min after carrageenin was 98 ± 3 Fos-LI neurons/section, in segments L4 and L5. The Fos-LI neurons were preferentially located in the superficial laminae (I and II) of the dorsal horn (66 ± 1%), whereas fewer neurons were observed in the nucleus proprius (3 ± 1%), the deep laminae (V and VI; 24 ± 3%) and the ventral horn (7 ± 1%) of the spinal cord.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of coadministration of RB101 (5 mg/kg i.v.) and CI988 (3 mg/kg i.p.) and reversal by naloxone (1 plus 1 mg/kg i.v.) on carrageenin-induced spinal c-Fos expression. A, each schema includes all labeled neurons in one 40-µm section; each dot represents one labeled neuron. The boundaries of the superficial laminae and of the reticular part of the neck of the dorsal horn are outlined. B, results are expressed as the mean ± S.E.M. of the number of Fos-LI neurons for the total number of Fos-LI neurons per section in L4 and L5 segments (Total) and per laminar distribution. Significance is expressed compared with the control carrageenin group, by analysis of variance and protected least-significant difference Fisher's test. Significant differences between pharmacological groups are indicated by arrows (*P < .05, ***P < .001).

Peripheral edema. Again, unilateral peripheral edema was associated with the intraplantar injection of carrageenin. Both the paw and ankle diameters of the injected hind paw were increased (189 ± 4% and 118 ± 2% of nonstimulated rat paw and ankle diameters, respectively), whereas the contralateral hind paw was unaffected (105 ± 0% and 100 ± 1% of nonstimulated rat paw and ankle diameters, respectively). None of the various drug combinations influenced the carrageenin-induced peripheral edema.

Lack of Effect of the Coadministration of RB101 and Devazepide, a CCK_A Receptor Antagonist, on Carrageenin-induced c-Fos Expression and Peripheral Edema

c-Fos expression. In the third experimental series, the total number of Fos-LI neurons observed 90 min after carrageenin was 102 ± 5 Fos-LI neurons/section, in segments L4 and L5. The Fos-LI neurons were preferentially located in the superficial laminae (I and II) of the dorsal horn (58 ± 2%), whereas fewer neurons were observed in the nucleus proprius (6 ± 1%), the deep laminae (V and VI; 28 ± 2%) and the ventral horn (8 ± 1%) of the spinal cord.

Peripheral edema. Again, unilateral peripheral edema was associated with the intraplantar injection of carrageenin. Both the paw and ankle diameters of the injected hind paw were increased (181 ± 5% and 113 ± 0% of nonstimulated rat paw and ankle diameters, respectively), whereas the contralateral hind paw was unaffected (101 ± 2% and 97 ± 0% of nonstimulated rat paw and ankle diameters, respectively). None of the various drug combinations influenced the carrageenin-induced peripheral edema.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

In the present study, we have demonstrated that RB101, a complete inhibitor of enkephalin-catabolizing enzymes (Fournié-Zaluski et al., 1992), dose-dependently decreases carrageenin-induced spinal c-Fos expression, in a naloxone-reversible manner. In addition, we have shown that CI988, a CCK_B receptor antagonist, reveals the antinociceptive effects of an inactive dose of RB101, in a naloxone-reversible manner. Furthermore, none of the drug combinations influenced the peripheral edema, suggesting that this interaction takes place at spinal or supraspinal levels.

Numerous Fos-LI neurons were observed, mainly in the superficial laminae of the dorsal horn of the L4 and L5 spinal segments, 90 min after intraplantar carrageenin injection. Both the laminar and rostrocaudal distributions of Fos-LI neurons after intraplantar carrageenin are in good agreement with our previous study (Honoré et al., 1995b). Indeed, in the latter study, we showed that, after intraplantar carrageenin injection, spinal c-Fos expression was observed mainly in the superficial laminae during the first 90 min after carrageenin injection. At later time points, c-Fos expression in deeper spinal laminae was detectable, i.e., at 2 hr after carrageenin injection, and an equal repartition of spinal Fos-LI neurons between superficial and deep laminae was observed at 3 hr after carrageenin (Honoré et al., 1995a,b). The total number of Fos-LI neurons observed in the two control carrageenin-treated groups was different in the two experimental series. Such variations are presumably due to the inherent variability among experimental series associated with this immunohistochemical technique. Overall, in the same experimental series, the data were very homogeneous, allowing the statistical analysis of the drug effects.

Administration of RB101 (10, 20 or 40 mg/kg i.v.), 10 min before intraplantar carrageenin injection, significantly and dose-dependently reduced the number of spinal Fos-LI neurons observed 90 min after intraplantar carrageenin injection, in a naloxone-reversible manner, and i.v. RB101 (40 mg/kg) did not induce spinal c-Fos expression in nonstimulated rats. Our results are in good agreement with previous studies, which showed that systemic enkephalin-catabolizing enzyme inhibitors such as kelatorphan (Tölle et al., 1994b) and RB101 (Abbadie et al., 1994c) dose-dependently reduced noxious heat-evoked spinal c-Fos expression, in a naloxone-reversible manner. Furthermore, our results are reminiscent of behavioral (Fournié-Zaluski et al., 1984, 1985, 1992; Kayser et al., 1989; Maldonado et al., 1993, 1994; Noble and Roques, 1995; Noble et al., 1992; Perrot et al., 1993; Schmidt et al., 1991; Valverde et al., 1994) and electrophysiological (Dickenson et al., 1987; Morton et al., 1987; Stanfa and Dickenson, 1994; Sullivan et al., 1989) studies, which showed that various enkephalin-catabolizing enzyme inhibitors exerted antinociceptive effects in animals (for review, see Roques et al., 1993). In the present study, none of the tested doses of RB101 reduced the development of the peripheral edema, suggesting that the effects of this compound observed in this study are mainly due to a spinal and/or supraspinal site of action. However, we cannot totally exclude a peripheral site of action, because the antinociceptive effects of systemic RB101 on responses to noxious pressure of the hind paw in rats with local inflammation are partially blocked by the administration of methylnaloxonium, which does not cross the blood-brain barrier (Maldonado et al., 1994).

Administration of the CCK_B receptor antagonist CI988 alone did not influence the number of spinal Fos-LI neurons observed after intraplantar carrageenin injection, even with very high doses. This result suggests that CCK itself does not influence the overall level of c-Fos expression evoked by intraplantar carrageenin and that CCK systems do not tonically influence nociceptive processing in this model. Our results are in good agreement with previous studies, which also showed that CCK receptor antagonists are without effect on nociceptive processes when administered alone (Chapman et al., 1995; Dourish et al., 1988, 1990; Kellstein and Mayer, 1990; Magnuson et al., 1990; Maldonado et al., 1993; Noble et al., 1995; Ossipov et al., 1994; Stanfa and Dickenson, 1993; Valverde et al., 1994; Watkins et al., 1985; Zhou et al., 1993). Furthermore, it has been shown that, under nonpathological conditions, the amount of spinal CCK released is very low (Zhou et al., 1993). However, CI988 has been shown to display significant antinociceptive effects after peripheral nerve injury (Xu et al., 1994a) and to relieve allodynia-like symptoms (Xu et al., 1994b). Thus, further studies would be necessary to determine whether there is a differential effect of CCK_B receptor antagonists on inflammatory pain vs. neuropathic pain.

Coadministration of inactive doses of RB101 and the selective CCK_B receptor antagonist CI988 significantly and strongly (59 ± 5% reduction) reduced the carrageenin-evoked spinal c-Fos expression, whereas coadministered RB101 and devazepide (a selective CCK_A receptor antagonist) did not influence spinal c-Fos expression. Interestingly, the peripheral edema was not influenced by coadministered RB101 and CI988, suggesting that the location of the potentiation between the effects of RB101 and CI988 is at the level of the spinal cord and/or higher brain structures, although we cannot exclude a peripheral effect. Our results are reminiscent of previous studies, which provided evidence for spinal (Ossipov et al., 1994; Stanfa and Dickenson, 1993; Wiesenfeld-Hallin and Duranti, 1987; Wiesenfeld-Hallin et al., 1990; Zhou et al., 1993) and supraspinal (Noble et al., 1993; Pu et al., 1994) sites for the potentiation between opioids and CCK_B receptor antagonists. It must be also emphasized that, in the present study, the effects of the combination of CCK_B receptor antagonist plus RB101 were mainly studied in the superficial dorsal horn of the spinal cord (laminae I and II), which is the main terminal site of nociceptive primary afferent fibers, which contains almost exclusively neurons receiving noxious inputs (laminae I and II outer) (Besson and Chaouch, 1987) and which is extremely rich in opioid receptors, endogenous opioids (see references in Basbaum and Besson, 1991) and CCK_B receptors (Ghilardi et al., 1992). Thus, the use of this new experimental strategy, the "c-Fos strategy," confirms the role of the superficial dorsal horn in nociceptive processes and shows that, at this level, various neurotransmitters involved in pain mechanisms bind to different receptor classes so that some of them interact synergistically and others interact antagonistically, which is the case for endogenous opioids and CCK.

This potentiation is in agreement with the well-documented potentiation between morphine and CCK_B receptor antagonists, as shown by behavioral and electrophysiological experiments (Dourish et al., 1988, 1990; Ossipov et al., 1994; Stanfa and Dickenson, 1993; Wiesenfeld-Hallin et al., 1990; Xu et al., 1994a,b; Zhou et al., 1993). It is interesting to note that Stanfa and Dickenson (1993) showed that, during carrageenin inflammation, a CCK_B receptor antagonist was unable to increase the effects of morphine. However, the action of morphine is enhanced in inflamed rats even without CCK _B receptor antagonists; this may be due to a decrease in CCK at the spinal level, which could also explain the lack of interaction between CCK_B receptor antagonists and morphine. In our study, the experimental conditions were different, because drugs were injected before the injection of carrageenin and covered the early stages of inflammation, during which CCK was not decreased, allowing us to observe an effect of the CCK_B receptor antagonist on the effects of RB101. Similarly, using noxiously evoked spinal c-Fos expression, a CCK_B receptor antagonist was shown to increase the antinociceptive effects of morphine (Chapman et al., 1995). In the present study, we also demonstrated that there is a good relationship between previous behavioral studies (Maldonado et al., 1993; Valverde et al., 1994; but see Noble et al., 1995) and the effects of coadministration of RB101 and a CCK_B receptor antagonist on noxiously evoked spinal c-Fos expression. In their behavioral studies, Valverde et al. (1994) suggested that the effectiveness of the antinociceptive responses induced by the coadministration of CCK_B receptor antagonist and RB101 is greater than that observed by other groups with CCK_B receptor antagonists and morphine. However, it is difficult to compare our present data with those previously obtained (Chapman et al., 1995) on the effects of a CCK_B receptor antagonist and morphine on noxiously evoked spinal c-Fos expression. Indeed, in the latter experiment, the effects of coadministered morphine and CCK_B receptor antagonist were not gauged at the time point corresponding to the peak effect of morphine (90 min to 2 hr) (Honoré et al., 1995b), as in the present study with coadministered RB101 and CCK_B receptor antagonist. However, the considerable decrease of noxiously evoked spinal c-Fos expression we observed at the superficial level suggests that the use of such drug associations to address cooperative interactions between drugs could be very promising for the relief of pain.