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Background Pain during labor is a common and severe phenomenon, but its clinical management remains haphazard because its neurophysiology is poorly understood. In the current study, the authors evaluate the parturient rat as a relevant model to study the pharmacology of labor pain. Methods Control of birth timing in term pregnant rats was achieved by gavage with RU 486 (5 mg/kg) the day before the expected day of parturition. The behavioral events preceding the expulsion of the first pup were analyzed, and immunodetection of the c-Fos protein was used to evaluate the spinal neuronal activity at the lumbosacral level where genital and perineal inputs terminate. Results Hind limb and abdominal stretches occurred during labor (mean number, 57 +/- 10), arbitrarily defined as the time elapsed between the first stretch and the expulsion of the first pup (mean duration, 62 +/- 5 min). Subcutaneous oxytocin increased the frequency of stretches, accounting for the fact that these manifestations are linked to uterine contractions. Finally, epidural morphine (30 microg/10 microl) in oxytocin-treated rats, although resulting in no change of labor duration, significantly decreased the number of stretches (8 +/- 2 vs. 57 +/- 12 for epidural saline) and the number of c-Fos-positive neurons in the lumbosacral spinal segments (80 +/- 25 vs. 165 +/- 17 for epidural saline). Conclusions These results indicate that stretches during labor in the rat correspond to a behavioral response to nociception associated with uterine contractions and suggest that parturition in the rat could be a relevant model to investigate nociceptive mechanisms associated with parturition in women.

We studied the effects of the high-efficacy 5-hydroxytryptamine1A (5-HT1A) receptor agonist, F 13640 on both formalin-induced spinal cord c-Fos protein expression and pain behaviours in the rat. Replicating earlier data, F 13640 (0.63 mg/kg, i.p.; t(-15 min)) completely inhibited the elevation and licking of the formalin-injected paw. In the same animals, and in spite of the agent as in earlier data increasing the number of c-Fos labelled nuclei when it was administered alone, F 13640 markedly reduced the number of formalin-induced c-Fos labelled nuclei. This was found in both the superficial (I-II) and deep (V-VI) dorsal horn laminae (2 h post-injection: 72+/-2% and 92+/-1% of reduction, respectively; P0.001 in either case), spinal areas that contain neurons responsive to nociceptive stimulation. Co-operation occurred so that after the co-administration of F 13640 and formalin, c-Fos expression was inferior to that induced when either stimulation was administered alone. The data provide initial evidence for the agent's inhibitory effects on noxiously evoked c-Fos expression. The results indicate that co-operation between 5-HT1A receptor activation and nociceptive stimulation powerfully inhibits responses to severe, tonic nociception.

The effects of the S enantiomer of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, alone or in combination with a functional NMDA receptor antagonist, (+)-HA966 were studied on the spinal c-Fos protein expression in the carrageenan model of inflammatory nociception. One hour 30min after intraplantar carrageenan in awake rats, c-Fos immunoreactive (c-Fos-IR) nuclei were preferentially located in the laminae I-II and V-VI of the spinal dorsal horn, i.e., spinal areas containing numerous neurons responding exclusively, or not, to peripheral nociceptive stimuli. RB101(S) (5, 10, 20 and 40mg/kg i.v.) dose-dependently reduced the total number of carrageenan-evoked c-Fos-IR nuclei (r=0.63, P0.01), with 49+/-3% reduction (P0.001) for the highest dose. Two highest doses of RB101(S) (20 and 40mg/kg) significantly reduced the number of carrageenan-evoked c-Fos-IR nuclei in both superficial I-II (32+/-7% and 36+/-5% reduction, respectively, P0.05 for both) and deep V-VI (42+/-6% and 61+/-2% reduction, respectively, P0.001 for both) laminae. The effects of RB101(S) were naloxone-reversible. Combination of low doses of RB101(S) (2.5 or 10mg/kg i.v.) and an inactive dose of (+)-HA966 (2.5mg/kg s.c.) produced supra-additive effects (39+/-4% and 51+/-5% reduction of the total number of c-Fos-IR nuclei, respectively, P0.001 for both). These effects were partially reversed by naloxone. These results provide evidence for the potent effects of combination of RB101(S) and (+)-HA966. Considering the absence of major opioid side effects of RB101(S) and the marked increase of its antinociceptive effects by NMDA receptor antagonist, this type of drug combination could have beneficial therapeutical application.

We have recently shown concurrent changes in behavioural responses and c-Fos protein expression in the central nervous system in both naive and morphine-dependent rats after systemic administration of the opioid antagonist naloxone. However, because naloxone acts on the three major types of opioid receptors, the present study aimed at determining, in the same animals, both changes in behaviour and c-Fos-like immunoreactivity after intravenous injection of selective opioid antagonists, such as mu (beta-funaltrexamine, 10 mg/kg), delta (naltrindole, 4 mg/kg) or kappa (nor-binaltorphimine, 5 mg/kg) opioid receptor antagonists, in naive or morphine-dependent rats. In a first experimental series, only beta-funaltrexamine increased c-Fos expression in the eight central nervous system structures examined, whereas no effect was seen after naltrindole or nor-binaltorphimine administration in naive rats. These results suggest a tonic activity in the endogenous opioid peptides acting on mu opioid receptors in normal rats. A second experimental series in morphine-dependent rats showed that beta-funaltrexamine had the highest potency in the induction of classical signs of morphine withdrawal syndrome, as well as the increase in c-Fos expression in the 22 central nervous system structures studied, suggesting a major role of mu opioid receptors in opioid dependence. However, our results also demonstrated that naltrindole and, to a lesser extent, nor-binaltorphimine were able to induce moderate signs of morphine withdrawal and relatively weak c-Fos protein expression in restricted central nervous system structures. Therefore, delta and kappa opioid receptors may also contribute slightly to opioid dependence.

We have previously shown that RB101, a dual inhibitor of enkephalin-degrading enzymes, decreased carrageenin-evoked c-Fos protein expression at the spinal cord level in awake rats. Moreover, we have also shown that c-Fos expression is a useful marker of the possible direct or indirect interactions between neural pathways, such as opioid and cholecystokinin systems. We now investigated the respective roles of the three main types of opioid receptors (μ, δ, or κ) and their possible interactions, in the depressive effects of RB101 in inflammatory nociceptive conditions induced by intraplantar carrageenin (6 mg/150 μl of saline). We used β-funaltrexamine (β-FNA), naltrindole (NTI), and nor-binaltorphimine (BNI) as specific antagonists for μ-, δ- and κ-opioid receptors, respectively. c-Fos protein-immunoreactivity (c-Fos-IR) was evaluated as the number of c-Fos-IR nuclei in the lumbar spinal cord 90 min after carrageenin. c-Fos-IR nuclei were preferentially located in the superficial (I–II) and deep (V–VI) laminae of segments L4–L5 (areas containing numerous neurons responding exclusively, or not, to nociceptive stimuli). RB101(S) (30 mg/kg, i.v.) significantly reduced the total number of carrageenin-evoked c-Fos-IR nuclei (30% reduction, P

Urethane anaesthesia strongly reduced the peripheral edema (31+/-5 and 96+/-8% reduction of carrageenan-enhanced paw and ankle diameters, respectively; P

This study examined the possibility that a tonic activity in the endogenous opioid systems (EO systems) exists in animals under normal conditions. In a first set of experiments, concurrent changes in behavioral responses and in the numbers of c-Fos-like immunoreactive (Fos-LI) neurons in 58 structures of the brain and lumbosacral spinal cord were analyzed in rats after systemic administration of the opioid antagonist naloxone (NAL; 2 mg/kg). Possible roles of the EO systems were inferred from changes in the numbers of Fos-LI neurons between normal rats that received either NAL or the same volume of saline. Free-floating sections were processed immunohistochemically for c-Fos protein using standard avidin-biotin complex methods. After NAL, the numbers of Fos-LI neurons were significantly increased in the area postrema; in the caudal, intermediate, and rostral parts of the nucleus tractus solitarii; in the rostral ventrolateral medulla; in the Kölliker-Fuse nucleus; in the supramammillary nucleus; and in the central nucleus of the amygdala. In a second set of experiments examining changes in c-Fos expression in the latter structures, similar increases were found after NAL but not after an equimolar dose of NAL-methiodide, a preferential, peripherally acting opioid receptor antagonist. Therefore, Fos-LI was likely triggered after blockade of central opioid receptors, but not peripheral opioid receptors, releasing neurons from EO system-mediated inhibition. The results of this study suggest the existence of a tonic activity of the EO systems exerted on a restricted number of brain regions in normal rats. This tonic activity of the EO systems may control part of the neural networks involved in cardiorespiratory functions and in emotional and learning processes.

Inflammatory pain involves the sensitization of both primary afferent and spinal cord neurons. To explore the neurochemical changes that contribute to inflammatory pain, we have examined the expression and ligand-induced internalization of the substance P receptor (SPR) in the spinal cord in acute, short-term, and long-term inflammatory pain states. These inflammatory models included unilateral injection of formalin (8–60 min), carrageenan (3 hr), and complete Freund’s adjuvant (CFA; 3 d) into the rat hindpaw as well as adjuvant-induced polyarthritis (21 d). In acute inflammatory pain there is ongoing release of substance P (SP) as measured by SPR internalization in lamina I neurons at both 8 and 60 min after formalin injection. Although there is no tonic release of SP in short-term inflammatory pain, at 3 hr after carrageenan injection, SP is released in response to both noxious and non-noxious somatosensory stimulation with SPR internalization being observed in neurons located in both laminae I and III-IV. In long-term inflammatory pain models (CFA and polyarthritis) the same pattern of SP release and SPR activation occurs as is observed in short-term inflammation with the addition that there is a significant upregulation of the SPR in lamina I neurons. These results suggest that SPR internalization might serve as a marker of the contribution of ongoing primary afferent input in acute and persistent pain states. These stereotypical neurochemical changes suggest that there are unique neurochemical signatures for acute, short-term, and long-term inflammatory pain.

This study investigated the contribution of NMDA receptors to the development of tolerance to the antinociceptive properties of morphine at the level of the spinal cord dorsal horn. The expression of c-Fos protein following intraplantar (i.pl.) injection of carrageenin (6 mg/150 microl of saline) was used. In naive rats, acute intravenous (i.v.) administration of morphine (3 mg/kg) decreased the total number per section of Fos-Like-Immunoreactive (Fos-LI) neurons by 51%, observed at 2 h after injection of carrageenin. In tolerant rats, acute morphine did not significantly modify the total number of Fos-like immunoreactive neurons/section. In rats receiving chronic morphine and chronic injections of the non-competitive ((+)-MK 801 maleate: (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine) or the competitive (LY 235959: [3S-(3alpha,4a alpha,6beta,8a alpha)]-Decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic+ ++ acid) NMDA receptor antagonists, only partial tolerance to the acute effects of morphine were observed (decrease of 42% and 38%, respectively). Administration of an antagonist at the strychnine-insensitive glycine site of the NMDA receptor ((+)-HA-966: R(+)-3-Amino-1-hydroxypyrrolidin-2-one) did not affect the development of morphine tolerance. These findings suggest that compounds attenuating the actions of the NMDA receptor via blockade of the glycine modulatory site may be substantially different from those acting at the ion channel of the NMDA receptor complex. This in vivo experiment in freely moving animals demonstrates for the first time an attenuation of tolerance at the cellular level.

In an attempt to study the anti-inflammatory and the antinociceptive effects of a kappa1-opioid receptor agonist (U-69,593: trans-3,4-dichloro-N-methyl-N-[7-(1-pyrrolidinyl)cycloexil]benzene acetamide methanesulfonate), we used a combination of the measurement of peripheral oedema (with a calliper) and Fos immunodetection in the carrageenin model of inflammation. The intraplantar injection of carrageenin-induced the development of a peripheral oedema, associated with an increase in Fos-like immunoreactivity at the level of the dorsal horn of the spinal cord. U-69,593 administered intravenously (i.v.) 10 min before carrageenin administration over the dose range 0.75, 1.5 and 3 mg/kg, reduced both paw and ankle oedema in a non dose-dependent manner. The maximal decrease was observed at the highest dose and did not exceed 21% and 20% for the paw and the ankle respectively. These effects were kappa-opioid receptor specific since the anti-inflammatory effect of 1.5 mg/kg i.v. of U-69,593 was antagonised by a specific kappa-opioid receptor antagonist nor-binaltorphimine. Pre-treatment with U-69,593 strongly decreased the number of Fos-like Immunoreactive neurones of the spinal cord in a dose-dependent, antagonist reversible manner; maximal effect was 65%. The disparate results between the anti-inflammatory effects and the depressive effects on Fos expression suggest that anti-inflammatory effects of kappa-opioid receptor agonist are of minor importance for the antinociceptive effects of this compound.

This study performed in freely moving rats evaluated the ability of specific opioid receptor antagonists to reverse the inhibitory effects of morphine on carrageenin-induced c-Fos expression in the spinal cord. Our study focused on the superficial dorsal horn (laminae I-II), which is the main termination site of nociceptive primary afferent fibers and is rich in opioid receptors. In order to replicate clinical routes of administration, all agents were administered intravenously (i.v.). As previously demonstrated, pre-administered i.v. morphine (3 mg/kg) produced a marked decrease (58+/-5%) in the number of Fos-LI neurones measured at 2 h after intraplantar (i.pl.) carrageenin (6 mg/150 microl) and yet was without influence on peripheral oedema. This decrease in c-Fos expression was completely blocked by combined administration of morphine with the mu-opioid receptor antagonist, [D-Phe-Cys-Tyr-D-Orn-Thr-Pen-Thr-NH2] (CTOP-1+1 mg/kg). Naltrindole (NTI-1+1 mg/kg), a delta-opioid receptor antagonist partially blocked the effects of systemic morphine, so that the inhibitory effects of morphine after NTI injection are now 40+/-4%. However, this effect of NTI was weak since the depressive effects of morphine were still highly significant (p

The organization of projections from the parabrachial (PB) area to the ventral posterior parvicellular (VPpc) "gustatory" and intralaminar nuclei of the thalamus was studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L), into subregions of the PB area. The present study is a follow-up of three former studies (Bernard et al. [1993] J. Comp. Neurol. 329:201-229; Alden et al. [1994] J. Comp. Neurol. 341:289-314; Bester et al. [1997a] J. Comp. Neurol. 383:245-281) that examined PB projections onto the amygdala, the bed nucleus of the stria terminalis, and the hypothalamus. Our data showed that (1) the region centered in the internal lateral PB subnucleus projects densely with a bilateral and symmetric pattern to the caudal portion of the paracentral and, to a lesser extent, to the adjacent portion of the central and parafascicular medial thalamic nuclei; (2) the mesencephalic PB region centered in the ventral lateral subnucleus and scattered neurons in the subjacent brachium conjunctivum project primarily, although diffusely, to the central medial thalamic nucleus. The third region includes two subgroups: (3a) the medial subgroup, including the medial, the waist area, and the ventral lateral subnuclei of the pontine PB area, projects bilaterally but with a weak ipsilateral predominance to the VPpc, terminals bearing large varicosities. Additionally, a diffuse projection with small varicosities spreads in the area between the two VPpc nuclei and the central medial nucleus. (3b) The lateral subgroup, centered in the external medial subnucleus, projects with a contralateral predominance in the periphery of the VPpc nuclei, most terminals being located around the dorsomedial tip. It is suggested that the PB projections to the intralaminar nucleus could be involved in cortical limbic arousal processing in relation with nociceptive, (somatic, visceral, and intraoral) and gustatory aversive stimuli. The projection with large varicosities inside the VPpc could process gustatory discrimination.

The long-term changes in Fos like-immunoreactivity (Fos-LI) in the dorsal horn of the spinal cord following various peripheral nerve lesions remain controversial. This study considers such an approach with chronic constriction injury rats (CCI: loose ligations of the sciatic nerve), at 2 weeks after the surgery, when changes in spontaneous and evoked behaviour were clearly described. All rats used for Fos studies displayed allodynia to mechanical stimulation (decrease of 32% of the vocalization threshold to paw pressure). In CCI rats, which displayed 'spontaneous pain-related behaviour', the number of Fos-LI neurones, in the absence of any intentional stimulation, was very low and comparable with that observed in normal and sham-operated rats (10 neurones/40 microm section). Thus, in this model, the expression of Fos protein is not a reliable index of spontaneous pain. Surprisingly, despite the fact that in this model numerous anatomical studies described a dramatic loss of large and unmyelinated primary afferent fibers, we were unable to detect changes in the number and distribution of Fos-LI evoked by various modalities of peripheral noxious stimulation (noxious thermal stimuli, noxious mechanical stimuli and carrageenin induced inflammation). For example, the stimulus-response curves for the number of Fos-LI neurones evoked by a series of heat stimuli (40, 45, 48, 52, 55 degrees C) were almost superimposable for CCI, sham-operated and normal rats. In contrast, stroking of the nerve-injured paw induced a significant expression of Fos-LI in the superficial laminae (I-II) of the dorsal horn of CCI rats (19.5 +/- 3/sections, P = 0.027) which was greater than that observed in sham-operated (6.5 +/- 3/sections) or in normal rats (3.5 +/- 2/section). These modifications may reflect mechanical allodynia observed in behavioural studies and could be related to A beta fibers, which are known to be severely affected after the constriction of the nerve. These results suggest that this approach could be useful to study, at the cellular level, in freely moving rats, some pharmacological aspects of neuropathic pain.

SUMMARYNumerous studies use the c-Fos protein immunoreactivity as a neuronal marker allowing quantitative and systematic physiopharmacological studies related to nociceptive processes. We have used c-Fos technique, at the spinal cord level, in combination with measurements of the peripheral edema to investigate effects of various substances implicated, or not, in the inflammatory nociceptive processes due to intraplantar injection of carrageenan. This article summarizes the studies performed with this approach to gauge the effects of various non-steroidal anti-inflammatory drugs [NSAIDs] and, in addtion, their association with substances having other mechanisms of action. Our results suggest that co-administration of various anti-inflammatory and/or analgesic substances may produce enhanced therapeutic effects with reduced doses, and thus a reduced side-effect liability.

1. We have evaluated the effects of intravenous or intraplantar racemic-, S(+)- and R(-)-flurbiprofen on both the carrageenan-evoked peripheral oedema and spinal c-Fos immunoreactivity, an indirect index of neurons involved in spinal nociceptive processes. 2. Three hours after intraplantar injection of carrageenan (6 mg in 150 microl of saline) in awake rats, a peripheral oedema and numerous c-Fos protein-like immunoreactive (c-Fos-LI) neurons in L4 L5 segments were observed. c-Fos-LI neurons were essentially located in the superficial (I-II) and deep (V-VI) laminae of the dorsal horn. 3. Intravenous racemic-flurbiprofen (0.3, 3 and 9 mg kg(-1)) dose-relatedly reduced the carrageenan-evoked oedema and spinal c-Fos expression (r=0.64, r=0.88 and r=0.84 for paw diameter, ankle diameter and number of c-Fos-LI neurons; P

The effect of graded inflammatory stimuli (intraplantar-carrageenan, 0.2, 1, and 6 mg/150 microl) on paw edema and c-Fos protein expression at two levels of the spinoparabrachial pathway, the spinal cord and parabrachial area (PB), were studied. The present study, in awake rats, is an extension of previous study (Bester et al. [1997] J. Comp. Neurol. 383:439-458) which evaluated, in anesthetized rats, the effect of graded cutaneous heat stimulation on c-Fos-expression at the same levels. At the spinal level, the c-Fos-protein-like-immunoreactive (c-Fos-LI) neurons were located primarily in superficial laminae ipsilateral to intraplantar carrageenan. The number of c-Fos-LI neurons increased dose dependently (r = 0.973, n = 24) for carrageenan, from a number close to zero for the saline injection. At the PB level, c-Fos was predominantly expressed contralateral to intraplantar carrageenan. c-Fos-LI neurons were located primarily around the pontomesencephalic junction in (i) a restricted pontine area, centered in the lateral crescent, and including an adjacent part of the outer portion of the external lateral subnucleus, and (ii) the mesencephalic superior lateral subnuclei. The number of c-Fos-LI neurons in the PB area was correlated with that in the superficial laminae (r = 0.935, n = 24) and with the paw edema (r = 0.931, n = 24). No significant changes in c-Fos expression were observed in the nucleus of the solitary tract and ventrolateral medulla. The close correlation between c-Fos expression at both the spinal and PB levels and inflammatory edema provides further evidence for the involvement of spinoparabrachial pathway in inflammatory nociceptive processes. The present results are congruent with the existence of electrophysiologically demonstrated spinoparabrachio-amygdaloid and -hypothalamic nociceptive pathways.

We have analysed in briefly anaesthetised rats (1% halothane for 18 minutes) the effects of innocuous and noxious heat, applied to the hindpaw, on evoked c-Fos immunoreactivity at the levels of the parabrachial area (PB), spinal cord, and nucleus of the solitary tract (NTS). After anaesthesia recovery, animals were left to move freely for 2 hours. At the spinal level, c-Fos was expressed primarily in the ipsilateral superficial laminae, increasing with the applied temperatures in a dependent manner in the noxious range (correlation coefficient r = 0.954, n = 20). At the NTS level, no noxiously evoked c-Fos expression was observed. At the PB level, c-Fos was expressed preferentially contralaterally, increasing with the applied temperatures in a dependent manner in the noxious range (r = 0.971, n = 25). The maximum expression was observed in the outer portion of the external lateral, the lateral crescent, and the superior lateral subnuclei around the pontomesencephalic junction. This was congruent with the densest supraspinal projection of lamina I neurones of the dorsal horn. Labelling in the PB area was highly correlated (r = 0.936, n = 20) with labelling in the superficial laminae. We conclude that, under our experimental procedures, noxious heat-induced c-Fos expression at the PB level depends on the intensity of the noxious stimulation. These data further support the relevance of the recently described spino-PB pain pathway. Because of their location, the Fos-immunoreactive neurones observed in the pontine and the mesencephalic divisions of the PB area were likely PB-amygdaloid and PB-hypothalamic nociceptive neurones, respectively.

The organization of projections from the parabrachial (PB) area to the hypothalamus was studied in the rat by using microinjections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into subregions of the PB area. The present study is a follow-up of two former studies (Bernard et al. [1993] J. Comp. Neurol. 329:201-229; Alden et al. [1994] J. Comp. Neurol. 341:289-314) that examined PB projections onto the amygdala and the bed nucleus of the stria terminalis. The results demonstrate that 1) the mesencephalic PB region, centered in the lateral portion of the superior lateral subnucleus projects extremely densely to almost the entire dorsomedial subdivision of the ipsilateral ventromedial hypothalamic nucleus; 2) the mesencephalic PB region, located in the medial portion of the superior lateral subnucleus and weakly overflowing into the rostralmost dorsal lateral pontine subnucleus, projects densely to the retrochiasmatic area and, to a lesser extent, to the ipsilateral ventromedial nucleus of the hypothalamus; 3) the PB region, including the central lateral, a portion of the superior lateral, and the outer external lateral subnuclei, projects densely to the ipsilateral median, anteroventral, and periventricular preoptic hypothalamic nuclei and projects more weakly to the dorsal border of the paraventricular nucleus (PVN). No consistent projection was found in the magnocellular PVN. All of these PB regions also project diffusely to the dorsomedial area and to a small tuberal subfornical hypothalamic area. In addition, the medial half of the PB area projects consistently to the posterior lateral hypothalamus. It is suggested that these pathways may be involved in aversive-defensive behavior, in autonomic and neuroendocrine aspects of pain, and in feeding and energy metabolism regulation. J. Comp. Neurol. 383:245-281, 1997. © 1997 Wiley-Liss, Inc.

The effects of intravenous (3 mg/kg i.v.) and intraplantar (50 micrograms/50 microliters i.pl.) morphine were investigated on spinal c-Fos expression induced 2 h after intraplantar carrageenin (6 mg/150 microliters of saline) and on carrageenin (2 mg/150 microliters of saline) induced mechanical hyperalgesia, at day 4, in both naive and chronic morphine treated (80 mg/kg/day s.c. on days 1, 2 and 3) rats. In naive rats, i.v. and i.pl. morphine significantly decreased spinal c-Fos expression (64 +/- 4% and 44 +/- 4% reduction of control carrageenin c-Fos expression, P < 0.0001 for both, respectively) and mechanical hyperalgesia (maximal increase: 326 +/- 29%, P < 0.0001 and 87 +/- 5%, P < 0.005 of control carrageenin paw pressure vocalisation threshold (VTPP), respectively), which only developed in the carrageenin injected paw. Both treatments were ineffective in chronic morphine treated rats (92 +/- 9% and 106 +/- 6% of control carrageenin c-Fos expression; 33 +/- 17% and 30 +/- 15% increase of control carrageenin VTPP, respectively). Furthermore, only i.v. morphine increased the VTPP in the contralateral paw, in naive rats (maximal increase: 90 +/- 8%, P < 0.0001 of control carrageenin VTPP), its effects being significantly less pronounced than for the inflamed paw (P < 0.0001). These studies based on spinal c-Fos expression as an indirect marker of spinal nociceptive processes and on behavioural experiments clearly revealed that chronic treatment with systemic morphine induced tolerance to both its systemic and peripheral effects.

Intraplantar co-injection of HOE140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), a selective bradykinin B2 receptor antagonist (0.1, 1 and 10 micrograms), with carrageenan dose-dependently (r = 0.66, P0.01) reduced the carrageenan-evoked total number of c-Fos protein-like immunoreactive (c-Fos-LI) neurones (23 +/- 5%, 35 +/- 6% and 50 +/- 5% reduction; P0.01, P0.001 and P0.001, respectively). These reducing effects were dose-dependent for the number of c-Fos-LI neurones in both superficial (r = 0.70, P0.01) and deep (r = 0.53, P0.05) laminae. Intraplantar co-injection of HOE140 (0.1, 1 and 10 micrograms) with carrageenan significantly reduced the carrageenan-evoked paw (25 +/- 7%, 41 +/- 6% and 41 +/- 3% reduction; P0.001 for all) and ankle (46 +/- 6%, 61 +/- 5% and 61 +/- 5% reduction; P0.001 for all) oedema. Our results provide further evidence for the involvement of peripheral bradykinin B2 receptors in carrageenan-induced inflammatory nociceptive transmission.

Understanding pain or, more precisely, the different types of pain, is above all a question of understanding its physiological mechanisms and, in this regard, the role of basic research has without doubt been to trigger the development of new therapeutic strategies. In an approach to these problems, the main international teams involved in pain research have attempted to develop models of experimental pain in rats. Clearly, research aimed at developing these models is controlled by certain ethical considerations; however, in this context, the end must surely justify the means. The main models used (acute or chronic inflammation, rheumatoid arthritis, peripheral neuropathy) certainly do not give a comprehensive representation of all the pain syndromes encountered in clinical practice, but they do provide new data concerning the physiological, behavioural and pharmacological aspects of pain. While giving a brief description of the complexity of the pain circuit, this article also makes reference to certain pharmacological approaches to the treatment of pain. Peripheral nociceptive messages are conveyed by a mosaic of unmyelinated free fibres distributed throughout cutaneous, muscular and articular tissue, and within the visceral walls. They are then transmitted via various nerve endings (polymodal nociceptors) by small diameter A delta and C fibres, which are activated by mechanical, thermal and chemical stimuli. It is nevertheless difficult to ascertain whether these small diameter fibres are involved only in nociception (specific nociceptors) or whether pain causes an excessive activation of these receptors, which under normal conditions have a role in the reflex that regulates various functions (nonspecific nociceptors). Numerous chemical substances play a part in generating nociceptive impulses (e.g. histamine, serotonin, prostaglandins). Furthermore, the role of neuropeptides, such as calcitonin gene-related peptide and particularly substance P, has been clearly demonstrated in the activation of early neurogenic inflammation. Other substances, such as bradykinin and cytokines, are involved in prolonging the sensation of pain. Nerve growth factor also prolongs the sensation of pain by increasing the cellular excitability of nociceptors and promoting the action of the sympathetic nervous system, which has a major role in controlling pain. The very great diversity of all these interacting substances makes the pharmacological treatment of pain extremely complex. Nevertheless, new therapeutic advances are providing interesting approaches, particularly the development of specific inhibitors of cyclo-oxygenase 2 (COX 2), which is produced by the inflammatory process. Such inhibitors would preserve COX 1, which is both constitutive and physiological, and thereby provide improved tolerability compared with currently used NSAIDs, which act upon both COX pathways. A major focus of research relating to new analgesics is the development of synthetic antagonists of bradykinin, substance P and N-methyl-D-aspartate receptors. An improved understanding of anatomical and electrophysiological processes has led to the discovery of new ascending pathways that transmit nociceptive messages to the reticular formation, the hypothalamus, and the amygdala, as well as to certain areas of the cortex. As a result the notion of one single pain centre is no longer valid. This idea is further reinforced by the knowledge that, at different stages of the pain pathway, different control systems constantly modulate the transmission of nociceptive information. Consequently, at a spinal level, activation of the large diameter cutaneous fibres (A alpha et beta) blocks pain stimuli transmitted by the small diameter fibres. Knowledge of this "gate control' mechanism of the posterior horn of the spinal cord is put to practical application in treatments involving transcutaneous electrical nerve stimulation. (ABSTRACT TRUNCATED)

We have assessed the effects of intraplantar local anaesthetics (bupivacaine and lignocaine) on carrageenan-induced oedema, mechanical allodynia and spinal c-fos protein expression. Mechanical allodynia was evaluated using the vocalization threshold to paw pressure (VTPP) every 30 min until 60, 180 or 240 min after administration of carrageenan. Peripheral oedema, mechanical allodynia and spinal c-fos protein expression were maximal 180 min after carrageenan. Lignocaine did not influence either oedema or VTPP, but reduced spinal c-fos expression at 60 min after carrageenan without later effects. Bupivacaine induced an increase in VTPP at 30 and 60 min, limitation of oedema at 60 min and a reduction in spinal c-fos expression at 60 and 180 min, but these effects were not present 240 min after carrageenan. Intraplantar infiltration with lignocaine and bupivacaine before carrageenan transiently limited signs of inflammatory pain but did not prevent them.

This study assesses the anti-inflammatory/analgesic effects of ketoprofen a non-steroidal anti-inflammatory drug, using the method of c-Fos immunoreactivity at the spinal cord level in two models of noxious stimulation: carrageenan-induced inflammatory pain or acute noxious heat. Ketoprofen was pre-administered intravenously or orally 25 min before an intraplantar injection of carrageenan (6 mg in 150 microliters of saline) in hindpaw of the non-anaesthetised rat or before a single noxious heat (52 degrees C, 15 sec) stimulation of hindpaw of the anaesthetised rat. Three hours after carrageenan or 2 h after noxious heat, the number of spinal c-Fos protein-like immunoreactive (c-Fos-LI) neurons in L4-L5 segments and both the ankle and paw diameter, the indicator of peripheral oedema, were assessed. Pre-administered ketoprofen (1, 3 and 10 mg/kg i.v.) dose-dependently blocks the development of the carrageenan-induced spinal c-Fos protein expression and peripheral oedema, with the highest dose influencing in parallel both parameters (75 +/- 2% diminution of total number of c-Fos-LI neurons per L4-L5 section; 64 +/- 4% and 82 +/- 6% diminution of paw and ankle oedema, respectively). The effect of ketoprofen was significantly greater on the number of c-Fos-LI neurons in deep, as compared to superficial, laminae. Furthermore, the dose-dependent effects of ketoprofen on the carrageenan-induced spinal c-Fos protein expression and both the paw and ankle oedema were correlated. Oral pre-administration of ketoprofen (20 mg/kg) produced the blockage of development of the carrageenan-induced spinal c-Fos protein expression (65 +/- 3% diminution of total number of c-Fos-LI neurons per L4-L5 section) and peripheral oedema (20 +/- 3% and 59 +/- 10% diminution of paw and ankle oedema, respectively). In contrast, the same doses of both the intravenous and oral pre-administration of ketoprofen did not influence either the spinal c-Fos protein expression nor slightly enhanced paw diameter induced by a single noxious heat stimulation. This study suggests a predominant peripheral site, without excluding a central site of action of ketoprofen in the carrageenan-induced inflammation. The method of c-Fos protein-like immunoreactivity revealed ketoprofen to be more potent in comparison to members of other families of non-steroidal anti-inflammatory drugs, previously studied in the same experimental conditions of carrageenan-induced inflammatory pain.

1. We have studied the effects of intraplantar administration of the same doses of morphine on intraplantar carrageenin (6 mg 150 microliters-1 of saline) and noxious heat (52 degrees C for 15 s) induced spinal c-Fos expression and inflammation. 2. Intraplantar carrageenin, in awake rats, induced numerous Fos-like immunoreactive (Fos-LI) neurones in the dorsal horn of L4-L5 lumbar segments of the spinal cord and extensive peripheral oedema. At 1 h 30 min, Fos-LI neurones were preferentially located in the superficial laminae (74 +/- 2%) whereas at 3 h, Fos-LI neurones were observed both in the superficial (45 +/- 2%) and deep (37 +/- 1%) laminae of the spinal dorsal horn. 3. Intraplantar morphine dose-dependently reduced c-Fos expression induced 1 h 30 min after carrageenin (r = 0.605, P < 0.02), these effects were completely blocked by intraplantar methiodide naloxone (20 micrograms) (121 +/- 22% of control carrageenin expression). The systemic injection of the highest dose of intraplantar morphine (50 micrograms) had no significant effect on the number of Fos-LI neurones (88 +/- 9% of control carrageenin expression). None of the drugs influenced unilateral peripheral oedema observed 1 h 30 min after carrageenin. 4. In the second series of experiments, intraplantar morphine dose-dependently reduced the number of superficial and deep Fos-LI neurones induced 3 h after carrageenin (r = 0.794, P < 0.0004 and r = 0.698, P < 0.004, respectively). Furthermore, the effects of the highest dose of intraplantar morphine were completely blocked by co-administration of intraplantar methiodide naloxone (20 micrograms). 5. In addition, intraplantar morphine dose-dependently reduced the ankle (r = 0.747, P < 0.002) and paw (r = 0.682, P < 0.005) oedema observed 3 h after carrageenin, with the effect of the highest dose of intraplantar morphine being completely blocked by co-administration of methiodide naloxone (98 +/- 4% and 102 +/- 8% of control paw and ankle oedema, respectively). 6. Brief noxious heat stimulation, in urethane anaesthetized rats, induced, 2 h after the stimulation, numerous Fos-LI neurones in the dorsal horn of L3-L4 lumbar segments of the spinal cord but no detectable peripheral oedema. Fos-LI neurones were preferentially located in superficial laminae (94 +/- 2%) of the spinal dorsal horn. None of the drugs influenced the noxious heat induced c-Fos expression. 7. Such results illustrate that peripheral effects of morphine preferentially occur during inflammatory states and outline the interest of extending clinical investigations of the possible use of local injection of morphine in various inflammatory pain states.

1. Thirty-five percent of 120 neurons recorded extracellularly in the parabrachial (PB) area of anesthetized rats responded to a peripheral cold stimulus (0 degrees C). The cold-sensitive neurons were located in the lateral PB area, and most of those exhibiting a strong response to cold stimuli were inside or in close vicinity to the area receiving a high density of projections from superficial neurons of the dorsal horn. 2. The receptive fields for cold stimulation often were restricted to one or two parts of the body with a contralateral predominance for the limbs. No side predominance was observed for the face. 3. From a low spontaneous activity (10th percentile < median < 90th percentile: 0.1 < 1.5 < 5 Hz), the PB neurons responded to cold noxious stimuli (0 degree C water bath or waterjet, 20 s), without observable delay, with a sustained discharge. The mean maximal response to the stimulus was 16.1 +/- 1.2 Hz (mean +/- SE; n = 42). 4. About one-half (45%) of these cold-sensitive neurons were activated specifically by cold stimulation and did not respond or were inhibited by noxious heat and/or pinch. The remaining (55%) cold-sensitive neurons were also driven by heat and/or pinch. 5. The cold-sensitive neurons exhibited a clear capacity to encode cold stimuli in the noxious range: the stimulus-response function was always positive and monotonic from 30 to 0 degrees C; the mean curve was linear between 20 and 0 degrees C before plateauing between 0 to -10 degrees C; the mean threshold to cold stimulation was 17.1 +/- 1 degrees C (n = 21) and the mean t50 was 10.7 +/- 1.1 degrees C (n = 13). 6. The cold-sensitive neurons responded to intense transcutaneous electrical stimulation with an early and/or a late peak of activation, the latencies of which were in the 15-50 ms and 80-170 ms ranges (n = 8), respectively, i.e., compatible with the activation of A delta and C fibers. Interestingly, the cold-specific neurons predominantly responded with a late peak, suggesting these neurons were primarily driven by peripheral C fibers. 7. The intravenous injection of morphine depressed the responses of PB neurons to cold noxious stimuli in a dose-related (1, 3, and 9 mg/kg) and naloxone reversible fashion. The ED50 value was estimated approximately 2 mg/kg. Furthermore, two populations of neurons could be separated according to their morphine sensitivity. 8. It is concluded that PB cold-nonspecific neurons could be involved in affective-emotional, autonomic and neuroendocrine reactions in response to noxious cold events. The PB cold-specific neurons could be, in addition, involved in some thermoregulatory processes.

Preadministered niflumic acid, a nonsteroidal anti-inflammatory drug (1, 3 and 9 mg/kg i.v.), dose-relatedly reduced carrageenan-evoked spinal c-Fos expression and the peripheral ankle oedema, with the highest dose reducing in parallel both parameters (55 ± 3% reduction of carrageenan c-Fos expression, 57 ± 13% reduction of carrageenan-evoked ankle oedema, respectively, P < 0.001 for both). Co-administration of low doses of niflumic acid and (+)-HA966, a low-efficacy partial agonist at the glycine site of the NMDA receptor (1 mg/kg i.v. + 2.5 mg/kg s.c., respectively) significantly reduced spinal c-Fos expression, this effect was significantly different from the lack of effect of niflumic acid alone or (+)-HA966 alone on spinal c-Fos expression (P < 0.01 for both drugs). Co-administered niflumic acid and (+)-HA966 did not influence the peripheral carrageenan-evoked oedema. Spinal interactions between prostaglandin- and NMDA receptor-mediated events during inflammatory nociceptive transmission are discussed.

Adjuvant-induced arthritis in rats is considered as a chronic pain model. In a search for a relevant behavioural marker of the chronic pain state, we studied social interactions between an arthritic rat and a healthy partner in a neutral cage. During the test, arthritic rats emitted ultrasonic 22-28 kHz vocalizations (USV), whereas control rats did not, and showed less exploration and more immobility, but did not differ from controls in terms of social behaviours (social investigation, allogrooming). Aspirin (200 mg kg (en)1) or morphine (3 mg kg (en)1) injected intraperitoneally before the test significantly decreased USV without affecting the animals' behaviour. These results suggest that USV could be associated with affect related to aversive or painful stimuli and may constitute a behavioural marker of chronic pain in arthritic rats.

This study, performed in freely moving rats, evaluates the effects of the two most prescribed analgesics, aspirin and acetaminophen, on carrageenin inflammation and the associated c-Fos expression in the rat lumbar spinal cord. Maximal dorsal horn c-Fos expression is observed 3 h after carrageenin (6 mg/150 microliters of saline), with Fos-like (Fos-LI) neurones being predominantly located in laminae I-II and V-VI (41 +/- 3% and 39 +/- 5% of the total number of Fos-LI neurones per section for the control group, respectively) of the dorsal horn. Pretreatment with aspirin (75 or 150 mg/kg, i.v.) reduced the number of Fos-LI neurones induced by carrageenin-inflammation (28 +/- 2% and 45 +/- 1% reduction, respectively; P < 0.001 for both). Acetaminophen (75 or 105 mg/kg, i.v.) reduced the number of Fos-LI neurones (19 +/- 1% and 43 +/- 1% reduction, respectively; P < 0.001 for both). When considering the lower dose (75 mg/kg), the effects of aspirin were significantly more marked than those of acetaminophen (P < 0.001). There was a tendency for both aspirin and acetaminophen to have a more pronounced effect on the number of Fos-LI neurones located in deeper laminae, these differential effects being significant for 75 mg/kg of aspirin (P < 0.01) and 150 mg/kg of acetaminophen (P < 0.01). Both the two doses of aspirin and acetaminophen greatly reduced the inflammatory signs associated with the intraplantar injection of carrageenin. Furthermore there was a positive correlation between the effects of aspirin and acetaminophen on the number of Fos-LI neurones and the inflammatory signs which developed after carrageenin. Our results suggest that the effects of both drugs are mainly due to peripheral site of action without rejecting an additional central site of action of systemic non-steroidal anti-inflammatory drugs (NSAIDs) and acetaminophen. In addition, our results suggest that the approach we used could be a useful tool to evaluate systematically and quantitatively the effects of NSAIDs. Finally, the effects obtained with the low dose of acetaminophen question the classical view of textbooks claiming that such a compound had no anti-inflammatory effect and are in agreement with previous observations in humans.

The effect of intravenous 7-nitro-indazole (7NI), a selective inhibitor of neuronal nitric oxide synthase, on intraplantar formalin evoked spinal expression of c-Fos was studied. The spinal expression of c-Fos in the superficial laminae of the dorsal horn was not significantly altered by 7NI. In contrast formalin evoked c-Fos expression in the deep laminae was reduced by 15 mg/kg of 7NI (42 +/- 8% reduction of control, P0.05). Formalin evoked inflammation was not influenced by 7NI. Our results implicate a functional relationship between c-Fos expression and nitric oxide, at the spinal level, under inflammatory conditions.

1. Intraplantar carrageenin (6 mg 150 microliters-1) evoked a high level of spinal c-Fos expression in the dorsal horn, of segments L4-L5 of the spinal cord, and an extensive peripheral oedema; both parameters were assessed 3 h after carrageenin. 2. Two series of experiments were performed, with the mean total number of Fos like-immunoreactive neurones (Fos-LI), after carrageenin, not being significantly different for the two series of experiments (266 +/- 17 and 332 +/- 31 Fos-LI neurones). For both series of experiments Fos-LI neurones were predominantly located in the superficial and deep laminae, only 10% of the total number of Fos-LI neurones were located in the nucleus proprius and 10% were located in the ventral horn. 3. Pre-administration of the N-methyl-D-aspartate (NMDA) receptor antagonist, (+)-HA966 (0.5 mg kg-1 and 2.5 mg kg-1, s.c.), 30 min before carrageenin, did not significantly influence the total number of Fos-LI neurones, as compared to control carrageenin expression. 4. Pre-administration of the highest dose of (+)-HA966 (10 mg kg-1) significantly reduced the number of deep laminae Fos-LI neurones (28 +/- 3% reduction of control number of Fos-LI neurones after carrageenin, P < or = 0.05), without influencing the number of superficial Fos-LI neurones. There was a tendency towards a reduction of the number of Fos-LI neurones in the nucleus proprius by the highest concentration of pre-administered (+)-HA966, (31 +/- 8% reduction), but this effect did not reach significance. 5. Pre-plus post-administered (+)-HA966 (0.5 mg kg-1), 30 min before and again 45 min afterintraplantar carrageenin, did not significantly influence the total number of Fos-LI neurones, as compared to control carrageenin expression.6. Pre- plus post-administration of 2.5 mg kg-1 (+)-HA966 significantly reduced the total number of Fos-LI neurones, as compared to control carrageenin expression. This effect was reflected by a significant reduction in the number of Fos-LI neurones in the nucleus proprius (36+/-7% reduction of control carrageenin c-Fos expression respectively, P

This study evaluated the 'evoked' expression of Krox-24 protein in the lumbar spinal cord after peripheral carrageenan-induced inflammation and its modification by preadministration of indomethacin, a non-steroidal anti-inflammatory drug, in freely moving rats. Three h after intraplantar carrageenan (6 mg/150 microliters saline) a maximal 'evoked' Krox-24 expression was observed in L2-L6 segments of the dorsal horn ipsilateral to carrageenan inflammation. A maximal number of 'evoked' Krox-24 neurons was observed in L4-L5 segments, predominantly in the superficial laminae (I-II) and to a lesser extent in the medial part of neck (laminae V-VI) of the dorsal horn. Such an increase was not observed after an intraplantar injection of control vehicle saline. increase doses of carrageenan (1, 3 and 6 mg) induced a dose-dependent increase (r2 = 0.617, P0.0001) in the number of evoked' Krox-24 neurons observed in the superficial dorsal horn 3 h after carrageenan. Systemic preadministration of indomethacin (1, 2.5 and 5 mg/kg) dose-dependently reduced (r2 = 0.508, P0.0001) the total number of carrageenan (6 mg at 3 h)-'evoked' Krox-24 neurons (29 +/- 5, 45 +/- 4 and 57 +/- 2% reduction as compared with control, respectively). Systemic indomethacin dose-dependently reduced the inflamed paw and ankle diameter (16 +/- 8, 34 +/- 12, 54 +/- 6% and 48 +/- 14,. 75 + 16, 90 +/- 7% reduction as compared with the control carrageenan inflammation, respectively). There was a positive correlation between the effect of systemic indomethacin on both 'evoked' Krox-24 expression in superficial laminae and the inflammatory signs (r2 = 0.25, P0.01 for the paw diameter; r2 = 0.22, P0.05 for the ankle diameter). In addition, the total number of 'evoked' Krox-24 neurons was significantly reduced (43 +/- 5% reduction as compared with control) by an oral pretreatment of indomethacin (10 + 10 mg/kg). Oral indomethacin totally blocked the ankle diameter and reduced the paw diameter (100 + 14 and 30 +/- 6% reduction of the control carrageenan inflammation, respectively).

1. Parabrachiohypothalamic (PB-H) neurons (n = 71) were recorded with extracellular micropipettes in the parabrachial (PB) area and were antidromically driven from the ventromedial nucleus (VMH) or the retrochiasmatic area (RCh) of the hypothalamus, in the anesthetized rat. The spontaneous activity of these neurons was very low, (10th percentile < median frequency < 90th percentile were 0.01 < 0.2 < 7 Hz). The axons of these neurons exhibited a very slow conduction velocity in the range of 0.2-1.4 m/s, i.e., corresponding to thin unmyelinated fibers. 2. Most PB-H neurons (89%) were located in the mesencephalic division of the PB area (mPB) mainly in the superior lateral (mPBsl) and external lateral (mPBel) subnuclei. 3. These units were separated in three groups: 1) a group of nociceptive-specific (NS) neurons (49%) activated by mechanical and/or thermal (heat) cutaneous stimuli only in noxious range; 2) a group of inhibited neurons (7%), not activated by any of the mechanical or thermal cutaneous stimuli but inhibited, by at least one of these stimuli, which had to be in noxious range; and 3) a group of nonresponsive neurons (44%). 4. The NS neurons responded exclusively to mechanical (pinch or squeeze) and/or thermal (waterbath or waterjet > 44 degrees C) noxious stimuli with a rapid onset, a marked and sustained activation, and generally no afterdischarge. The magnitude of the responses was between 2 and 30 Hz with a mean value of 14.8 +/- 1.4 Hz (mean +/- SE, n = 49). These neurons exhibited a clear capacity to encode thermal stimuli in the noxious range: 1) the stimulus-response function was always positive and monotonic; 2) the slope of the mean curve increased up to a maximum (between 46 and 50 degrees C) then beyond the slope decreased; and 3) the mean threshold was 44.3 +/- 2.2 degrees C. 5. The excitatory receptive fields of the NS neurons were often large including all (22% of the population) or several (67% of the population) parts of the body. In the few remaining cases (11%) they were restricted to one part of the body. In addition, in several cases, noxious stimuli applied outside the excitatory receptive field were found to strongly inhibit the discharge of NS neurons. 6. Most NS neurons responded to intense transcutaneous electrical stimulation with two peaks of activation.(ABSTRACT TRUNCATED AT 400 WORDS)

This study evaluated, in awake rats, the time course of the expression of c-Fos in spinal cord neurons, in the L4-L5 segments, at various time points after intraplantar carrageenin (0.5 h, 1 h, 1.5 h, 2 h and 2.5 h). In addition, the effects of pre-administered morphine (3 mg/kg, i.v.) on the c-Fos expression, at the various time points, were studied. Very few Fos-like immunoreactive (Fos-LI) neurons were observed 0.5 h after carrageenin. However, spinal c-Fos expression increased initially (at 1 h), in the superficial laminae (I-II) of the spinal dorsal horn, and incrementally increased both in the superficial and deep (V-VI) laminae at later time points after carrageenin. Systemic morphine did not significantly decrease the number of superficial Fos-LI neurons observed 1 h after carrageenin, whereas it significantly reduced the number of superficial Fos-LI neurons induced at 1.5 h and 2 h after carrageenin (58 +/- 3% and 57 +/- 10% reduction, P < 0.001, respectively). In addition, morphine reduced the number of deep Fos-LI neurons at 1.5 h and 2 h after carrageenin (86 +/- 4%, P < 0.01 and 82 +/- 8%, P < 0.001 reduction as compared to control carrageenin expression, respectively). In contrast, morphine was less efficacious in decreasing the number of Fos-LI neurons observed in the superficial and deep laminae at 2.5 h after carrageenin (34 +/- 6% and 59 +/- 6% reduction, P < 0.001, respectively). Thus, the peak effect of pre-administered morphine on carrageenin evoked c-Fos expression was observed 1.5 h and 2 h after intraplantar carrageenin, with a weaker effect observed at 2.5 h after carrageenin. The pharmacokinetic complications between the time course of the antinociceptive effects of morphine and c-Fos expression is discussed. These results clearly demonstrate that studies of c-Fos expression with pharmacological investigations should take into consideration this finding since one delay after the stimulation does not give a full indication of the full potential of the drug tested.

We previously showed that pathological adjuvant-induced arthritis (AIA), a chronic pain model, is associated with an increase in the number of Fos-like immunoreactivity (Fos-LI) in lumbar spinal cord neurons; maximal Fos-LI corresponds to the peak of pathological and behavioral signs of the disease. On the other hand, AIA is prevented in rats that have previously been injected with diluted complete Freund adjuvant (CFA). Here, the effects of pretreatment with CFA were studied in parallel on both pathological parameters and pain-related tests, and on Fos-LI, 3 weeks after the inoculation of the arthritogenic solution during the acute phase of the disease, that is, when hyperalgesia is maximal. One group of rats (vaccinated group) was pretreated with diluted CFA, 3 weeks and 1 week before inoculation (AIA) (with the concentrated arthritogenic solution), while a second group (arthritic group) was pretreated with the vehicle for the CFA solution and then inoculated with the concentrated arthritogenic solution. Control groups consisted of rats treated with various combinations of either saline or vehicle solutions during the pretreatment periods or at the time of inoculation. The arthritic group developed all pathological symptoms of AIA (i.e., loss of weight, difficulties in walking, increases in paw diameter, and decreases in the vocalization thresholds to mechanical pressure of the hindpaw, and finally an increase in the vocalization response to flexion and extension). In contrast, the vaccinated group did not develop AIA symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

The organization of efferent projections from the pontine parabrachial (pPB) area to the forebrain rostral to the central nucleus of the amygdala (Ce) was studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L), into subregions of the pPB area. The present study is a follow-up of a former study (Bernard et al. [1993] J. Comp. Neurol. 329:201-229) which examines pPB projections onto the Ce. The results demonstrate that: (1) the pPB(m) region (the medial, the ventral lateral subnuclei and the waist area) diffusely projects to the lateral division (BSTL) of the bed nucleus of the stria terminalis (BST), the Ce-BSTL continuum (including, the dorsal portion of substantia innominata, the ventral portion of globus pallidus, the fundus striatum, and the substriatal area) and to a lesser extent the agranular insular cortex; (2) the pPB(1) region [the central lateral (pPBcl) and the outer portion of external lateral subnuclei] densely projects to the dorsal lateral subnucleus of BST (BSTdl); only the pPBcl subnucleus projects to the median, the anteroventral and the periventricular nuclei of the preoptic hypothalamus; and (3) the remaining pPB area (the dorsal lateral, part of the external lateral and the external medial subnuclei) projects to the nucleus of horizontal limb of diagonal band but does not project onto the BST and the preoptic hypothalamus. It is suggested that the pPB(m)-BSTL "diffuse pathway" is mainly implicated in motivational and autonomic aspects of taste. The pPB(1)-BSTdl and hypothalamic "concentrated pathways" could be implicated in autonomic and nociceptive processes.

It has recently been demonstrated that the nucleus centralis of the amygdala contains numerous neurons specifically driven by noxious stimuli. The aim of the present study was to investigate the effect of i.v. morphine on responses of neurons located in the nucleus centralis of the amygdala to noxious mechanical or thermal stimuli. It was observed, in halothane-anesthetized rats, that i.v. morphine caused a marked depression of responses induced by noxious thermal (waterbath, 50 degrees C) and mechanical (pinch) stimuli and caused a moderate depression of spontaneous activity in a dose-related (1, 3, 9 mg/kg) and naloxone reversible fashion. The ED50 value was 1.2 and 9 mg/kg for i.v. morphine for the evoked activity and spontaneous activity, respectively. The strong depressive effect of morphine on evoked activity probably reflects a direct action of this drug at both spinal and parabrachial levels. These results could account, at least in part, for the effect of morphine on the emotional-affective aspects of pain.

Our previous data reported a maximal expression of the c-fos immediate-early gene in the lumbar spinal cord of the non-stimulated polyarthritic rat neurons, three weeks after Freund's adjuvant injection. The present study utilises c-fos expression to judge the reactivity of spinal neurons to calibrated mechanical pressure applied to the ankle joint, in both normal and arthritic rats under ketamine anesthesia. The results indicate that the number of Fos-like immunoreactive neurons (1) is slightly decreased in ketamine-anesthetized non-stimulated arthritic rats as compared to the non-anesthetized non-stimulated ones, (2) is significantly higher in both stimulated normal and arthritic animals as compared to non-stimulated animals, particularly in laminae I, II, V and VI of L3 and L4, and (3) is significantly increased in stimulated arthritic as compared to stimulated normal rats, in all laminae of lumbar spinal segments. The appearance of 'basal' Fos labeling during the adjuvant-induced arthritic disease and the increased number of Fos-like immunoreactive neurons in stimulated arthritic rats compared to stimulated normal animals indirectly suggests that these neurons are abnormally active and thus involved in the hyperalgesia of arthritic disease. Therefore the use of Fos-like immunoreactivity in the chronic pain model seems to be an appropriate tool to study possible effects of various pharmacological compounds, such as analgesics and anti-inflammatory drugs.

The organization of the efferent projections from the pontine parabrachial (pPB) area to the amygdala has been studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L), a sensitive and selective anterograde axonal marker, into restricted subregions of the pPB area. The results confirmed that the pPB area primarily projected onto the ipsilateral nucleus centralis of the amygdala (Ce), and to a lesser extent onto the ipsilateral posterior basolateral (BLP), anterior basomedial (BMA), and amygdaloid cortical (ACo) nuclei of the amygdala. Substantial projections were also found in the substantia innominata dorsal/ventral portion of the globus pallidus (SId/GPv), substriatal (SStr), and fondus striatal (FStr) regions which continue the amygdala rostrally. The results demonstrated that the projections of the pPB area onto the Ce were topically organized: 1) The region of the pPB area mainly including the medial subnucleus (pPBm), the waist area (pPBwa), and a thin rostral lamina of the ventral lateral subnucleus (pPBvl) projects primarily to the medial portion of the Ce (CeM). Dense projections were also found in the BLP, BMA, and ACo nuclei of the amygdala, and in the SId/GPv, SStr, and FStr rostral areas. 2) The region of the pPB mainly including the rostral portion of the central lateral subnucleus (pPBcl) and the outer-rostral portion of the external lateral subnucleus (pPBel) projects primarily to the lateral portion of the Ce (CeL). 3) The region of the pPB mainly including the dorsolateral subnucleus (pPBdl), the remaining pPBel, and the external medial (pPBem) subnuclei projects primarily to the lateral capsular portion of the Ce (CeLC) and bilaterally to its rostral portion. Dense projections were also found in the regions which extend the CeLC rostrally and in the SId/GPv, SStr, and FStr rostral areas. The possible role of each of the three parabrachio-amygdaloid pathways described is discussed. It was suggested that the pPB-CeM pathway is mainly implicated in gustatory processes; the pPB-CeL pathway is mainly implicated in visceral and chemosensitive processes; and the pPB-CeLC pathway is mainly implicated in respiratory, cardiovascular, and nociceptive processes.

I n their paper, Maier and colleagues proposed the existence of an “antianalgesia” system triggered by “safety” signals and mediated by the release of cholecystokinin (CCK) in the spinal cord. They also proposed the existence of a hyperalgesia system activated by illness. These proposals are fascinating, and there is no doubt that they will lead to extensive experimental investigation in both animals and humans. This new insight in the domain of pain modulation reminds us of the initial enthusiastic studies of the 1970s related to the stimulation-producing analgesia phenomenon. In fact, until the end of the 1980s the use of deep brain stimulation in the relief of intractable pain in humans seemed to be effective according to various neurosurgical groups. However, actually, this technique is less and less used, notably in Europe, and the controversies concerning it are wellexplained by the numerous side effects obtained by periaqueductal gray electrical stimulation in the rat9,” This was a good illustration of how the results collected by different groups in behavioral research are sometimes not homogeneous and that extensive interpretation may sometimes be premature. In this respect, Maier and colleagues, with great prudence, wrote that “the suggestions made in this paper are tentative,” and they indicated that “they are going well beyond the data.” The most exciting finding of this article, which has triggered such enthusiasm and lack of moderation, was that a “safety” signal was able to reduce morphine analgesia when the opiate effects were gauged by means of the tail-flick test and given systemically

A parallel clinical and behavioral study of adjuvant-induced arthritis in the rat showed four stages in the time-course of the disease: preclinical (first week), acute (weeks 2–4), post-acute (weeks 5–8) and recovery weeks 9–11) [Calvino et al. (1987) Behav. Brain Res.24, 11–29]. As several studies have reported the expression of the proto-oncogene c-fos in spinal cord neurons following acute noxious peripheral stimuli, the aim of this study was to quantitatively assess Fos-like immunoreactivity in lumbar spinal cord neurons at various times of adjuvant-induced arthritis development, i.e. one, two, three, 11 and 22 weeks post-inoculation. The total number of Fos-like immunoreactive neurons in the lumbar enlargement correlated with the observed development of adjuvant-induced arthritis, i.e. Fos-like immunoreactivity was absent at one week, moderate at two weeks, greatly increased at three weeks, decreased at 11 weeks and returned to control values at 22 weeks. At three weeks, at the peak of Fos-like immunoreactivity distribution and acute stage of hyperalgesia, maximal labeling was observed in L3 and L4 spinal segments. In these segments, the most densely labeled region was the neck (laminae V and VI) of the dorsal horn (55%) and the ventral horn (35%) as compared to the superficial laminae (laminae I and II; 5%) and the nucleus proprius (laminae III and IV; 5%). These data indicate that c-fos expression induced by chronic inflammation is better expressed in deeper laminae than in the superficial ones, and that the number of Fos-positive cells correlates with behavioral studies. Thus, the use of Fos-like immunoreactivity in the chronic inflammatory pain model seems to be an interesting tool to study possible effects of various pharmacological compounds such as analgesic or anti-inflammatory drugs.

The high density of GABA-benzodiazepine receptors in the superficial dorsal horn suggests a possible involvement of benzodiazepines (BZs) in the modulation of spinal pain processes. In this electrophysiological study we have examined the effects of midazolam (MZ), a water-soluble short-acting BZ, on the activities of 57 nociceptive non-specific dorsal horn cells, one in each animal. Recordings were performed at lumbar level in unanesthetized decerebrate spinal rats before and following intravenous injection of MZ (1, 2 or 5 mg/kg). The spontaneous activity was weakly and significantly but not dose dependently reduced by MZ. For the total neuronal population MZ induced no significant effect on C-fiber evoked responses, whatever the dose used. More precise analysis shows that for 45/55 neurons the responses were slightly depressed, but this effect was not dose dependent. In contrast, A delta-fiber evoked responses were markedly and dose dependently depressed. These effects of MZ were reversed by intravenous administration of the antagonist flumazenil (FZ). Despite the fact that MZ displays a very weak effect on responses due to C-fiber stimulation, the possible involvement of BZs in the modulation of nociceptive transmission at the level of the dorsal horn is discussed on the basis of clinical and experimental findings, taking into account the role of GABAergic mechanisms in sensory events.

The effects of the analgesic agent tramadol (0.1-1 mg/kg i.v.) were compared to those of the mixed agonist-antagonist analgesics nalbuphine (1 mg/kg i.v.) and buprenorphine (3 micrograms/kg i.v.) in the vocalization threshold to paw pressure test. Normal and Freund's adjuvant-induced arthritic rats were used. We have shown previously that these animals used as a model of clinical pain exhibit an enhanced sensitivity to morphine (0.1-1 mg/kg i.v.), with a rapid development of tolerance after repetitive low doses, a response not observed in normal rats. In the present study, the antinociceptive effects of tramadol, buprenorphine and nalbuphine were enhanced (by 2- to 5-fold) in arthritic compared to normal rats. In this model, these effects were significantly reduced by a dose of naloxone (0.1 mg/kg i.v.) that completely antagonized the effect of morphine. In this model, the antinociceptive effect of tramadol (1 mg/kg i.v.) was comparable to that of nalbuphine (1 mg/kg i.v.), buprenorphine (3 micrograms/kg i.v.) and morphine (1 mg/kg i.v.). Repeated administration of low doses of tramadol twice daily for 4 days to arthritic rats did not induce tolerance, in contrast to nalbuphine, buprenorphine, and morphine. In addition, no cross-tolerance between tramadol and morphine was observed in these animals.

We studied the effects of sodium valproate, a widely used antiepileptic drug and a hyperammonemic agent, on L-[1-14C]glutamine and L-[1-14C]glutamate metabolism in isolated human kidney-cortex tubules. Valproate markedly stimulated glutamine removal as well as the formation of ammonia, 14CO2, pyruvate, lactate and alanine, but it inhibited glucose synthesis; the increase in ammonia formation was explained by a stimulation by valproate mainly of flux through glutaminase (EC 3.5.1.2) and to a much lesser extent of flux through glutamate dehydrogenase (EC 1.4.1.3). By contrast, valproate did not stimulate glutamate removal or ammonia formation, suggesting that the increase in flux through glutamate dehydrogenase observed with glutamine as substrate was secondary to the increase in flux through glutaminase. Accumulation of pyruvate, alanine and lactate in the presence of valproate was less from glutamate than from glutamine. Inhibition by aminooxyacetate of accumulation of alanine from glutamine caused by valproate did not prevent the acceleration of glutamine utilization and the subsequent stimulation of ammonia formation. It is concluded from these data, which are the first concerning the in vitro metabolism of glutamine and glutamate in human kidney-cortex tubules, that the stimulatory effect of valproate is primarily exerted at the level of glutaminase in human renal cortex.

1. Neurons were recorded in the parabrachial (PB) area, located in the dorsolateral region of the pons (with the use of extracellular micropipette), in the anesthetized rat. Parabrachioamygdaloid (PA) neurons (n = 67) were antidromically identified after stimulation in the centralis nucleus of the amygdala (Ce). The axons of these neurons exhibit a very slow conduction velocity, between 0.26 and 1.1 m/s, i.e., in the unmyelinated range. 2. These PA neurons were located in a restricted region of the PB area: the subnuclei external lateral (PBel) and external medial (PBem). A relative somatotopic organization was found in this region. 3. These units were separated into two groups: 1) a group of nociceptive-specific (NS) neurons (69%), which responded exclusively to noxious stimuli, and 2) a group of nonresponsive (NR) neurons (31%). 4. The NS neurons exhibited low or lacked spontaneous activity. They responded exclusively to mechanical (pinch or squeeze) and/or thermal (waterbath or waterjet greater than 44 degrees C) noxious stimuli with a marked and sustained activation with a rapid onset and generally without afterdischarge. Noxious thermal stimuli generally induced a stronger response than the noxious mechanical stimuli. These neurons exhibited a clear capacity to encode thermal stimuli in the noxious range: 1) the stimulus-response function was always positive and monotonic; 2) the slope of the curve progressively increased up to a maximum where it was very steep, then the steepness of the slope decreased close to the maximum response; and 3) the mean threshold was 44.1 +/- 2 degrees C, and the point of steepest slope of the mean curve was around 47 degrees C. 5. The excitatory receptive fields of the NS neurons were large in the majority (70%) of the cases and included several areas of the body. A more marked activation was often obtained from stimuli applied to one part of the body, denoted as the preferential receptive field (PRF). In the other cases (30%), the excitatory receptive field was relatively small (SRF) and restricted to one part of the body (the tail, a paw, a hemiface, or the tongue). Both the PRF and SRF were more often located on the contralateral side. In addition, noxious stimuli applied outside the excitatory receptive field were found to strongly inhibit the responses of NS neurons. 6. All the NS neurons responded to intense transcutaneous electrical stimulation applied to the PRF or SRF with two peaks of activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Pain during labor is a common and severe phenomenon, but its clinical management remains haphazard because its neurophysiology is poorly understood. In the current study, the authors evaluate the parturient rat as a relevant model to study the pharmacology of labor pain.Control of birth timing in term pregnant rats was achieved by gavage with RU 486 (5 mg/kg) the day before the expected day of parturition. The behavioral events preceding the expulsion of the first pup were analyzed, and immunodetection of the c-Fos protein was used to evaluate the spinal neuronal activity at the lumbosacral level where genital and perineal inputs terminate.Hind limb and abdominal stretches occurred during labor (mean number, 57 +/- 10), arbitrarily defined as the time elapsed between the first stretch and the expulsion of the first pup (mean duration, 62 +/- 5 min). Subcutaneous oxytocin increased the frequency of stretches, accounting for the fact that these manifestations are linked to uterine contractions. Finally, epidural morphine (30 microg/10 microl) in oxytocin-treated rats, although resulting in no change of labor duration, significantly decreased the number of stretches (8 +/- 2 vs. 57 +/- 12 for epidural saline) and the number of c-Fos-positive neurons in the lumbosacral spinal segments (80 +/- 25 vs. 165 +/- 17 for epidural saline).These results indicate that stretches during labor in the rat correspond to a behavioral response to nociception associated with uterine contractions and suggest that parturition in the rat could be a relevant model to investigate nociceptive mechanisms associated with parturition in women.

We have recently shown concurrent changes in behavioural responses and c-Fos protein expression in the central nervous system in both naive and morphine-dependent rats after systemic administration of the opioid antagonist naloxone. However, because naloxone acts on the three major types of opioid receptors, the present study aimed at determining, in the same animals, both changes in behaviour and c-Fos-like immunoreactivity after intravenous injection of selective opioid antagonists, such as mu (beta-funaltrexamine, 10 mg/kg), delta (naltrindole, 4 mg/kg) or kappa (nor-binaltorphimine, 5 mg/kg) opioid receptor antagonists, in naive or morphine-dependent rats. In a first experimental series, only beta-funaltrexamine increased c-Fos expression in the eight central nervous system structures examined, whereas no effect was seen after naltrindole or nor-binaltorphimine administration in naive rats. These results suggest a tonic activity in the endogenous opioid peptides acting on mu opioid receptors in normal rats. A second experimental series in morphine-dependent rats showed that beta-funaltrexamine had the highest potency in the induction of classical signs of morphine withdrawal syndrome, as well as the increase in c-Fos expression in the 22 central nervous system structures studied, suggesting a major role of mu opioid receptors in opioid dependence. However, our results also demonstrated that naltrindole and, to a lesser extent, nor-binaltorphimine were able to induce moderate signs of morphine withdrawal and relatively weak c-Fos protein expression in restricted central nervous system structures. Therefore, delta and kappa opioid receptors may also contribute slightly to opioid dependence.

The very-high-efficacy, selective 5-HT(1A) receptor agonist, F 13640 produces uniquely powerful analgesia in rat models of chronic pain by novel neuroadaptive mechanisms (inverse tolerance and co-operation with nociception) [Neuropharmacology 43 (2002) 945-958]. A signal transduction theory and evidence suggest that F 13640 initiates these mechanisms, paradoxically, by mimicking the central effects of nociceptive stimulation. We report that the i.p. injection of F 13640 induces c-Fos protein expression in the L3-L5 segments of the spinal cord. Some 65% of c-Fos protein immunoreactive (c-Fos-IR) nuclei occurred bilaterally in the dorsal horn laminae I-II and V-VI, spinal areas that contain neurons responsive to nociceptive stimulation. This pattern is not unlike that found earlier in arthritic rats, a model of somatotopically widespread nociception. Dose-response studies indicated that c-Fos protein expression was induced at doses (0.63 and 2.5 mg/kg, i.p.) at which previous studies had found F 13640 to produce hyperalgesia. Time-response studies found that c-Fos-IR nuclei appeared within 1-4 h after 0.63 mg/kg of F 13640, with a maximum at 2 h. This parallels literature evidence that c-Fos expression reaches peak late after, and outlasts, nociceptive stimulation. Similar to opioids counteracting noxiously induced c-Fos expression, 10 mg/kg (s.c.) of morphine reduced the number of c-Fos-IR nuclei induced by 0.63 mg/kg of F 13640 (by 45+/-5%; P0.001). The induction by F 13640 of c-Fos protein expression may relate to the initial hyperalgesia which earlier data indicate the agent to produce early upon its administration.

We have previously shown that RB101, a dual inhibitor of enkephalin-degrading enzymes, decreased carrageenin-evoked c-Fos protein expression at the spinal cord level in awake rats. Moreover, we have also shown that c-Fos expression is a useful marker of the possible direct or indirect interactions between neural pathways, such as opioid and cholecystokinin systems. We now investigated the respective roles of the three main types of opioid receptors (mu, delta, or kappa) and their possible interactions, in the depressive effects of RB101 in inflammatory nociceptive conditions induced by intraplantar carrageenin (6 mg/150 microl of saline). We used beta-funaltrexamine (beta-FNA), naltrindole (NTI), and nor-binaltorphimine (BNI) as specific antagonists for mu, delta- and kappa-opioid receptors, respectively. c-Fos protein-immunoreactivity (c-Fos-IR) was evaluated as the number of c-Fos-IR nuclei in the lumbar spinal cord 90 min after carrageenin. c-Fos-IR nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of segments L4-L5 (areas containing numerous neurons responding exclusively, or not, to nociceptive stimuli). RB101(S) (30 mg/kg, i.v.) significantly reduced the total number of carrageenin-evoked c-Fos-IR nuclei (30% reduction, P0.01). This effect was completely blocked by beta-FNA (10 mg/kg, i.v.), or NTI (1 mg/kg, i.v.). In contrast, BNI (2.5 mg/kg, i.v.) did not reverse the reducing effects of RB101(S) on carrageenin-evoked c-Fos protein expression. These results suggest that functional interactions occur between mu- and delta-opioid receptors in enkephalin-induced antinociceptive effects.

In behavioural tests, RB101 (N-[(S)-2-benzyl-3[(S)(2-amino-4-methyl-thio)butyldithio]-1-oxopropyl]-L-phenylalanine benzyl ester), a mixed inhibitor of enkephalin-degrading enzymes, induces antinociceptive effects without producing tolerance, or cross-tolerance with morphine. In the present experiments, the acute or chronic effects of enantiomer RB101(S) were examined on the response of spinal cord neurons to nociceptive inflammatory stimulation (intraplantar injection of carrageenin) using c-Fos studies in awake rats. The number of c-Fos immunoreactive nuclei was evaluated in the lumbar spinal cord 90 min after carrageenin. c-Fos-immunoreactive nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of segments L4-L5 (areas containing numerous neurones responding exclusively, or not, to nociceptive stimuli). In the first experimental series, acute RB101(S) (30 mg/kg, i.v.), morphine (3 mg/kg, i.v.), or respective vehicles were injected in rats chronically treated with RB101(S) (160 mg/kg/day for 4 days, s.c.). In chronically treated RB101(S) rats, both acute RB101(S) and morphine reduced the total number of carrageenin-evoked c-Fos-immunoreactive nuclei. In the second experimental series, acute RB101(S) (30 mg/kg, i.v.) reduced the total number of carrageenin-evoked c-Fos-immunoreactive nuclei with similar magnitude in naive and in morphine-tolerant (100 mg/kg/day for 3 days, s.c.) rats. These data provide further evidence that different cellular mechanisms occurred after chronic stimulation of opioid receptors by morphine or endogenous enkephalins.