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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; P<0.001 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 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%; P<0.001). 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 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 (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, P<0.01). 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.

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, P<0.01), with 49+/-3% reduction (P<0.001) 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, P<0.05 for both) and deep V-VI (42+/-6% and 61+/-2% reduction, respectively, P<0.001 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, P<0.001 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.

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.

We have evaluated the effects of nefopam on the spinal c-Fos protein expression in the model of acute (noxious heat) and persistent (intraplantar injection of formalin) nociception in the rat. One and two hours after i.pl. formalin injection, c-Fos immunoreactive (c-Fos-IR) nuclei were preferentially located in the superficial (I-II) and deep (V-VI) laminae of the spinal dorsal horn of segments L4-L5, i.e. spinal areas containing numerous neurons responding exclusively, or not, to peripheral nociceptive stimuli. The doses of 15 and 30 mg/kg (s.c.) of nefopam had significant reducing effects on the formalin-evoked spinal c-Fos protein expression (36+/-14% and 47+/-9% reduction of the total number of c-Fos-IR nuclei per section, respectively, P<0.05 for both). These reducing effects of nefopam were not detectable 2 h after formalin. These results provide evidence that the significant effects of nefopam are time-limited in the formalin model of persistent nociception. One hour after noxious heat stimulation (52 degrees C for 15 s), c-Fos-IR nuclei were principally located in the superficial laminae I-II of the spinal dorsal horn (about 90% of the total number of c-Fos-IR nuclei per section). Nefopam (15 mg/kg s.c.) significantly reduced the noxious heat-evoked spinal c-Fos protein expression (33+/-3% reduction of the total number of c-Fos-IR nuclei, P<0.0001). The present results provide first evidence for the reducing effects of nefopam on the noxiously evoked spinal c-Fos protein expression, principally in acute nociceptive processes. These results suggest that nefopam may produce antinociceptive effects mainly in acute pain states.

Opioid compounds have powerful analgesic properties when administered to the spinal cord. These effects are exerted through mu and delta opioid receptors, and both pre- and postsynaptic mechanisms have been implicated. To specifically address the relative pre- and postsynaptic contribution to spinal opioid analgesia, we have quantitatively assessed the pre- vs. postsynaptic distribution of the mu-opioid (MOR-1, MOP(1)) and delta-opioid receptors (DOR-1, DOP(1)). We also examined the rostro-caudal arborization of MOR-1 and DOR-1 immunoreactive primary sensory neurons, using an isolated dorsal root preparation. These results were compared to those obtained by labeling for calcitonin gene-related peptide (CGRP), a neuropeptide whose expression in the spinal cord is restricted to the terminals of small diameter primary sensory neurons. We estimate that approximately one half of MOR-1 and two thirds of DOR-1 immunoreactivity in the cervical spinal cord is located on primary afferent fibers. These fibers have a broad rostro-caudal distribution, extending at least three segments rostral and caudal to their segment of entry. Regardless of marker used, the rostral projection was greatest, however, the distribution of CGRP-immunoreactive fibers differed somewhat in that they had a much smaller projection to the most caudal segments examined. Our results suggest that presynaptic delta opioid actions predominate, but that there are mixed pre- and postsynaptic inhibitory effects exerted by opioid analgesics that act at the spinal cord mu opioid receptor.

Using in vivo voltammetric detection of nitric oxide (NO) a previous study demonstrated an increased NO release at the lumbar dorsal horn level of the spinal cord by peripheral inflammatory processes in decerebrated-spinalized rats. This study concerns the effects of acute peripheral stimulations. Gentle non-noxious or isolated nociceptive stimulation did not modify the oxidation current due to NO. However, transient transcutaneous repetitive electrical stimulation of the hindpaw at mild to high intensities (1, 3, and 6 mA) provoked increases in the voltammograms, which were prolonged after stimulation ceased. These effects were absent in animals pretreated with the N-methyl-D-aspartate channel blocker MK-801. The data provide novel direct evidence that NO participates, at least during repetitive stimulation, in the transmission of noxious afferent messages in the dorsal horn of the spinal cord.

The paper describes in vivo voltammetric detection of nitric oxide with carbon fibre microelectrodes at the lumbar spinal dorsal horn level of decerebrated-spinalized rats during peripheral noxious inflammatory processes. At the lumbar (L3-L4) dorsal horn level, a nitric oxide dependent peak of oxidation current (650 mV), remaining stable for up to 4h ((92 +/- 5)% of control) could be detected indicating that significant amounts of nitric oxide are produced continuously. Following subcutaneous injection in the hindpaw of 50 microl of 0.5% formalin the oxidation current rapidly increased ((115 +/- 5)% of control at 25 min) and reached (120 +/- 6)% of control 1h later. Subsequently the voltammograms stabilized for up to 90 min and decreased ((107 +/- 4)% at 124 min). After an injection in the hindpaw of 150 microl of 4% carrageenan, the voltammograms remained at control level for 1h and then the oxidation current increased continuously for up to 4h ((145 +/- 16)% of control at 240 min); such an increase was reversed by ketamine. In these two models of inflammation, the delay in onset and the duration of the increases in NO release within the dorsal horn relate, to some extent, to the time course of the peripheral inflammatory processes, since they are shorter after formalin than after carrageenan. The results provide a direct in vivo demonstration that the intercellular messenger nitric oxide participates in the transmission of noxious afferent messages within the dorsal horn of the spinal cord following peripheral inflammation., (Copyright 2002 European Federation of Chapters of the International Association for the Study of Pain)

In a model of mononeuropathic pain (chronic constriction injury of the sciatic nerve, CCI), we have demonstrated that light touch stimuli (stroking) to the paw induced Fos-like immunoreactivity (Fos-LI) in the superficial and deep dorsal horn of the rat spinal cord (Catheline et al., Pain 80 (1999a) 347). The efficacy of opioids in neuropathic pain being controversial, we have tested the effects of morphine (0.3, 1 and 3 mg/kg intravenous, i.v.) on this spinal Fos-LI evoked by light tactile stimuli, which could be related to mechanical allodynia. Morphine did not change the level of spinal Fos-LI observed following light touch stimuli in the CCI rats (43 +/- 3, 38 +/- 7, and 37 +/- 4 Fos-LI neurones/40 microm L4-L5 section, respectively, for the three doses versus 32 +/- 4 in the control group). In contrast, the administration of 3 mg/kg of i.v. morphine reduced by 30% the number of Fos-LI neurones induced by heat stimulation (52 degrees C, 15 s duration) in CCI rats (P < 0.05) as in sham-operated rats. These effects were reversed by the systemic administration of naloxone. The lack of effect of morphine on touch-evoked Fos-LI in the superficial dorsal horn reinforces the assertion that dynamic mechanical allodynia is related to information transmitted by A-beta fibres, since opioid receptors are mainly located on thin primary afferent fibres. Our results provide a basis for a certain form of allodynia that is insensitive to morphine.

Several studies have used c-Fos expression to delineate the neural substrate underlying naloxone-precipitated morphine withdrawal (MW). However, because behavioral manifestations of MW depend on both the degree of dependence and the doses of naloxone (NAL), a comprehensive study would require examining c-Fos expression in relation with the degree of MW. Here, changes in behavior and in c-Fos-like immunoreactivity (FLI) were studied in the same rats after injection of three doses of NAL to precipitate various degrees of MW. Fifteen established signs of MW were examined for 1 hour after NAL injection, and FLI was quantified in 52 regions of the brain and in the lumbosacral spinal cord. Linear regression analyses were used to examine changes in numbers of signs and FLI neurons with the doses of NAL, and data were considered dose-related for a statistical level of significance of P < 0.05. In summary, autonomic signs of MW increased in a dose-related manner, whereas somatomotor signs did not. After MW, 33 central nervous system regions exhibited significant increases in FLI and were, thus, considered as important neural correlates of MW. Twenty of them displayed dose-related increases in c-Fos expression and correspond to regions related to autonomic functions. Low c-Fos expression was detected in some regions involved in motor control or in reward, suggesting either their minor role in MW or a limitation of the technique. This dose-response analysis suggests that the increase in the severity of autonomic manifestations of MW is associated with a gradual activation of major structures of the autonomic nervous system., (Copyright 2001 Wiley-Liss, Inc.)

Urethane anaesthesia strongly reduced the peripheral edema (31+/-5 and 96+/-8% reduction of carrageenan-enhanced paw and ankle diameters, respectively; P<0.001 for both) and the spinal c-Fos protein expression (71+/-4% reduction of the number of c-Fos protein-labeled nuclei; P<0.001), 3 h after intraplantar injection of carrageenan in rats. In urethane anaesthetised rats, i.v. injection of racemic-flurbiprofen (0.3, 3 and 9 mg/kg) has weaker effects on carrageenan-evoked spinal c-Fos protein expression and peripheral edema than those previously described in the same inflammatory nociceptive model in awake rats.

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., (Copyright 2000 Wiley-Liss, Inc.)

We have evaluated the effects of either intravenous or intraplantar administration of racemic-, S(+)- and R(-)-flurbiprofen on the spinal c-Fos protein expression after a single noxious heat stimulation (52 degrees C for 15 s) of the rat hindpaw in urethane anaesthetized rats. Two hours after noxious heat, numerous c-Fos protein immunoreactive (c-Fos-IR) nuclei (>70 c-Fos-IR nuclei per section at the level of L4-L5 segments) were observed with essential localization in the superficial (I-II) laminae of the spinal dorsal horn, i.e. areas containing numerous neurons driven exclusively by noxious stimuli. Considering the number of c-Fos-IR nuclei in laminae I-II, the intravenous injection of racemic-flurbiprofen (0.3, 3 and 9 mg/kg) was inefficacious and S(+)-flurbiprofen had weak and non-dose-related effects. The same doses of R(-)-flurbiprofen produced dose-related effects (r=0.58, P<0.05) with weak, but significant, effects for doses of 3 and 9 mg/kg (18+/-6% and 26+/-5% reduction of the number of noxious heat-evoked c-Fos-IR nuclei in laminae I-II, P<0.05 and P<0.01, respectively). The weak effects of R(-)-flurbiprofen are probably due to the central site of action since the intraplantar injection of a relatively high dose of 30 microg is inefficacious. These results provide further evidence for weak effects of non-steroidal anti-inflammatory drugs and their enantiomers on the acute responses to nociceptive stimulus which are very efficacious upon inflammatory nociception, but not upon brief noxious heat-evoked nociception.

The full structure of the high-pressure cubic phase I' of hydrogen sulfide has been solved using neutron diffraction data. The molecules are partially rotationally disordered about the <111> axes, as in phase II at ambient pressure but with markedly greater nonuniformity of the toroidal D distribution. The changes in structure at the II-->I' transition signal the onset of significant pressure-induced hydrogen bonding.

Single-unit extracellular recordings of spino-parabrachial (spino-PB) neurons (n = 53) antidromically driven from the contralateral parabrachial (PB) area were performed in the lumbar cord in anesthetized rats. All the spino-PB neurons were located in the lamina I of the dorsal horn. Their axons exhibited conduction velocities between 2.8 and 27.8 m/s, in the thin myelinated fibers range. They had an extremely low spontaneous activity (median = 0. 064 Hz) and a small excitatory receptive field (

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.

We have demonstrated that pre-administered RB101 (40 mg/kg, i.v.), a mixed inhibitor of enkephalin-catabolizing enzymes, decreased spinal c-Fos expression induced 1 h and 30 min after intraplantar (i.pl.) carrageenin (41% reduction, p<0.01). These effects were completely blocked by pre-administered beta-funaltrexamine (10 mg/kg, i.v., 24 h prior to stimulation), a selective long-lasting mu-opioid receptor antagonist. In conclusion, these results clearly demonstrate that the effects of endogenous enkephalins on noxiously evoked spinal c-Fos expression are essentially mediated via mu-opioid receptors., (Copyright 1999 Elsevier Science B.V.)

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<0.001). In contrast, nor-binaltorphimine (nor-BNI-1+1 mg/kg), a kappa-opioid receptor antagonist, had no significant effect on the effects of morphine. These results indicate the major contribution of mu-opioid receptors to the antinociceptive effects of systemic morphine at the level of the superficial dorsal horn. The observed effect of NTI is not necessarily related to a direct action of morphine on delta-opioid receptors and some possible actions of this antagonist are discussed., (Copyright 1999 Published by Elsevier Science B.V.)

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; Aldén 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.

Glutamate acts as a neurotransmitter of primary afferent messages in the spinal cord. Through glutamatergic mechanisms nitric oxide (NO) is also a potential intermediary in the transmission of sensory messages, particularly nociceptive, at the spinal level. The aim of the present study was, by using electrochemical monitoring of NO, to determine if the activation of glutamatergic transmission, particularly through NMDA receptors, could increase NO production within the dorsal horn of the lumbar spinal cord in the rat. 30 micrometers diameter treated carbon fiber electrodes coated with nickel-porphyrine and Nafion(R), and associated with differential normal pulse voltammetry, have been used in vivo to monitor NO within the dorsal horn of the lumbar spinal cord of decerebrated-spinalized rats. A NO-dependent peak of oxidation current (650 mV vs. Ag-AgCl), remaining stable for up to 3 h (+/-5%) could be detected under basal conditions, which indicates that significant amounts of NO are produced continuously. The non-competitive N-methyl-d-aspartate (NMDA) channel blockers, Ketamine (100 mg kg-1 i.p.) and MK-801 (10 mg kg-1 i.p.), decreased the voltammograms to 70+/-5% and 69+/-2% of controls at 120 min, respectively. Glutamate (10 mM), when directly superfused upon the spinal cord (20 min at 50 microliters min-1) induced a rapid and significant increase of the 650 mV peak, with a maximum at around 90 min (148+/-6% of control) followed by a slow decay (138+/-4% of control at 150 min). This increase could be totally reversed or blocked by i.p. injection of 100 mg kg-1 of Ketamine. NMDA (30 mg kg-1 i.p.) induced a long-lasting increase in the peaks (149+/-11% at 90 min and 162+/-20% at 120 min), which was also fully reversed by Ketamine or MK-801. These results provide in vivo direct evidence of a glutamate- and/or NMDA-induced release of NO at the spinal level, and is discussed in relation to the glutamatergic transmission of primary afferent messages., (Copyright 1999 Elsevier Science B.V.)

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.

The development of tolerance to the antinociceptive effects of morphine was investigated in rats using carrageenin-induced spinal c-Fos expression. We took advantage of this technique to especially study, at the cellular level, in freely moving animals, the development of tolerance based on the visualization of dorsal horn spinal cord neurons which play a major role in nociceptive processes. Two hours after intraplantar injection of carrageenin (6 mg/150 microliter of saline), c-Fos-like immunoreactivity (FLI) was observed predominantly in the superficial and deep laminae of the dorsal horn in segments L4 and L5 of the spinal cord. In naive rats, acute intravenous morphine (3 mg/kg, i.v.) reduced the number of superficial and deep FLI neurons; 49% and 59% reduction respectively (p<0.0001 for both). In morphine-pretreated rats (daily administration of subcutaneous morphine: 1, 3, 5, 10, 20 or 40 mg/kg once a day for 4 days), antinociceptive tolerance tested on day 5 by acute morphine (3 mg/kg, i.v.) was manifest in those groups pretreated with the highest doses of morphine (10, 20 or 40 mg/kg). From regression analysis, it appeared that tolerance to the antinociceptive effect of morphine developed progressively as a function of the chronic morphine dose used on neurons involved in spinal nociceptive processes (superficial and deep dorsal horn neurons). Similarly, in rats pretreated with 10 mg/kg of morphine over 1, 2, 3 or 4 days, tolerance progressively developed, for both spinal neuronal populations, as a function of the duration of the pretreatment. These results are discussed in the context of the several possible sites of action of morphine., (Copyright 1998 Published by Elsevier Science B.V.)

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<0.05. P<0.001 and P<0.001 respectively). 4. Similar effects to those of intravenous racemic-flurbiprofen were obtained with intravenous S(+)-flurbiprofen (0.3, 3 and 9 mg kg(-1)) which dose-relatedly reduced the number of c-Fos-LI neurons (r=0.69, P<0.01) and diameters of paw and ankle (r=0.56 and r=0.52 respectively, P<0.05 for both). 5. For the dose of 0.3 mg kg(-1) i.v., R(-)-flurbiprofen did not modify the number of c-Fos-LI neurons and produced a weak reduction of oedema at only the ankle level (23+/-12% reduction, P<0.05). However, a ten times higher dose of R(-)-flurbiprofen (3 mg kg(-1) i.v.) was necessary to obtain effects comparable to those of S(+)- or racemic-flurbiprofen (0.3 mg kg(-1) i.v.). 6. Intraplantar racemic-flurbiprofen (1, 10 and 30 microg) dose-relatedly reduced the carrageenan-enhanced ankle diameter (r=0.81, P<0.001) and the number of c-Fos-LI neurons in L4-L5 segments (r=0.83, P<0.001). with a 60+/-3% reduction of the number of c-Fos-LI neurons (P<0.001), and 30+/-3 and 67+/-7% reduction of paw and ankle diameter respectively (P<0.001 for both) for the dose of 30 microg. 7. For intraplantar S(+)-flurbiprofen (1, 10 and 30 microg) the dose-related effects (r=0.77, r=0.60 and r=0.59 for c-Fos-LI neurons, paw and ankle diameters respectively, P<0.001, P<0.01 and P<0.01) were similar to those of racemic-flurbiprofen. In contrast, intraplantar R(-)-flurbiprofen (1, 10 and 30 microg) did not have detectable effects on all studied parameters. 8. The present study provides clear evidence for potent anti-inflammatory and antinociceptive effects of both intravenous or intraplantar racemic- and S(+)-flurbiprofen. These results further demonstrate marked anti-inflammatory and antinociceptive effects of intravenous, but not intraplantar, R(-)-flurbiprofen. These results suggest that the main site of action of racemic- and S(+ )-flurbiprofen is in the periphery and indicate that the site of action of R(-)-flurbiprofen is mainly of central origin.

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.

A new method for density measurements by means of X-ray absorption under high pressure and high temperature using synchrotron radiation has been developed. The method has been modified for a large-volume Paris-Edinburgh press and combined with intense high-energy X-rays at the ESRF. In order to overcome effects of deformation of sample shape under pressure, a ruby cylinder was used as a sample container. The density was determined from the intensity profile of transmitted X-rays. The densities of crystalline and liquid Bi were successfully measured up to 750 K at 1 GPa.

The effects of oral administration of UP 202-56, an adenosine analogue, were assessed on carrageenan-induced spinal c-Fos protein expression and peripheral oedema. Three hours after intraplantar injection of carrageenan (6 mg/150 microl of saline), in awake rats, numerous c-Fos-like immunoreactive (c-Fos-LI) neurons in the dorsal horn of L4-L5 lumbar segments of the spinal cord (191 +/- 8; 184 +/- 10; 205 +/- 7 c-Fos-LI neurons per 40 microm section, for carrageenan controls in three experimental series performed in this study, respectively) and an extensive peripheral oedema were observed. Oral UP 202-56 (10, 30 or 50 mg/kg) dose-dependently reduced the number of carrageenan-induced c-Fos-LI neurons (r = 0.931. P < 0.0001), with the highest dose of UP 202-56 producing 72 +/- 4% reduction of the total number of carrageenan-induced spinal c-Fos-LI neurons, and 12 +/- 3% and 33 +/- 6% of reduction of control carrageenan oedema at paw and ankle levels, respectively. DPCPX (1 mg/kg i.p.), a selective adenosine A1 receptor antagonist, which injected alone had no effect on carrageenan-induced spinal c-Fos expression and peripheral oedema, blocked the effects of UP 202-56 (30 mg/kg p.o.) on the number of carrageenan-induced c-Fos-LI neurons. In addition, DPCPX did not modify the effects of UP 202-56 on carrageenan-induced peripheral oedema. DMPX (1 mg/kg i.p.), a somewhat selective adenosine A2 receptor antagonist, which injected alone had no significant effect on carrageenan-induced spinal c-Fos protein expression and peripheral oedema, did not influence the effects of UP 202-56 (30 mg/kg p.o.) on both carrageenan-induced spinal c-Fos expression and peripheral oedema. Our results demonstrate that UP 202-56 dose-dependently reduced the spinal c-Fos protein expression in carrageenan model of inflammatory pain. The ability of DPCPX to block the effect of UP 202-56, in contrast to the lack of effect of DMPX, increased evidence for a predominant role of adenosine A1 receptors activation in the mechanism of action of UP 202-56. These results increase evidence for a role of adenosine in the modulation of nociceptive transmission and support the antinociceptive action of adenosine analogues, such as UP 202-56, in inflammatory pain processes.

Objective: To evaluate the anti-inflammatory/analgesic effects of lornoxicam in the carrageenan model of inflammatory nociception., Material and Methods: Three hours after intraplantar carrageenan (6 mg/150 microl of saline), we assessed the effects of pre-administered lornoxicam (0.1, 0.3, 1, 3 and 9 mg/kg i.v., n=10 rats for each group) on both the peripheral oedema and number of c-Fos-protein-like immunoreactive (c-Fos-LI) neurons in the lumbar L4-L5 segments, in the awake rat., Results: Lornoxicam dose-relatedly reduced both the carrageenan evoked oedema (r=0.63 and r=0.53 for paw and ankle diameter respectively; p<0.001 for both) and total number of spinal c-Fos-LI neurons (r=0.79; p<0.001), with the strongest effect corresponding to a 75 +/- 2% reduction of the number of c-Fos-LI neurons (p<0.001) for the highest dose (9 mg/kg), and a 45 +/- 3% reduction (p<0.001) for the low dose of 0.3 mg/kg. Reductions of both the peripheral oedema and spinal c-Fos expression were correlated (r=0.74 and r=0.57 for the paw and ankle diameter respectively; p<0.001 for both)., Conclusions: Our results demonstrate that lornoxicam reduces in parallel both the carrageenan-evoked oedema and spinal c-Fos expression, with clear evidence for a potent effect of low doses of lornoxicam. Correlated reductions in c-Fos expression and paw oedema suggest a predominantly peripheral site of action of lornoxicam.

NO synthase (NOS) is largely distributed in the superficial and deep laminae of the dorsal horn as well as in dorsal root ganglion cells. It has been proposed that nitric oxide (NO) participates in the transmission of sustained, and possibly brief, nociceptive, inputs at the spinal level. The aim of this study was to check the ability of in vivo electrochemical monitoring of NO within the dorsal horn of the lumbar spinal cord (L3-L4 level) of chloral hydrate anesthetized or decerebrated spinalized rats. 30 microm diameter and 450 microm length treated carbon fiber electrodes coated with nickel(II) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrine and NafionR, and associated with differential normal pulse voltammetry, gave a peak of oxidation current around 650 mV (vs. Ag-AgCl) in vitro in NO solutions between 0.125 and 1.25 microM. In vivo, a 650 mV peak appeared which was stable (recording interval 2 min) for up to 3 h (+/-6%). Comparison between in vitro calibration and in vivo voltammograms gave an estimated in vivo extracellular concentration of 0.50 microM. In vivo, peaks decreased by 95% at 90 min and for up to 3 h after an i.p. injection of 100 mg/kg of the NOS inhibitor (NOSI) L-arginine-p-nitroanilide (L-ANA). At the same dose i.p., N(G)-nitro-L-arginine methyl ester (L-NAME) was almost ineffective after 90 min in animals paralyzed with pancuronium bromate or gallamine trethiodide. However, in non-curarized decerebrated spinalized animals, L-NAME depressed the voltammograms by 36% at 90 min. S-Ethylthiourea (80 mg/kg i.p.), also decreased the voltammograms by 45% at 140 min, and finally, 7-nitroindazole (7-NI, 90 mg/kg i.p), induced a important decrease of the 650 mV peak (23% of control) at 120 min. These results are in agreement with biochemical data showing the decrease of NOS activity within the lumbar spinal cord by L-NAME (45% of control at 90 min) and 7-NI (20% of control at 90 min). The NO donor hydroxylamine (30 mg/kg i.p.) significantly increased the peaks (140% at 90 min), and sodium nitroprusside (SNP, 20 mM) when directly superfused upon the spinal cord (200-300 microl min(-1)) induced a large increase in the peak (300% at 90 min). Moreover, SNP 60 min after L-ANA, or 90 min after L-NAME, rapidly restored the 650 mV peak up to control values. These results demonstrate the validity of electrochemical monitoring of NO within the dorsal horn of the spinal cord. The in vivo electrochemical detection of NO is in progress to study the implication of this messenger in the transmission of nociceptive messages at the spinal level.

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.