Signal transduction and functional selectivity of F15599, a preferential post-synaptic 5-HT1A receptor agonist.

BACKGROUND AND PURPOSE: Activation of post-synaptic 5-HT(1A) receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT(1A) receptor agonist. EXPERIMENTAL APPROACH: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. KEY RESULTS: F15599 was highly selective for 5-HT(1A) receptors in binding experiments and in [(35)S]-GTPgammaS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT(1A) receptors. In cell lines expressing h5-HT(1A) receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT(1A) receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [(35)S]-GTPgammaS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated G(alphai) than G(alphao) activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT(1A) receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT(1A) receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT(1A) receptors in vivo almost as potently as F13714. CONCLUSIONS AND IMPLICATIONS: F15599 showed a distinctive activation profiles for 5-HT(1A) receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT(1A) receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition.

Urethane anaesthesia could partly mask antinociceptive effects of non-steroidal anti-inflammatory drugs: a spinal c-Fos protein study.

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.

Effects of flurbiprofen and its enantiomers on the spinal c-Fos protein expression induced by noxious heat stimuli in the anaesthetized rat.

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.

Peripheral and/or central effects of racemic-, S(+)- and R(-)-flurbiprofen on inflammatory nociceptive processes: a c-Fos protein study in the rat spinal cord.

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.

Involvement of the spinoparabrachial pathway in inflammatory nociceptive processes: a c-Fos protein study in the awake rat.

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.

UP 202-56, an adenosine analogue, selectively acts via A1 receptors to significantly decrease noxiously-evoked spinal c-Fos protein expression.

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.

Dose-related anti-inflammatory/analgesic effects of lornoxicam: a spinal c-Fos protein study in the rat.

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.

Antinociceptive effects of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, are enhanced by a cholecystokinin type B receptor antagonist, as revealed by noxiously evoked spinal c-Fos expression in rats.

The effects of RB101, a complete inhibitor of enkephalin-catabolizing enzymes, alone or with a selective cholecystokinin (CCK)B receptor antagonist (CI988) or CCK(A) receptor antagonist (devazepide), on carrageenin-induced spinal c-Fos expression were investigated. Spinal c-Fos expression was observed 90 min after intraplantar carrageenin (6 mg/150 microl saline), with Fos-like-immunoreactive neurons preferentially located in the superficial laminae of the spinal dorsal horn. Intravenous RB101 (10, 20 and 40 mg/kg) dose-dependently reduced the number of superficial Fos-like-immunoreactive neurons (r2 = 0.739, P < .0001), with 63 +/- 2% (P < .0001) reduction for the highest dose. These effects were completely blocked by coadministered naloxone. Coadministration of inactive doses of i.v. RB101 (5 mg/kg) and i.p. CI988 (3 mg/kg) significantly and strongly reduced the number of carrageenin-induced, superficial, Fos-like-immunoreactive neurons (55 +/- 5% reduction of control carrageenin c-Fos expression, P < .0001). This effect was blocked by coadministered naloxone. It is important to note that coadministered RB101 and devazepide did not influence spinal c-Fos expression. None of the various drug combinations influenced the carrageenin-induced peripheral edema. These results show that RB101 dose-dependently decreases carrageenin-evoked spinal c-Fos expression. In addition, the effectiveness of RB101 can be revealed by preadministration of the CCK(B) receptor antagonist CI988. Considering the weak opioid side effects obtained with RB101 treatment and the strong increase of its effects by the CCK(B) receptor antagonist, this type of drug combination could have promising therapeutic application in the management of pain in humans.

The contribution of peripheral bradykinin B2 receptors to carrageenan-evoked oedema and spinal c-Fos expression in rats.

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, P < 0.01) reduced the carrageenan-evoked total number of c-Fos protein-like immunoreactive (c-Fos-LI) neurones (23 +/- 5%, 35 +/- 6% and 50 +/- 5% reduction; P < 0.01, P < 0.001 and P < 0.001, respectively). These reducing effects were dose-dependent for the number of c-Fos-LI neurones in both superficial (r = 0.70, P < 0.01) and deep (r = 0.53, P < 0.05) 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; P < 0.001 for all) and ankle (46 +/- 6%, 61 +/- 5% and 61 +/- 5% reduction; P < 0.001 for all) oedema. Our results provide further evidence for the involvement of peripheral bradykinin B2 receptors in carrageenan-induced inflammatory nociceptive transmission.

Effects of local anaesthetics on carrageenan-evoked inflammatory nociceptive processing in the rat.

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.

Ketoprofen produces profound inhibition of spinal c-Fos protein expression resulting from an inflammatory stimulus but not from noxious heat.

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.

Physiological contributions of neurokinin 1 receptor activation, and interactions with NMDA receptors, to inflammatory-evoked spinal c-Fos expression.

1. Intraplantar injection of formalin (5%, 100 microliters in saline) was associated with a high level of spinal c-Fos immunoreactivity and a peripheral paw and ankle edema, as assessed at 3 h after formalin administration. For the two experimental series, the control number of formalin-evoked Fos-like immunoreactive (Fos-LI) neurons were 174 +/- 6 and 193 +/- 18 (means +/- SE) Fos-LI neurons per 40-microns section of the lumbar segment L4-L5 of the rat spinal cord. For both series of experiments, Fos-LI neurons were located predominantly in the superficial (I-II; 40 and 44% of the total number of Fos-LI neurons for the two experimental series) and deep (V-VI; 37 and 40% of the total number of Fos-LI neurons for the two experimental series) laminae of the dorsal horn of the spinal cord. The small number of remaining Fos-LI neurons were located in the nucleus proprius (laminae III-IV) and the ventral horn. 2. Prior intravenous administration of RP67580 (0.05, 0.5, and 1.5 mg/kg), a selective neurokinin 1 (NK1) receptor antagonist, dose-relatedly reduced the total number of formalin evoked Fos-LI neurons (88 +/- 5%, 80 +/- 4%, P < 0.01 and 64 +/- 4%, P < 0.0001, of the control number of formalin-evoked Fos-LI neurons). Laminar analysis of the regional effect of RP67580 on formalin-evoked Fos-LI neurons illustrated that the number of superficial and deep laminae Fos-LI neurons were attenuated to a similar extent by RP67580. 3. Prior intravenous administration of RP68651 (1.5 mg/kg), the inactive isomer of RP67580, produced only a small reduction in the total number of formalin-evoked Fos-LI neurons (84 +/- 5% of the control number of formalin-evoked Fos-LI neurons (P < 0.05). The effect of RP68651 on the number of formalin-evoked Fos-LI neurons was significantly smaller (P < 0.01) than the effect of the equivalent concentration of RP67580, the active isomer. 4. Prior coadministration of intravenous RP67580 (0.5 mg/kg) and subcutaneous (+)-HA966 (2.5 mg/kg), an antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor, significantly reduced the number of formalin-evoked Fos-LI neurons (64 +/- 4% of the control number of formalin-evoked Fos-LI neurons, P < 0.01). The attenuating effect of coadministered RP67580 and (+)-HA966 was significantly greater than the effect of RP67580 alone (P < 0.01) and the effect of (+)-HA966 alone (P < 0.05). Laminar analysis illustrated that coadministered RP67580 and (+)-HA966 reduced the number of formalin-evoked Fos-LI neurons in the superficial and deep laminae to a similar extent. 5. Intraplantar injection of formalin was associated with a peripheral paw (0.92 +/- 0.02 cm) and ankle (0.92 +/- 0.02 cm) edema, as compared with the paw (0.46 +/- 0.02 cm) and ankle (0.67 +/- 0.14 cm) diameters of saline-stimulated rats. Neither prior administration of intravenous RP67580 (0.05, 0.5, and 1.5 mg/kg) or RP68651 (1.5 mg/kg) or prior coadministration of RP67580) (0.5 mg/kg) and (+)-HA966 (2.5 mg/kg) influenced the extent of the paw or ankle-edema at 3 h after intraplantar injection of formalin. 6. Our results illustrate that NK1-receptor activation contributes to inflammatory-evoked spinal c-Fos expression and thus supports the current contention that NK1-receptor activation, and by inference SP, plays a role in spinal nociceptive processing. The second part of our study suggests that the previously reported NK1/NMDA-receptor interactions contribute to formalin-evoked spinal c-Fos expression and consequently may contribute to the longer term spinal neuroplasticity associated with inflammatory nociceptive processing.

The effects of morphine on carrageenin-induced spinal c-Fos expression are completely blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist.

We have demonstrated that pre-administered morphine (3 mg/kg, i.v.) decreased spinal c-Fos expression induced 2 h after intraplantar carrageenin (55 +/- 5% reduction, P < 0.0001). 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 morphine on noxiously-evoked spinal c-Fos expression are essentially mediated via mu-opioid receptors.

The contribution of GABAB receptor-mediated events to inflammatory pain processing: carrageenan oedema and associated spinal c-Fos expression in the rat.

In this pharmacological study we have assessed the effect of baclofen, a selective GABAB receptor agonist, on spinal expression of the immediate early gene c-Fos and the peripheral oedema evoked by a prolonged peripheral inflammation due to intraplantar carrageenan. Baclofen was administered intravenously 30 min before intraplantar injection of carrageenan in freely moving rats. Three hours after carrageenan the number of spinal c-Fos protein-like immunoreactive neurons and peripheral (ankle and paw) oedema were assessed. For the two series of experiments the total number of control carrageenan-evoked c-Fos protein-like immunoreactive neurons in segments L4-L5 of the spinal cord was 176 +/- 6 and 177 +/- 9 c-Fos protein-like immunoreactive neurons per section, for carrageenan control with intravenous and intraplantar saline, respectively. c-Fos protein-like immunoreactive neurons were predominantly located in laminae I-II and V-VI of the dorsal horn of the spinal cord in carrageenan controls receiving intravenous (68 +/- 3 and 69 +/- 2 c-Fos protein-like immunoreactive neurons, respectively) and intraplantar (62 +/- 4 and 71 +/- 5 c-Fos protein-like immunoreactive neurons, respectively) saline. Pre-administered systemic baclofen (0.05, 1.5 and 3 mg/kg i.v.) dose dependently reduced the total number of c-Fos protein-like immunoreactive neurons (81 +/- 3, 66 +/- 4 and 49 +/- 4% of control total number of c-Fos protein-like immunoreactive neurons, respectively), with strongest effects on the number of deep (74 +/- 3, 60 +/- 3 and 43 +/- 4% of control, respectively) as compared with superficial (90 +/- 4, 77 +/- 5 and 59 +/- 5% of control, respectively) c-Fos protein-like immunoreactive neurons. The effects of systemic baclofen on the carrageenan-induced spinal c-Fos expression and both the paw and ankle oedema were positively correlated (r = 0.479, P < 0.05 and r = 0.733, P < 0.001, respectively). Intraplantar baclofen (50 and 100 micrograms in 50 microliters of saline), simultaneously injected with intraplantar carrageenan, did not significantly influence carrageenan-evoked spinal c-Fos expression or ankle oedema. Despite the fact that the highest dose of intraplantar baclofen significantly reduced paw oedema (23 +/- 3% reduction of control paw oedema), our results are clearly in favour of a spinal site of action of systemic baclofen.

To what extent do spinal interactions between an alpha-2 adrenoceptor agonist and a mu opioid agonist influence noxiously evoked c-Fos expression in the rat? A pharmacological study.

Three hours after the intraplantar injection of carrageenin (6 mg/150 microliters of saline) Fos-like immunoreactivity (Fos-LI) was observed in both superficial and deep laminae of the dorsal horn segments L4 and L5 of the spinal cord. Systemic medetomidine, an alpha-2 adrenoceptor agonist (12.5, 25 or 75 micrograms/kg i.v.), dose-dependently reduced the number of superficial and deep Fos-LI neurons; 75 micrograms/kg produced a 66 +/- 4% and a 90 +/- 4% reduction of superficial and deep Fos-LI neurons, respectively, P < .0001 for both. In addition, systemic medetomidine dose-relatedly reduced the carrageenin-evoked paw and ankle edema; medetomidine 75 micrograms/kg resulted in a 70 +/- 3% reduction of paw edema and in a blockade of the development of ankle edema. The effects of medetomidine were blocked by systemic atipamezole (75 micrograms/kg, i.v.), which, when injected alone, had no effect on the number of Fos-LI neurons or the peripheral edema. Co-administration of a low dose of medetomidine (12.5 micrograms/kg i.v.) with an ineffective dose of morphine (1.5 micrograms/kg i.v.) strongly decreased the number of superficial and deep Fos-LI neurons (40 +/- 5%, P < .0001 and 62 +/- 11%, P < .0001 reduction as compared with control group) without altering the effects of medetomidine on the peripheral edema. Both atipamezole and a combined injection of atipamezole and naloxone blocked the effects of medetomidine plus morphine on both the total number of Fos-LI neurons (86 +/- 11% and 86 +/- 6% of control, respectively) and carrageenin inflammation (87 +/- 6%, P < .05 and 84 +/- 3%, P < .05 of control for the paw edema; 75 +/- 8%, P < .01 and 81 +/- 7%, P < .05 of control for the ankle edema, respectively). Naloxone alone blocked the effects of the co-administered agonists on the total number of Fos-Li neurons (91 +/- 6% of the control carrageenin group) without influencing the effect on the peripheral edema. Our results demonstrate, for the first time, that co-administration of alpha-2 adrenoceptor and mu opioid agonists substantially reduces inflammatory evoked expression of c-Fos, one of the long-term consequences of sustained nociceptive processing.

Concomitant administration of morphine and an N-methyl-D-aspartate receptor antagonist profoundly reduces inflammatory evoked spinal c-Fos expression.

BACKGROUND: After intraplantar injection of carrageenin, peripheral inflammation and spinal c-Fos expression are extensive, with the latter being sensitive to both large doses of morphine or N-methyl-D-aspartate receptor antagonism. The authors investigated the effects of coadministered morphine and (+)-HA966, a functional antagonist at the glycine site of the N-methyl-D-aspartate receptor, on the two parameters. METHODS: The effects of morphine, (+)-HA966 and coadministration of morphine and (+)-HA966 on spinal c-Fos expression in segments L4-L5 of the spinal cord and peripheral edema, induced at 1.5 h and 3 h after intraplantar carrageenin (6 mg/150 microliters) were studied. RESULTS: Previous coadministration of 0.3 mg/kg systemic morphine and 2.5 mg/kg subcutaneous (+)-HA966 significantly reduced c-Fos expression induced 1.5 h, but not 3 h, after carrageenin administration. However, coadministration of a larger dose of morphine (3 mg/kg) with (+)-HA966 (2.5 mg/kg) reduced c-Fos expression at 3 h after carrageenin administration, in a partially naloxone-reversible manner. CONCLUSIONS: Concurrent mu-opioid receptor activation and N-methyl-D-aspartate receptor antagonism reduces nociceptive transmission at the level of the spinal cord, as shown by the reduction of carrageenin-evoked c-Fos expression.

Intraplantar morphine depresses spinal c-Fos expression induced by carrageenin inflammation but not by noxious heat.

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.

Interactions between NMDA- and prostaglandin receptor-mediated events in a model of inflammatory nociception.

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.

Selective cyclooxygenase-2 inhibition reduces carrageenan oedema and associated spinal c-Fos expression in the rat.

Pre-administered NS-398 (0.1, 1 and 10 mg/kg p.o.), a selective cyclooxygenase-2 inhibitor without gastro-intestinal side-effects, dose-dependently reduced carrageenan evoked spinal c-Fos expression (16 +/- 4%, 32 +/- 3% and 56 +/- 5% reduction, respectively) at 3 h after intraplantar carrageenan. The effects of NS-398 on carrageenan induced peripheral oedema and spinal c-Fos expression were correlated, thus demonstrating the beneficial relief of inflammatory pain.

When is the maximal effect of pre-administered systemic morphine on carrageenin evoked spinal c-Fos expression in the rat?

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.