Region-specific changes in 5-HT1A agonist-induced Extracellular signal-Regulated Kinases 1/2 phosphorylation in rat brain: a quantitative ELISA study.

Brain serotonin 5-HT(1A) receptor, a traditional target for the treatment of mood disorders, modulates intracellular signalling pathways, such as the Extracellular signal-Regulated Kinases 1/2 (ERK1/2) pathway. The present studies are the first to determine levels of phospho-ERK1/2 (pERK1/2) in brain using a quantitative Enzyme Linked-Immuno-Sorbent Assay. We examined pERK1/2 levels in rat brain following administration of (+)8-OH-DPAT, buspirone as well as of the more selective, high-efficacy 5-HT(1A) agonists F13640 and F13714. Intraperitoneal injection of these compounds increased pERK1/2 in prefrontal cortex and hypothalamus, with a maximum at 5-15min and a significant effect lasting until 30-60min post-injection. However, these compounds reduced hippocampal pERK1/2 with a maximum effect at 30min, persisting until 60min post-injection. In hippocampus, F13640, F13714 and buspirone inhibited pERK1/2 in a dose-dependent manner as of 0.04, 0.04 and 2.5mg/kg, respectively. Given these low doses, this response is likely related to activation of sensitive presynaptic 5-HT(1A) receptors in the raphe nucleus. 4- and 16-fold higher doses of these compounds were necessary to stimulate pERK1/2 in prefrontal cortex and hypothalamus, respectively, via direct 5-HT(1A) receptor activation. In contrast, (+)8-OH-DPAT was active at similar doses (0.63mg/kg) in these different regions. Pretreatment with the 5-HT(1A) antagonist, WAY100635, completely blocked the effects of these compounds, with the exception of buspirone-induced pERK1/2 increases in hypothalamus. Thus, 5-HT(1A) agonist-induced changes in pERK1/2 in rat brain are time- and dose-dependent and region-specific. Furthermore, F13640, F13714, buspirone, but not (+)8-OH-DPAT, exert their effects via preferential activation of presynaptic 5-HT(1A) receptors.

Effects of the high-efficacy 5-HT1A receptor agonist, F 13640 in the formalin pain model: a c-Fos study.

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 novel analgesic and high-efficacy 5-HT1A receptor agonist, F 13640 induces c-Fos protein expression in spinal cord dorsal horn neurons.

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.

Antinociceptive effects of RB101(S), a complete inhibitor of enkephalin-catabolizing enzymes, are enhanced by (+)-HA966, a functional NMDA receptor antagonist: a c-Fos study in the rat spinal cord.

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.

Further evidence for the interaction of mu- and delta-opioid receptors in the antinociceptive effects of the dual inhibitor of enkephalin catabolism, RB101(S). A spinal c-Fos protein study in the rat under carrageenin inflammation.

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.

RB101(S), a dual inhibitor of enkephalinases does not induce antinociceptive tolerance, or cross-tolerance with morphine: a c-Fos study at the spinal level.

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.

Effects of nefopam on the spinal nociceptive processes: a c-Fos protein study in the rat.

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.

Enhanced effects of co-administered dexamethasone and diclofenac on inflammatory pain processing and associated spinal c-Fos expression in the rat.

This study determines the effects of dexamethasone versus co-administered dexamethasone and diclofenac, on carrageenan-evoked spinal c-Fos expression and peripheral oedema in the freely moving rat. Drugs were administered intravenously 25 min before intraplantar injection of carrageenan (6 mg/150 microliters of saline). Three hours later the number of spinal c-Fos-LI neurones and peripheral oedema were assessed. The total number of control carrageenan-evoked c-Fos-LI neurones in the lumbar spinal cord was 121 +/- 5 labelled neurones per section, segments L4-L5, which were predominantly located in the superficial and deep laminae (41 +/- 3% and 40 +/- 2% of the total number of c-Fos-LI neurones per section, respectively) of the dorsal horn of the spinal cord. Pre-administered dexamethasone (0.05, 0.10 and 0.50 mg/kg i.v.) dose-dependently reduced the total number of c-Fos-LI neurones (30 +/- 4%, 52 +/- 3% and 58 +/- 2% reduction, respectively), with effects of the higher doses being strongest on the deep laminae c-Fos-LI neurones. The effects of dexamethasone on the total number of c-Fos-LI neurones and the peripheral oedema were positively correlated. Co-administration of low doses of dexamethasone and diclofenac (0.025 + 1.5 mg/kg i.v. respectively), which had negligible effects when administered separately, greatly reduced both the total number of carrageenan-evoked c-Fos-LI neurones (61 +/- 5% reduction as compared to control value) and the peripheral oedema (80 +/- 8% and 60 +/- 5% reduction for ankle and paw oedema, respectively). The attenuation by co-administered dexamethasone and diclofenac, of both c-Fos expression and the peripheral oedema, was significantly greater than the effect of dexamethasone alone (P < 0.001 for both) and diclofenac alone (P < 0.001 for both). Our study illustrates enhanced attenuating effects of co-administered dexamethasone and diclofenac on both inflammatory oedema and the associated spinal expression of c-Fos, an indicator of nociceptive transmission at the spinal level. The apparent interactions between the mechanisms of action of NSAIDs and steroids suggest that co-therapy may produce beneficial inflammatory and pain relief in the absence of excessive side effects.

Aspirin and acetaminophen reduced both Fos expression in rat lumbar spinal cord and inflammatory signs produced by carrageenin inflammation.

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.