Effects of coadministration of cannabinoids and morphine on nociceptive behaviour, brain monoamines and HPA axis activity in a rat model of persistent pain

European Journal of Neuroscience, Vol. 19, pp. 678±686, 2004 ß Federation of European Neuroscience Societies Effects of coadministration of cannabinoids and morphine on nociceptive behaviour, brain monoamines and HPA axis activity in a rat model of persistent pain D. P. Finn, S. R. G. Beckett, C. H. Roe, A. Madjd, K. C. F. Fone, D. A. Kendall, C. A. Marsden and V. Chapman Institute of Neuroscience, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK Keywords: cannabidiol, corticosterone, D9-tetrahydrocannabinol, formalin, morphine Abstract The antinociceptive effects of D9-tetrahydrocannabinol (D9-THC) have been widely described; however, its therapeutic potential may be limited by secondary effects. We investigated whether coadministration of low doses of cannabinoids or cannabinoids and morphine produced antinociception in the absence of side-effects. Effects of preadministration (i.p.) of D9-THC (1 or 2.5 mg/kg), cannabidiol (5 mg/kg), morphine (2 mg/kg), D9-THC ‡ morphine, D9-THC ‡ cannabidiol or vehicle on formalin-evoked nociceptive behaviour were studied over 60 min. Trunk blood and brains were collected 60 min after formalin injection and assayed for corticosterone and tissue levels of monoamines and metabolites, respectively. Drug effects on locomotor activity, core body temperature and grooming were assessed. D9-THC reduced both phases of formalin-evoked nociceptive behaviour, enhanced the formalin-evoked corticosterone response and increased the 4-hydroxy-3-methoxyphenylglycol : noradrenaline ratio in the hypothalamus. Cannabidiol alone had no effect on these indices and did not modulate the effects of D9-THC. Morphine reduced both phases of formalin-evoked nociceptive behaviour. Coadministration of D9-THC and morphine reduced the second phase of formalin-evoked nociceptive behaviour to a greater extent than either drug alone, and increased levels of thalamic 5-hydroxytryptamine. While the antinociceptive effects of D9-THC and morphine alone occurred at doses devoid of effects on locomotor activity, coadministration of D9-THC and morphine inhibited locomotor activity. In conclusion, coadministration of a low dose of morphine, but not cannabidiol, with D9-THC, increased antinociception and 5hydroxytryptamine levels in the thalamus in a model of persistent nociception. Nevertheless, these enhanced antinociceptive effects were associated with increased secondary effects on locomotor activity. Introduction Cannabinoid receptor agonists such as D9-tetrahydrocannabinol (D9THC) produce a tetrad of behaviour consisting of antinociception, hypolocomotor activity, hypothermia and catalepsy (Martin et al., 1991). Antinociceptive effects of D9-THC in models of acute pain (for review see Pertwee, 2001) and tonic persistent pain (Moss & Johnson, 1980) have been described. Cannabis extracts rich in D9-THC and another natural cannabinoid, cannabidiol (CBD), are effective analgesics in chronic pain patients (Wade et al., 2003). CBD also blocks anxiety, and other subjective alterations, induced by D9-THC in humans (Zuardi et al., 1982). Thus, CBD, or cannabis extracts rich in CBD, may have a reduced side-effect pro®le. The data from animal studies are, however, equivocal, with reports of CBD potentiating (Karniol & Carlini, 1973) or antagonizing (Welburn et al., 1976) the antinociceptive effects of D9-THC. In addition, systemic administration of CBD is not antinociceptive in models of acute pain (Karniol & Carlini, 1973; So®a et al., 1975; Welburn et al., 1976; Sanders et al., 1979). To date, animal studies of CBD have used models of acute pain, which may explain discrepancies between clinical and animal-based research. Correspondence: Dr David P. Finn, as above. E-mail: David.Finn@Nottingham.ac.uk Received 21 August 2003, revised 6 November 2003, accepted 4 December 2003 doi:10.1111/j.1460-9568.2004.03177.x Opioid receptor agonists are extremely effective analgesics, but are associated with undesirable side-effects (Nicholson, 2003). Coadministration of opioid and cannabinoid receptor agonists produces enhanced antinociceptive effects, compared with either drug alone, in models of acute pain (Welch & Eads, 1999; Cichewicz & McCarthy, 2003). Synergism occurs at subeffective or submaximal doses of cannabinoid or opioid agonists and these effects are blocked by cannabinoid receptor 1 (CB1) and opioid receptor antagonists (Reche et al., 1996; Smith et al., 1998). However, it is unclear whether enhanced antinociceptive effects of opioids and cannabinoids occur in models of persistent pain, and whether secondary effects are also enhanced. The formalin model of tonic, persistent pain (Dubuisson & Dennis, 1977; Abbott et al., 1995) is characterized by biphasic paw-directed nociceptive behaviour, hypothalamo-pituitary-adrenal (HPA) axis activation and alterations in brain monoamines (Pacak et al., 1995; Taylor et al., 1998; Palkovits et al., 1999). Intraplantar injection of formalin activates supra-spinal nociceptive pathways and increases immediate± early gene expression in the periaqueductal grey (PAG), thalamus and hypothalamus (Finn et al., 2003a; Pertovaara et al., 1993; Liu et al., 1998; Baulmann et al., 2000; Keay et al., 2002). Antinociceptive effects of systemically administered D9-THC and morphine are mediated by CB1 receptors and m-opioid receptors located in these brain regions (Lichtman & Martin, 1997; Manning & Franklin, 1998). Furthermore, CB1 receptors and m-opioid receptors modulate Antinociceptive effects of cannabinoids and morphine monoamine release and turnover at these supra-spinal sites (Sawynok & Reid, 1987; Murphy et al., 1990; Heijna et al., 1991; Tzavara et al., 2001). The neuroendocrine and central monoaminergic correlates of the enhanced antinociceptive effects of coadministered cannabinoids and opioids are poorly understood. The aim of the present studies was to investigate the effects of coadministration of D9-THC with CBD or morphine on behavioural, HPA axis and brain monoaminergic responses in the formalin model of persistent pain. Materials and methods Animals Adult male Lister-Hooded rats (250±300 g, Charles River, UK) were used in these experiments. Rats were group housed in cages of four for a minimum of 5 days prior to experimentation. Rats were kept on a 12-h light : 12-h dark cycle (lights on at 07.00 h) and food and water were available ad libitum. All experiments were carried out in the light period according to the UK Home Of®ce Animals (Scienti®c Procedures) Act 1986 under Project Licence number 40/1955 and under the ethical guidelines of the International Association for the Study of Pain. Effects of coadministration of CBD or morphine with D9-THC on formalin-evoked nociceptive behaviour Rats received either D9-THC (1 or 2.5 mg/kg), CBD (5 mg/kg), D9-THC (1 or 2.5 mg/kg) ‡ CBD (5 mg/kg), morphine (2 mg/kg), D9-THC (1 mg/kg) ‡ morphine, or vehicle (ethanol±cremophor±saline; 1 : 1 : 18, 1 mL/kg). Rats were injected i.p. 30 min prior to intraplantar injection of formalin (50 mL, 2.5%) into the right hindpaw. A control group received vehicle (i.p.) and intraplantar injection of saline (0.9% NaCl). A preliminary experiment using identical methodology also investigated the effects of a higher dose (50 mg/kg, i.p.) of CBD on formalin-evoked nociceptive behaviour. Drug doses and time of administration were chosen on the basis of previous studies investigating these compounds alone, or in combination, in animal models of pain (Karniol & Carlini, 1973; Welburn et al., 1976; Abbott et al., 1995; Reche et al., 1996). Doses were chosen to ensure submaximal antinociceptive effects of D9-THC and morphine administered alone so that interactions between the effects of the two drugs on formalinevoked behaviour could be detected. The dose of CBD was chosen on the basis of previous studies in the literature demonstrating its anxiolytic (Guimaraes et al., 1990), antiarthritic (Malfait et al., 2000), anticonvulsant (Wallace et al., 2001) and antiemetic (Parker et al., 2002) effects. The protocol used for induction and assessment of formalin-evoked nociceptive behaviour was similar to that previously employed in our laboratory (Finn et al., 2003a, 2003b). Brie¯y, intraplantar injections were carried out under brief iso¯urane anaesthesia to minimize stress to the animals. Behaviour was computertracked for 10 min postdrug and 60 min postformalin in a transparent perspex box (30  30  30 cm) and scored using Ethovision software (Noldus, Netherlands) by a trained experimenter, blind to the treatments. The maximum time taken to recover from anaesthesia, as judged by regain of the righting re¯ex, was 3 min, and this initial period of the formalin trial was excluded from the analysis. The video image was obtained from a camera positioned under the perspex box enabling clear examination of paw-directed behaviour throughout the experiment. Nociceptive behaviour was scored according to the weighted composite pain scoring technique (CPS-WST0,1,2) described by Watson et al. (1997). According to this method, pain behaviours were categorized as time spent raising the paw above the ¯oor without contact with any other surface (C1) and holding, licking, biting, shaking and ¯inching (C2) to obtain a composite pain score 679 CPS ˆ [C1 ‡ (2  C2)]/[total duration of analysis period]. The number of rears and the duration of grooming during the 10-min preformalin trial were scored. Hindpaw oedema was assessed by measuring the paw diameter pre- and postformalin injection with Vernier callipers. The sequence of testing was randomized throughout the experiment so as to minimize any confounding effects of the order of testing. Rats were killed by stunning and decapitation 60 min after formalin injection. Brains were quickly removed, frozen and stored at 80 8C prior to assay of monoamines in discrete brain regions by high performance liquid chromatography with electrochemical detection (HPLC-ED). Trunk blood was collected into ethylenediaminetetraacetic acid-coated tubes on ice and centrifuged at 1600 g for 10 min to extract plasma which was removed and stored at 80 8C prior to radioimmunoassay for corticosterone. Assessment of drug effects on locomotor activity and body temperature Separate groups of male Lister-Hooded rats were injected with CBD (5 or 50 mg/kg, i.p.), D9-THC (1 or 2.5 mg/kg) or vehicle 30 min prior to placement in computer-controlled infra-red activity monitor chambers (Medical Physics Department, University of Nottingham; Clemett et al., 1998). Each chamber consisted of a clear acrylic box (40  20  25 cm) with a wire mesh lid. Five parallel infra-red beams, 7.5 cm apart, crossed the chamber at three different heights but only activity from the middle row was used in the current study. This layer of beams (3.5 cm above ¯oor level) recorded locomotion. To prevent false recording of locomotion by small movements such as grooming, a count was only registered when two adjacent beams in the middle layer were broken in pairs and in a consecutive sequence. Ambulatory activity was recorded for 60 min using `Activity Monitor' software (Medical Physics Department, University of Nottingham) on an Apple Macintosh IIcx computer. Immediately following removal of rats from the locomotor activity boxes, rectal temperature was measured using a thermocouple probe with a digital readout (Portec Instrumentation Ltd, P9005) inserted 6±8 cm into the rectum using light restraint. The effect of morphine administered alone, or in combination with D9-THC, on locomotor activity (distance moved), was assessed during the 10-min preformalin trial (i.e. 20±30 min after drug injection) using Ethovision tracking software. The number of rears and the duration of grooming during the 10-min preformalin trial were scored by an experimenter blind to the treatment groups. Assay of brain monoamines by high performance liquid chromatography Brains were allowed to thaw and the hypothalamus, thalamus and PAG were dissected out rapidly on an ice-cold plate. The brain regions were weighed prior to sonication in 1 mL 0.1 M perchloric acid and homogenates were centrifuged at 18 000 g at 4 8C for 10 min. Homogenization in perchloric acid served to remove sulphate conjugated to the noradrenaline metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG). A 20-mL sample of supernatant was injected via a Rheodyne sample injector onto a reverse-phase analytical column (Nucleosil C18, length 15 cm and internal diameter 4.6 mm; Phenomenex, UK). Detection was via an electrochemical detector (Antec CU-04 with a VT-03 ¯ow cell) in which the electrode was maintained at a potential of ‡0.75 V. The mobile phase consisted of 0.05 M potassium dihydrogen orthophosphate, 1.6 mM 1-octanesulphonic acid and 1 mM ethylenediaminetetraacetic acid in 1 L deionized H2O to which methanol (14% v/v) was added. The pH of the mobile phase was adjusted to 4.0 and it was pumped through the HPLC system at a ¯ow rate of 0.6 mL/min. Standard curves for noradrenaline, MHPG, 5-hydroxyindoleacetic acid ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 680 D. P. Finn et al. (5-HIAA), 5-hydroxytryptamine (5-HT), dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were obtained each day prior to injection of samples. Corticosterone radioimmunoassay Plasma corticosterone levels were measured in nine experimental groups: vehicle±saline, vehicle±formalin, D9-THC (1 mg/kg)±formalin, D9-THC (2.5 mg/kg)±formalin, CBD (5 mg/kg)±formalin, D9-THC (1 mg/kg) ‡ CBD (5 mg/kg)±formalin, D9-THC (2.5 mg/kg) ‡ CBD (5 mg/kg)±formalin, morphine (2 mg/kg)±formalin, D9-THC (1 mg/ kg) ‡ morphine (2 mg/kg)±formalin. Total corticosterone was measured directly in plasma (diluted 1 : 10 in 0.9% saline) in duplicate using a commercially available corticosterone radioimmunoassay kit (Immunodiagnostic Systems Ltd, UK). The tracer was [125I]corticosterone with a speci®c activity of 6.7 kBq/mL. The limit of sensitivity of the assay was 0.39 ng/mL. Drug preparation D9-THC was purchased as 28 mg/mL stock solution in ethanol (Sigma, UK). CBD was obtained as a kind gift from Professor Raphael Mechoulam (Hebrew University of Jerusalem, Israel). Both drugs were ®rst diluted or dissolved in 100% ethanol. Surfactant (Cremophor, Sigma) and 0.9% saline were then added to produce ®nal concentrations of 1 or 2.5 mg/mL of D9-THC and 5 or 20 mg/mL CBD in ethanol±cremophor±saline (1 : 1 : 18) vehicle. An emulsion mixture of D9-THC and CBD was obtained by dissolving CBD in ethanolic D9-THC solution followed by addition of cremophor and saline in the ratio of 1 : 1 : 18 (ethanol±cremophor±saline). Morphine sulphate (Queen's Medical Centre Pharmacy, Nottingham, UK) was dissolved either alone or with D9-THC in ethanol±cremophor±saline (1 : 1 : 18) vehicle to produce a ®nal concentration of 2 mg/mL. All the drug solutions were freshly prepared on experimental days and stored on ice and in the dark during the experiments. Drugs or vehicle were administered i.p. in a volume of 1 or 2.5 mL/kg (for 50 mg/kg CBD). Data analysis All data are presented as means  SEM. The CPS data were analysed in 5-min time bins by two-way ANOVA, with treatment and time as the two factors, followed by one-way ANOVA on the ®rst phase (3±8 min; T1), interphase (8±18 min; T2±T3) and second phase (18±60 min; T4± T11) of formalin-evoked nociceptive behaviour. Fisher's Protected Least Signi®cant Difference (PLSD) post hoc test was used when appropriate. Plasma corticosterone and brain monoamine data from formalin-injected animals were analysed by one-way ANOVA followed by Fisher's PLSD test for post hoc comparison of pairs of groups. Comparisons between saline- and formalin-treated controls were made using Student's unpaired two-tailed t-test. The level of signi®cance was set at P < 0.05. Fig. 1. Effect of D9-THC and CBD on formalin-evoked nociceptive behaviour in rats. Data are means  SEM (n ˆ 5±11). P < 0.05 for D9-THC (1 or 2.5 mg/ kg) or D9-THC (1 or 2.5 mg/kg) ‡ CBD (5 mg/kg) vs. vehicle at T1. yyP < 0.01 for D9-THC (1 mg/kg) or D9-THC (1 or 2.5 mg/kg) ‡ CBD (5 mg/kg) vs. vehicle from T2±T3; ‡P < 0.05 for D9-THC (1 or 2.5 mg/kg) and D9-THC (1 or 2.5 mg/ kg) ‡ CBD (5 mg/kg) vs. vehicle from T4±T11 (ANOVA, F5,50 ˆ 22.85, P ˆ 0.0001 and Fisher's PLSD post hoc test). Results Formalin-evoked nociceptive behaviour Intraplantar injection of formalin produced robust licking, ¯inching, shaking and elevation of the injected paw. The early phase (3±8 min) and late phase (18±60 min) marked two distinct periods of nociceptive behaviour (measured as CPS) following injection of formalin in vehicle-treated control rats (Fig. 1). Saline-injected animals did not exhibit nociceptive behaviour (data not shown). Intraplantar injection of formalin evoked a signi®cant oedema of the hindpaw (change in paw diameter 1.6  0.07 mm) compared to rats receiving intraplantar injection of saline (change in paw diameter 0.4  0.0 mm). Intraplantar injection of formalin had no signi®cant effect on rearing or grooming (Table 1). Effects of cannabinoids on formalin-evoked nociceptive behaviour Systemic administration of D9-THC (1 or 2.5 mg/kg) alone, and in combination with CBD (5 mg/kg), signi®cantly reduced the ®rst and second phases of formalin-evoked nociceptive behaviour, and reduced nociceptive behaviour during the interphase period compared with the vehicle-treated formalin group (P < 0.05 for all, Fig. 1). There was no signi®cant difference between the effects of D9-THC alone and D9THC in combination with CBD on formalin-evoked nociceptive behaviour. CBD (5 mg/kg) administered alone had no signi®cant effect on the formalin-evoked nociceptive behaviour (Fig. 1). A higher dose Table 1. Effects of D9-THC, D9-THC ‡ CBD or CBD on non-nociceptive behaviour in rats measured either before saline or formalin (rearing, grooming) or over 60 min in a separate group of rats not treated with saline or formalin (ambulatory activity) Vehicle ‡ saline Vehicle ‡ formalin D9-THC (1) ‡ formalin D9-THC (2.5) ‡ formalin D9-THC (1) ‡ CBD (5) ‡ formalin D9-THC (2.5) ‡ CBD (5) ‡ formalin CBD (5) ‡ formalin Rears (n) Grooming (s) Ambulations (n) 33.8  4.0 39.5  3.0 25.7  5.9 31.4  10.0 15.7  2.7 20.7  3.9 39.8  3.2 30.6  14.0 51.6  11.6 12.4  3.1 25.4  5.6 21.2  4.2 27.0  9.5 75.2  17.7 NM 345.3  38.1 264.5  61.1 150.8  23.9 NM NM 358.4  38.6 Data are means  SEM, n ˆ 4±11. (1), 1 mg/kg, i.p.; (2.5), 2.5 mg/kg, i.p.; (5), 5 mg/kg, i.p. P < 0.05 vs. vehicle± formalin (t-test or ANOVA ‡ Fisher's PLSD post hoc test). CBD, cannabidiol; D9-THC, D9-tetrahydrocannabinol; NM, not measured. ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 Antinociceptive effects of cannabinoids and morphine of CBD (50 mg/kg, i.p.) also failed to modulate formalin-evoked nociceptive behaviour (data not shown). 9 Table 2. Effects of D -THC, D -THC ‡ Morph or Morph on non-nociceptive behaviour in rats measured before saline or formalin (rearing, grooming, distance moved) Effects of D9-THC and morphine on formalin-evoked nociceptive behaviour Systemic administration of morphine (2 mg/kg) alone signi®cantly reduced the ®rst phase, interphase and early second phase of nociceptive behaviour (P < 0.05, Fig. 2). Systemic administration of D9THC (1 mg/kg) alone, and in combination with morphine (2 mg/kg), signi®cantly reduced the ®rst phase, interphase and second phase of formalin-evoked nociceptive behaviour, compared with the vehicletreated formalin group (P < 0.05 for all, Fig. 2). Effects of coadministered D9-THC and morphine on the ®rst phase of formalin-evoked nociceptive behaviour were not signi®cantly different from the effects of either drug alone. However, coadministered D9-THC and morphine signi®cantly reduced the second phase of formalin-evoked nociceptive behaviour to a greater extent than morphine alone, but not D9-THC alone (Fig. 2). Effects of drug treatments on other physiological parameters Formalin-evoked oedema of the hindpaw was not altered by any of the drug treatments studied (data not shown). Rearing and grooming were not signi®cantly altered by CBD (5 mg/kg) alone, D9-THC (2.5 mg/kg) or morphine (2 mg/kg) alone. Systemic administration of D9-THC alone (1 mg/kg), or in combination with CBD (5 mg/kg), signi®cantly reduced rearing and grooming during the 10-min preformalin trial, compared with vehicle-treated formalin rats (Table 1, P ˆ 0.05). In addition, 2.5 mg/kg of D9-THC, but not 1 mg/kg of D9-THC or 5 mg/kg of CBD, reduced locomotor activity at later time points, 30±90 min postinjection (Table 1), and also reduced body temperature (data not shown; F3,15 ˆ 3.1, P ˆ 0.049). A higher dose of CBD (50 mg/kg) did not alter hindpaw oedema, rearing, grooming, locomotor activity or body temperature compared with vehicle-treated formalin controls (data not shown). Systemic administration of D9-THC alone (1 mg/kg), or in combination with morphine (2 mg/kg), signi®cantly reduced distance moved, rearing and grooming during the 10-min preformalin trial compared with vehicle-treated formalin rats (Table 2) and formalin-treated rats receiving morphine alone. Fig. 2. Effect of D9-THC (1 mg/kg i.p.) and morphine (2 mg/kg i.p.) on formalin-evoked nociceptive behaviour in male Lister-Hooded rats. Data are means  SEM (n ˆ 6±7). P < 0.05 for D9-THC (1 mg/kg), morphine (2 mg/ kg) and D9-THC (1 mg/kg) ‡ morphine (2 mg/kg) vs. vehicle at T1; yP < 0.05 for D9-THC (1 mg/kg), morphine (2 mg/kg) and D9-THC (1 mg/kg) ‡ morphine (2 mg/kg) vs. vehicle from T2±T3; ‡P < 0.05 for D9-THC (1 mg/kg) and D9THC (1 mg/kg) ‡ morphine (2 mg/kg) vs. vehicle from T4±T11; zP < 0.05 for morphine (2 mg/kg) vs. vehicle from T4±T6; $P < 0.05 for D9-THC (1 mg/kg) ‡ morphine (2 mg/kg) vs. morphine (2 mg/kg) from T7±T9 (ANOVA, F3,30 ˆ 24.3; P ˆ 0.0001 and Fisher's PLSD post hoc test). 681 9 Vehicle ‡ saline Vehicle ‡ formalin D9-THC (1) ‡ formalin Morph (2) ‡ formalin D9-THC (1) ‡ morphine (2) ‡ formalin Rears (n) Grooming (s) Distance moved (cm) 33.8  4.0 39.5  3.0 25.7  5.9 39.0  5.8y 13.7  5.3 30.6  14.0 51.6  11.6 12.4  3.1 25.9  8.8 6.7  4.8 NM 1577  108 1595  142y 1805  242y 962  68 Data are means  SEM, n ˆ 4±11. (1), 1 mg/kg, i.p.; (2), 2 mg/kg, i.p.  P < 0.05 vs. vehicle ‡ formalin; yP < 0.05 vs. D9-THC (1) ‡ morphine (2) ‡ formalin (t-test or ANOVA ‡ Fisher's PLSD post hoc test). NM, not measured. Effects of cannabinoids and opioids on formalin-evoked increases in plasma corticosterone levels in rats Intraplantar injection of formalin signi®cantly increased plasma corticosterone levels (60 min postinjection), compared with salineinjected controls (Fig. 3a). Systemic administration of D9-THC (2.5 mg/kg) produced a signi®cant enhancement of formalin-evoked Fig. 3. (a) Effect of intraplantar injection of formalin and D9-THC (1 or 2.5 mg/kg, i.p.) and CBD (5 mg/kg), alone or in combination, on plasma corticosterone levels in rats. Data are means  SEM (n ˆ 5±11). P ˆ 0.05 vs. Vehicle ‡ saline (Student's unpaired t-test). ‡‡P < 0.01, ‡P < 0.05 vs. Vehicle ‡ Form (ANOVA [F5,39 ˆ 4.8; P ˆ 0.002] and Fisher's PLSD post hoc test). (b) Effect of D9-THC (1 mg/kg, i.p.) and morphine (2 mg/kg, i.p.), alone or in combination, on the formalin-evoked increase in plasma corticosterone levels in rats. Data are means  SEM (n ˆ 5±6). Form, formalin; Sal, saline. ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 682 D. P. Finn et al. Table 3. Effect of intraplantar formalin injection and systemic administration of D9-THC (1 mg/kg) alone, or coadministered with CBD (5 mg/kg) or morphine (2 mg/kg), on tissue levels of monoamines and their metabolites in the rat PAG, thalamus and hypothalamus Vehicle ‡ saline PAG Dopamine DOPAC DOPAC/Dopamine Thalamus 5-HT 5-HIAA 5-HIAA/5-HT Hypothalamus Noradrenaline MHPG MHPG/Noradrenaline Vehicle ‡ formalin D9-THC ‡ formalin D9-THC ‡ CBD ‡ formalin D9-THC ‡ morphine ‡ formalin Morphine ‡ formalin 66.2  8.9 42.8  11.9 1.11  0.49 36.8  2.4 21  4.5 0.58  0.13 39.2  2.5 29.9  15.4 0.8  0.44 39.8  2.5 21.3  7.0 0.61  0.26 38.5  4.5 23.1  6.7 0.73  0.24 38.9  2.6 19.2  2.2 0.5  0.08 37.8  2.9 75.7  12.7 2  0.3 49.9  7.3y 76  6.8 1.6  0.2y 50.2  7.1y 69.6  10.0 1.4  0.1y 44.9  6.2 71.6  7.7 1.7  0.1 153.9  23.2 76.3  11.1 0.6  0.2 57.8  4.9y 77.6  6.9 1.3  0.1y 650.6  41.4 32.6  4.3 0.05  0.01 562.6  22.9 25.3  3.0 0.05  0 520.2  9.5 75.3  8z 0.15  0.01z 571.4  27.5 103.2  22.7z 0.18  0.04z 512  50.3 85.1  6.3z 0.18  0.03z 507.8  44.2 29.2  2.4y 0.06  0.01y All concentrations are expressed as nmol/mg wet weight of tissue. Values are mean  SEM, n ˆ 4±7. P < 0.01 vs. vehicle ‡ saline; yP < 0.05 vs. D9-THC ‡ morphine ‡ formalin; zP < 0.05 vs. vehicle ‡ formalin. increases in plasma corticosterone levels (Fig. 3a). Neither CBD (5 mg/kg) alone nor the low dose of D9-THC (1 mg/kg) altered the formalin-evoked increases in plasma corticosterone levels (Fig. 3a). Coadministration of CBD with both the low and high dose of D9-THC enhanced formalin-evoked increases in corticosterone levels (Fig. 3a). Neither morphine (2 mg/kg) alone nor morphine (2 mg/kg) in combination with D9-THC (1 mg/kg) altered formalin-evoked increases in plasma corticosterone levels (Fig. 3b). Changes in brain tissue levels of monoamines associated with formalin-induced nociception and drug treatments Following intraplantar injection of formalin, a signi®cant decrease in tissue levels of dopamine in the PAG, but not the thalamus or hypothalamus, was observed, compared with vehicle-treated rats receiving intraplantar saline (Table 3). In contrast, levels of 5-HT, 5-HIAA, noradrenaline, MHPG and DOPAC were not altered in the PAG, thalamus or hypothalamus following intraplantar injection of formalin. D9-THC alone, or in combination with CBD, signi®cantly increased tissue levels of MHPG and the MHPG/noradrenaline ratio (Table 3) in the hypothalamus of formalin-treated rats, compared with the vehicletreated formalin group (Table 3). There was no signi®cant difference between the effects of D9-THC alone and D9-THC ‡ CBD on hypothalamic MHPG levels or the MHPG/noradrenaline ratio. Neither D9THC nor D9-THC ‡ CBD altered tissue levels of noradrenaline, MHPG or the MHPG/noradrenaline ratio in the PAG and thalamus of formalin-treated rats (data not shown). Administration of D9-THC alone, or in combination with CBD, had no signi®cant effect on tissue levels of dopamine, DOPAC or the DOPAC/dopamine ratio in the PAG (Table 3), thalamus and hypothalamus (data not shown) of formalin-treated rats. Similarly, there was no effect of D9-THC, or D9-THC and CBD, on 5-HT, 5-HIAA or the 5HIAA/5-HT ratio in the thalamus (Table 3), PAG or hypothalamus (data not shown) of formalin-treated rats. In formalin-treated rats, administration of neither D9-THC nor morphine alone altered thalamic 5-HT levels or the 5-HIAA/5-HT ratio, compared with vehicle-treated formalin controls. However, coadministration of D9-THC and morphine signi®cantly increased tissue levels of 5-HT and reduced the 5-HIAA/5-HT ratio in the thalamus of formalin-injected rats, compared with the vehicle-treated formalin group and formalin-treated rats receiving either drug alone (Table 3). There was no effect of D9-THC or morphine, alone or in combination, on tissue levels of 5-HT or 5-HIAA or on the 5-HIAA/5HT ratio in the PAG or hypothalamus of formalin-treated rats (Table 3). In formalin-treated rats, morphine alone did not alter hypothalamic MHPG levels or the MHPG/noradrenaline ratio, compared with vehicle-treated formalin controls. However, administration of D9-THC alone, or with morphine, signi®cantly elevated tissue levels of MHPG and the MHPG/noradrenaline ratio in the hypothalamus of formalin-injected rats, compared with formalin-treated rats receiving vehicle or morphine alone (P < 0.05 for both, Table 3). In contrast, systemic administration of D9THC and morphine, alone or in combination, had no signi®cant effect on tissue levels of noradrenaline or MHPG or on the MHPG/noradrenaline ratio in the PAG and thalamus (data not shown). Finally, systemic administration of D9-THC and morphine, alone or in combination, did not alter tissue levels of dopamine, DOPAC or the DOPAC/dopamine ratio in the PAG (Table 3), thalamus or hypothalamus (data not shown). Discussion Intraplantar injection of formalin produced a characteristic biphasic pro®le of nociceptive behaviour, which was accompanied by an increase in plasma corticosterone levels and a reduction in levels of dopamine in the PAG. Systemic coadministration of low doses of D9THC and morphine reduced the second phase of formalin-evoked nociceptive behaviour more effectively than administration of either drug alone. The enhanced antinociceptive effects of coadministered D9-THC and morphine were accompanied by increased levels of 5-HT in the thalamus. In contrast, CBD alone did not display antinociceptive activity in the formalin test and did not alter D9-THC-induced antinociception in the formalin test. Our data demonstrating that systemic administration of D9-THC reduces both phases of formalin-evoked nociceptive behaviour supports a previous study demonstrating antinociceptive effects of orally administered D9-THC in this test (Moss & Johnson, 1980). In the present study, D9-THC-induced antinociception occurred at a dose (1 mg/kg) devoid of effects on ambulatory activity, body temperature and formalin-evoked HPA axis activity. These results are in agreement with reports in the literature for the effects of this dose of D9-THC on horizontal locomotor activity (SanÄudo-PenÄa et al., 2000; Jarbe et al., 2002). While the higher dose of D9-THC (2.5 mg/kg) also reduced formalin-evoked nociceptive behaviour, decreased ambulatory activity ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 Antinociceptive effects of cannabinoids and morphine and body temperature and enhancement of formalin-evoked HPA axis activity were also observed at this dose. The inhibitory effects of D9THC on vertical locomotor activity (rearing) and grooming in the present study were not dose-related. It is of interest to note that Jarbe et al. (2002) have demonstrated that the inhibitory effects of D9-THC on rearing in an open-®eld arena were only partially antagonized by the CB1 receptor antagonist SR141716A. Systemic administration of morphine also signi®cantly inhibited the ®rst phase and early second phase of formalin-evoked nociceptive behaviour at a dose (2 mg/kg) devoid of effects on vertical or horizontal locomotor activity. Previous studies have demonstrated that the antinociceptive effects of morphine in the formalin test are dependent on the concentration of formalin and the timing of drug administration (Abbott et al., 1995; Sevostianova et al., 2003). Our ®ndings are consistent with previous studies using comparable dosing regimens of systemically administered morphine (Oluyomi et al., 1992; Sevostianova et al., 2003). We have demonstrated that coadministration of submaximal doses of D9-THC and morphine reduced formalin-evoked nociceptive behaviour to a signi®cantly greater extent than either drug alone. Our data, generated using the formalin model of tonic pain, corroborate earlier studies which have demonstrated synergy between subeffective doses of D9-THC and morphine following systemic (Reche et al., 1996; Smith et al., 1998; Cichewicz et al., 1999; Cichewicz & McCarthy, 2003), intrathecal (Welch & Stevens, 1992; Pugh et al., 1996) and intracerebroventricular (Welch et al., 1995) routes of administration in models of acute pain. Although not replicated in the present report, previous studies have con®rmed the involvement of m-opioid receptors and CB1 receptors in the synergistic interactions between D9-THC and morphine (Reche et al., 1996; Smith et al., 1998; Cichewicz et al., 1999). Opioid±cannabinoid interactions are not exclusive to D9-THC and morphine and have also been demonstrated in studies employing the synthetic cannabinoid agonist CP55,940 (Smith et al., 1998) and mopioid receptor agonist DAMGO (Reche et al., 1996). It is important to note, however, that although we report enhanced antinociceptive effects of coadministered submaximal doses of D9-THC and morphine during the second phase of the formalin test, these effects were associated with enhanced suppression of locomotor activity, compared with the effects of either drug alone. This ®nding suggests that any potential therapeutic bene®ts of coadministered of D9-THC and morphine are likely to be hindered by associated increases in side-effects. In the present study, CBD alone did not produce antinociceptive effects in the formalin model of tonic pain, supporting previous studies (see Introduction). Recently, cannabis medicinal extract rich in CBD has been shown to signi®cantly alleviate pain in chronic pain states associated with nerve damage (Wade et al., 2003). Thus, current studies suggest that, although CBD lacks antinociceptive activity in animal models of acute and tonic pain, it may have therapeutic use in the treatment of neuropathic pain states. In the present study, CBD also had no effect on locomotor activity or body temperature. Thus, the pharmacological pro®le of the effects of CBD differs markedly from that of D9-THC, which produced the classic tetrad of CB1 receptormediated effects. The lack of effect of CBD on the classic cannabinoid receptor-mediated effects corroborates previous studies demonstrating that CBD does not act at the CB1 receptor (Thomas et al., 1998; Jarai et al., 1999; Bisogno et al., 2001; Patel & Hillard, 2001; Wallace et al., 2001; Pertwee et al., 2002). In contrast to the enhanced antinociceptive effects of coadministered D9-THC and morphine, we did not ®nd any evidence that CBD alters the antinociceptive effects of D9-THC in the formalin model of tonic pain. This ®nding opposes previous studies reporting potentiation (Karniol & Carlini, 1973) or antagonism (Welburn et al., 1976) by CBD of the antinociceptive effects of D9-THC in models of acute 683 pain. The basis for these differences remains unknown, but may be related to the different mechanisms underlying acute vs. tonic pain states. Intraplantar injection of formalin is a potent stressor and here we demonstrate formalin-evoked activation of the HPA axis, as indexed by increased levels of plasma corticosterone. This ®nding is consistent with previous studies demonstrating that acute injection of formalin increases plasma levels of corticosterone and adrenocorticotrophic hormone in rats (Pacak et al., 1995; Taylor et al., 1998; Palkovits et al., 1999). In the present study systemic administration of D9-THC (2.5 mg/kg) enhanced the formalin-evoked corticosterone response. This ®nding supports previous studies demonstrating that acute systemic administration of the synthetic D9-THC analogue HU210 enhances the plasma corticosterone response to novel arena stress (Finn et al., in press) and that cannabinoid agonists induce a CB1 receptor-mediated elevation of plasma corticosterone and adrenocorticotrophic hormone in nonstressed rats (RodrõÂguez de Fonseca et al., 1995; 1996; Martin-Calderon et al., 1998; Romero et al., 2002). In contrast to D9-THC, systemic administration of CBD alone did not alter formalin-evoked corticosterone responses. Zuardi et al. (1984) reported that systemic administration of CBD elevated serum corticosterone levels in nonstressed rats, albeit at higher doses than that administered in the present study and at an earlier time point post-CBD administration. Nevertheless, when taken together these ®ndings suggest that the effects of CBD on HPA axis activity are dependent on the basal homeostatic state of the animal. In contrast to the effect of D9-THC alone, administration of morphine alone, or with D9-THC, had no effect on the formalin-evoked corticosterone response. By contrast, coadministration of D9-THC and CBD was associated with enhancement of the formalin-evoked corticosterone response. Thus, there is a clear dissociation between the direction of effect of D9-THC and morphine vs. D9-THC and CBD on corticosterone levels and nociceptive behaviour. These data suggest that changes in the formalin-evoked corticosterone responses are not necessary for the reported antinociceptive behaviour, as depicted by the effects of D9-THC and morphine. These ®ndings corroborate previous studies demonstrating that increased levels of endogenous corticosterone are not antinociceptive in the formalin test (Taylor et al., 1998). Thus, it appears that although D9-THC plus morphine treatment and D9-THC plus CBD treatment produce a comparable level of antinociception, in the case of D9-THC and CBD this is associated with an increased stress response as indicated by the rise in corticosterone. The lack of effect of morphine on HPA axis activity in the present study may be due to the relatively low dose of morphine used, because the same dose of systemic morphine has also been reported not to modulate corticosterone levels in nonstressed rats (Jezova et al., 1982). Morphine has, however, also been shown to activate the HPA axis in a dose-related manner in nonstressed rats (Jezova et al., 1982; Buckingham & Cooper, 1984; Iyengar et al., 1986; Suemaru et al., 1986). An alternative explanation for the lack of effect of coadministered D9-THC and morphine on corticosterone is that the effects of morphine on HPA axis activity are attenuated in stressed formalintreated rats, this theory is supported by previous studies demonstrating differential effects of morphine on HPA axis activity in stressed and nonstressed rats (Buckingham & Cooper, 1984; Suemaru et al., 1986; Tanaka et al., 1991; Debreceni et al., 1994; Zhou et al., 1999). The effects of D9-THC and morphine on nociceptive processing have been shown to be mediated by brain CB1 receptors and m-opioid receptors, respectively (Lichtman & Martin, 1997; Manning & Franklin, 1998). Key sites of action of systemically administered D9-THC and morphine include the PAG, thalamus and hypothalamus, and one of their downstream effects is modulation of monoamines ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 684 D. P. Finn et al. (Sawynok & Reid, 1987; Murphy et al., 1990; Heijna et al., 1991; Tzavara et al., 2001). Intraplantar injection of formalin has been shown to increase c-fos expression in the PAG, thalamus and hypothalamus of rats (Pertovaara et al., 1993; Liu et al., 1998; Baulmann et al., 2000) and to produce alterations in central monoaminergic transmission (Pacak et al., 1995; Taylor et al., 1998; Palkovits et al., 1999). In the present study, intraplantar injection of formalin reduced tissue levels of dopamine in the PAG. This ®nding supports a previous study demonstrating that extracellular levels of dopamine are increased in the PAG in the carrageenan model of short-term in¯ammatory nociception (Gao et al., 2001). These ®ndings underscore the need for future studies aimed at de®ning the role of dopamine in the PAG in mediating behavioural responses to nociceptive or aversive stimuli. Intraplantar injection of formalin did not alter tissue levels of noradrenaline and MHPG. Previously, increases in extracellular levels of noradrenaline in the hypothalamic paraventricular nucleus of rats have been reported following formalin injection (Pacak et al., 1995; Palkovits et al., 1999). The discrepancy between these studies may be due to differences in sample collection (i.e. dialysates vs. tissue homogenates) and region of analysis (PVN vs. whole hypothalamus). Systemic administration of D9-THC alone, or in combination with CBD or morphine, did not alter the formalin-evoked decrease in PAG dopamine levels. These ®ndings suggest that while activation of the PAG dopaminergic system may be one of the mechanisms underlying automodulatory processes initiated during persistent pain and in¯ammation, it is unlikely to mediate or contribute to the antinociceptive effects of systemically administered D9-THC and CBD or morphine. Coadministered D9-THC and morphine signi®cantly enhanced thalamic levels of 5-HT and reduced the 5-HIAA/5-HT ratio, compared with either drug alone or vehicle, as well as producing enhanced antinociceptive effects in formalin-treated rats. By contrast, CBD did not enhance the antinociceptive effects of D9-THC and levels of 5-HT in the thalamus were also unaltered by this treatment. These data suggest that modulation of thalamic 5-HT levels may either contribute to, or result from, the enhanced antinociceptive effects of coadministered D9-THC and morphine reported in the present study. In the present study, administration of D9-THC alone, or in combination with CBD or morphine, increased the MHPG/noradrenaline ratio, an index of hypothalamic noradrenaline turnover, in formalin-treated rats. However, morphine alone, which signi®cantly inhibited formalinevoked nociceptive behaviour, did not modify hypothalamic noradrenaline turnover. The possible link between modulation of the hypothalamic noradrenergic system and cannabinoid receptor-mediated behavioural effects remains to be determined. It is interesting to note that, in the present study, the stimulatory effect of D9-THC on noradrenergic neurotransmission in the hypothalamus was accompanied by increased corticosterone release while morphine had no effect on either of these parameters. These data are consistent with strong evidence indicating that noradrenaline acts at the level of the hypothalamus to stimulate HPA axis activity (Plotsky, 1987). In summary, the present study demonstrates antinociceptive effects of systemically administered D9-THC, but not CBD, in the formalin test. In addition, antinociceptive effects of D9-THC were unaltered by CBD. We report here enhanced antinociceptive effects of D9-THC and morphine, which were associated with increased levels of thalamic 5HT and an enhanced suppression of locomotor activity. These data support previous studies demonstrating antinociceptive effects of D9THC but not CBD in animal models of acute or tonic pain. The ®ndings also suggest that the potential therapeutic bene®ts of coadministered D9-THC and morphine may be offset by an increased side-effect pro®le. Acknowledgements The authors wish to thank Professor Raphael Mechoulam for kindly providing the compound cannabidiol and The Wellcome Trust for supporting this work. The technical assistance of Karen Turner with HPLC is also gratefully acknowledged. Abbreviations 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine; CB1, cannabinoid receptor 1; CBD, cannabidiol; CPS, composite pain score; D9-THC, D9-tetrahydrocannabinol; DOPAC, 3,4-dihydroxyphenylacetic acid; HPA, hypothalamo-pituitary-adrenal; MHPG, 4-hydroxy-3-methoxyphenylglycol; PAG, periaqueductal grey; PLSD, protected least signi®cant difference. References Abbott, F.V., Franklin, K.B.J. & Westbrook, R.F. (1995) The formalin test: Scoring properties of the ®rst and second phases of the pain response in rats. Pain, 60, 91±102. Baulmann, J., Spitznagel, H., Herdegen, T., Unger, T. & Culman, J. (2000) Tachykinin receptor inhibition and c-Fos expression in the rat brain following formalin-induced pain. Neuroscience, 95, 813±820. Bisogno, T., Hanus, L., De Petrocellis, L., Tchilibon, S., Ponde, D.E., Brandi, I., Moriello, A.S., Davis, J.B., Mechoulam, R. & Di Marzo, V. (2001) Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Br. J. Pharmacol., 134, 845±852. Buckingham, J.C. & Cooper, T.A. (1984) Differences in hypothalamo-pituitaryadrenocortical activity in the rat after acute and prolonged treatment with morphine. Neuroendocrinology, 38, 411±417. Cichewicz, D.L., Martin, Z.L., Smith, F.L. & Welch, S.P. (1999) Enhancement mu opioid antinociception by oral delta9-tetrahydrocannabinol: dose± response analysis and receptor identi®cation. J. Pharmacol. Exp. Ther., 289, 859±867. Cichewicz, D.L. & McCarthy, E.A. (2003) Antinociceptive synergy between delta (9)-tetrahydrocannabinol and opioids after oral administration. J. Pharmacol. Exp. Ther., 304, 1010±1015. Clemett, D.A., Cockett, M.I., Marsden, C.A. & Fone, K.C. (1998) Antisense oligonucleotide-induced reduction in 5-hydroxytryptamine7 receptors in the rat hypothalamus without alteration in exploratory behaviour or neuroendocrine function. J. Neurochem., 71, 1271±1279. Debreceni, L., Hartmann, G. & Debreceni, B. (1994) Effect of morphine on hypothalamic catecholamine and serotonin level in relation to the stressinduced pituitary-adrenocortical activation in the rat. Exp. Clin. Endocrinol., 102, 307±312. Dubuisson, D. & Dennis, S.G. (1977) The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain, 4, 161±174. Finn, D.P., Chapman, V., Jhaveri, M.D., Samanta, S., Manders, T., Bowden, J., Matthews, L., Marsden, C.A. & Beckett, S.R. (2003b) The role of the central nucleus of the amygdala in nociception and aversion. Neuroreport, 14, 981±984. Finn, D.P., Jhaveri, M.D., Beckett, S.R.G., Kendall, D.A., Marsden, C.A., & Chapman, V. (2004) Cannabinoids modulate ultrasound-induced aversive responses in rats. Psychopharmacology (Berl.), [DOI: 10.1007/s00213-0031629-1.] in press. Finn, D.P., Jhaveri, M.D., Beckett, S.R.G., Roe, C.H., Kendall, D.A., Marsden, C.A. & Chapman, V. (2003a) Effects of direct periaqueductal grey administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology, 45, 594±604. Gao, X., Zhang, Y.Q., Zhang, L.M. & Wu, G.C. (2001) Effects of intraplantar injection of carrageenan on central dopamine release. Brain. Res. Bull., 54, 391±394. Guimaraes, F.S., Chiaretti, T.M., Graeff, F.G. & Zuardi, A.W. (1990) Antianxiety effect of cannabidiol in the elevated plus-maze. Psychopharmacology (Berl.), 100, 558±559. Heijna, M.H., Padt, M., Hogenboom, F., Schoffelmeer, A.N. & Mulder, A.H. (1991) Opioid-receptor-mediated inhibition of [3H]dopamine but not [3H]noradrenaline release from rat mediobasal hypothalamus slices. Neuroendocrinology, 54, 118±126. Iyengar, S., Kim, H.S. & Wood, P.L. (1986) Kappa opiate agonists modulate the hypothalamic-pituitary-adrenocortical axis in the rat. J. Pharmacol. Exp. Ther., 238, 429±436. ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 Antinociceptive effects of cannabinoids and morphine Jarai, Z., Wagner, J.A., Varga, K., Lake, K.D., Compton, D.R., Martin, B.R., Zimmer, A.M., Bonner, T.I., Buckley, N.E., Mezey, E., Razdan, R.K., Zimmer, A. & Kunos, G. (1999) Cannabinoid-induced mesenteric vasodilation through an endothelial site distinct from CB1 or CB2 receptors. Proc. Natl Acad. Sci. USA, 96, 14136±14141. Jarbe, T.U., Andrzejewski, M.E. & DiPatrizio, N.V. (2002) Interactions between the CB1 receptor agonist Delta 9-THC and the CB1 receptor antagonist SR-141716 in rats: open-®eld revisited. Pharmacol. Biochem. Behav., 73, 911±919. Jezova, D., Vigas, M. & Jurcovicova, J. (1982) ACTH and corticosterone response to naloxone and morphine in normal, hypophysectomized and dexamethasone-treated rats. Life Sci., 31, 307±314. Karniol, I.G. & Carlini, E.A. (1973) Pharmacological interaction between cannabidiol and delta 9-tetrahydrocannabinol. Psychopharmacology (Berl.), 33, 53±70. Keay, K.A., Clement, C.I., Matar, W.M., Heslop, D.J., Henderson, L.A. & Bandler, R. (2002) Noxious activation of spinal or vagal afferents evokes distinct patterns of fos-like immunoreactivity in the ventrolateral periaqueductal gray of unanaesthetised rats. Brain Res., 948, 122±130. Lichtman, A.H. & Martin, B.R. (1997) The selective cannabinoid antagonist SR 141716A blocks cannabinoid-induced antinociception in rats. Pharmacol. Biochem. Behav., 57, 7±12. Liu, R.J., Qiang, M. & Qiao, J.T. (1998) Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station. Neuroscience, 85, 1073±1087. Malfait, A.M., Gallily, R., Sumariwalla, P.F., Malik, A.S., Andreakos, E., Mechoulam, R. & Feldmann, M. (2000) The nonpsychoactive cannabis constituent cannabidiol is an oral anti-arthritic therapeutic in murine collagen-induced arthritis. Proc. Natl Acad. Sci. USA, 97, 9561±9566. Manning, B.H. & Franklin, K.B. (1998) Morphine analgesia in the formalin test: reversal by microinjection of quaternary naloxone into the posterior hypothalamic area or periaqueductal gray. Behav. Brain. Res., 92, 97±102. Martin, B.R., Compton, D.R., Thomas, B.F., Prescott, W.R., Little, P.J., Razdan, R.K., Johnson, M.R., Melvin, L.S., Mechoulam, R. & Ward, S.J. (1991) Behavioral, biochemical, and molecular modeling evaluations of cannabinoid analogs. Pharmacol. Biochem. Behav., 40, 471±478. Martin-Calderon, J.L., Munoz, R.M., Villanua, M.A., del Arco, I., Moreno, J.L., RodrõÂgueZ. de Fonseca, F. & Navarro, M. (1998) Characterization of the acute endocrine actions of (-)-11-hydroxy-Delta (8)-tetrahydrocannabinoldimethylheptyl (HU-210), a potent synthetic cannabinoid in rats. Eur. J. Pharmacol., 344, 77±86. Moss, D.E. & Johnson, R.L. (1980) Tonic analgesic effects of Delta9-tetrahydrocannabinol as measured with the formalin test. Eur. J. Pharmacol., 61, 313±315. Murphy, L.L., Steger, R.W., Smith, M.S. & Bartke, A. (1990) Effects of delta-9tetrahydrocannabinol, cannabinol and cannabidiol, alone and in combinations, on luteinizing hormone and prolactin release and on hypothalamic neurotransmitters in the male rat. Neuroendocrinology, 52, 316±321. Nicholson, B. (2003) Responsible prescribing of opioids for the management of chronic pain. Drugs, 63, 17±32. Oluyomi, A.O., Hart, S.L. & Smith, T.W. (1992) Differential antinociceptive effects of morphine and methylmorphine in the formalin test. Pain, 49, 415±418. Pacak, K., Palkovits, M., Kvetnansky, R., Yadid, G., Kopin, I.J. & Goldstein, D.S. (1995) Effects of various stressors on in vivo norepinephrine release in the hypothalamic paraventricular nucleus and on the pituitary-adrenocortical axis. Ann. NY Acad. Sci., 771, 115±130. Palkovits, M., Baf®, J.S. & Pacak, K. (1999) The role of ascending neuronal pathways in stress-induced release of noradrenaline in the hypothalamic paraventricular nucleus of rats. J. Neuroendocrinol., 11, 529±539. Parker, L.A., Mechoulam, R. & Schlievert, C. (2002) Cannabidiol, a nonpsychoactive component of cannabis and its synthetic dimethylheptyl homolog suppress nausea in an experimental model with rats. Neuroreport, 13, 567±570. Patel, S. & Hillard, C.J. (2001) Cannabinoid CB (1) receptor agonists produce cerebellar dysfunction in mice. J. Pharmacol. Exp. Ther., 297, 629±637. Pertovaara, A., Bravo, R. & Herdegen, T. (1993) Induction and suppression of immediate-early genes in the rat brain by a selective alpha-2-adrenoceptor agonist and antagonist following noxious peripheral stimulation. Neuroscience, 54, 117±126. Pertwee, R.G. (2001) Cannabinoid receptors and pain. Prog. Neurobiol., 63, 569±611. Pertwee, R.G., Ross, R.A., Craib, S.J. & Thomas, A. (2002) (-)-cannabidiol antagonizes cannabinoid receptor agonists and noradrenaline in the mouse vas deferens. Eur. J. Pharmacol., 456, 99±106. 685 Plotsky, P.M. (1987) Facilitation of immunoreactive corticotrophin-releasing factor secretion into the hypophysial-portal circulation after activation of catecholaminergic pathways or central norepinephrine injection. Endocrinology, 121, 924±930. Pugh, G. Jr, Smith, P.B., Dombrowski, D.S. & Welch, S.P. (1996) The role of endogenous opioids in enhancing the antinociception produced by the combination of delta 9-tetrahydrocannabinol and morphine in the spinal cord. J. Pharmacol. Exp. Ther., 279, 608±616. Reche, I., Fuentes, J.A. & Ruiz-Gayo, M. (1996) Potentiation of delta 9tetrahydrocannabinol-induced analgesia by morphine in mice: involvement of mu- and kappa-opioid receptors. Eur. J. Pharmacol., 318, 11±16. RodrõÂguez de Fonseca, F., Rubio, P., Menzaghi, F., MerloPich, E., Rivier, J., Koob, G.F. & Navarro, M. (1996) Corticotropin-releasing factor (CRF) antagonist [D- Phe (12),Nle (21,38),C (alpha) MeLeu (37) ]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats. J. Pharmacol. Exp. Ther., 276, 56±64. RodrõÂguez de Fonseca, F., Villanua, M.A., Munoz, R.M., Sanmartinclark, O. & Navarro, M. (1995) Differential effects of chronic treatment with either dopamine D-1 or D-2 receptor agonists on the acute neuroendocrine actions of the highly potent synthetic cannabinoid Hu-210 in male rats. Neuroendocrinology, 61, 714±721. Romero, E.M., Fernandez, B., Sagredo, O., Gomez, N., Uriguen, L., Guaza, C., De Miguel, R., Ramos, J.A. & Viveros, M.P. (2002) Antinociceptive, behavioural and neuroendocrine effects of CP 55,940 in young rats. Dev. Brain Res., 136, 85±92. Sanders, J., Jackson, D.M. & Starmer, G.A. (1979) Interactions among the cannabinoids in the antagonism of the abdominal constriction response in the mouse. Psychopharmacology (Berl.), 61, 281±285. SanÄudo-PenÄa, M.C., Romero, J., Seale, G.E., Fernandez-Ruiz, J.J. & Walker, J.M. (2000) Activational role of cannabinoids on movement. Eur. J. Pharmacol., 391, 269±274. Sawynok, J. & Reid, A. (1987) Effect of 6-hydroxydopamine-induced lesions to ascending and descending noradrenergic pathways on morphine analgesia. Brain Res., 419, 156±165. Sevostianova, N., Zvartau, E., Bespalov, A. & Danysz, W. (2003) Effects of morphine on formalin-induced nociception in rats. Eur. J. Pharmacol., 462, 109±113. Smith, F.L., Cichewicz, D., Martin, Z.L. & Welch, S.P. (1998) The enhancement of morphine antinociception in mice by delta9-tetrahydrocannabinol. Pharmacol. Biochem. Behav., 60, 559±566. So®a, R.D., Vassar, H.B. & Knobloch, L.C. (1975) Comparative analgesic activity of various naturally occurring cannabinoids in mice and rats. Psychopharmacologia, 40, 285±295. Suemaru, S., Hashimoto, K. & Ota, Z. (1986) Effect of morphine on hypothalamic corticotropin-releasing factor (CRF) and pituitary-adrenocortical activity. Endocrinol. Jpn., 33, 441±448. Tanaka, M., Tsuda, A., Yokoo, H., Yoshida, M., Mizoguchi, K. & Shimizu, T. (1991) Psychological stress-induced increases in noradrenaline release in rat brain regions are attenuated by diazepam, but not by morphine. Pharmacol. Biochem. Behav., 39, 191±195. Taylor, B.K., Akana, S.F., Peterson, M.A., Dallman, M.F. & Basbaum, A.I. (1998) Pituitary-adrenocortical responses to persistent noxious stimuli in the awake rat: endogenous corticosterone does not reduce nociception in the formalin test. Endocrinology, 139, 2407±2413. Thomas, B.F., Gilliam, A.F., Burch, D.F., Roche, M.J. & Seltzman, H.H. (1998) Comparative receptor binding analyses of cannabinoid agonists and antagonists. J. Pharmacol. Exp. Ther., 285, 285±292. Tzavara, E.T., Perry, K.W., Rodriguez, D.E., Bymaster, F.P. & Nomikos, G.G. (2001) The cannabinoid CB (1) receptor antagonist SR141716A increases norepinephrine out¯ow in the rat anterior hypothalamus. Eur. J. Pharmacol., 426, R3±R4. Wade, D.T., Robson, P., House, H., Makela, P. & Aram, J. (2003) A preliminary controlled study to determine whether whole-plant cannabis extracts can improve intractable neurogenic symptoms. Clin. Rehabilitation, 17, 21±29. Wallace, M.J., Wiley, J.L., Martin, B.R. & DeLorenzo, R.J. (2001) Assessment of the role of CB1 receptors in cannabinoid anticonvulsant effects. Eur. J. Pharmacol., 428, 51±57. Watson, G.S., Sufka, K.J. & Coderre, T.J. (1997) Optimal scoring strategies and weights for the formalin test in rats. Pain, 70, 53±58. Welburn, P.J., Starmer, G.A., Chesher, G.B. & Jackson, D.M. (1976) Effect of cannabinoids on the abdominal constriction response in mice: within cannabinoid interactions. Psychopharmacologia, 46, 83±85. Welch, S.P. & Eads, M. (1999) Synergistic interactions of endogenous opioids and cannabinoid systems. Brain Res., 848, 183±190. ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686 686 D. P. Finn et al. Welch, S.P. & Stevens, D.L. (1992) Antinociceptive activity of intrathecally administered cannabinoids alone, and in combination with morphine, in mice. J. Pharmacol. Exp. Ther., 262, 10±18. Welch, S.P., Thomas, C. & Patrick, G.S. (1995) Modulation of cannabinoidinduced antinociception after intracerebroventricular versus intrathecal administration to mice: possible mechanisms for interaction with morphine. J. Pharmacol. Exp. Ther., 272, 310±321. Zhou, Y., Spangler, R., Maggos, C.E., Wang, X.M., Han, J.S., Ho, A. & Kreek, M.J. (1999) Hypothalamic-pituitary-adrenal activity and pro-opiomelanocortin mRNA levels in the hypothalamus and pituitary of the rat are differentially modulated by acute intermittent morphine with or without water restriction stress. J. Endocrinol., 163, 261±267. Zuardi, A.W., Shirakawa, I., Finkelfarb, E. & Karniol, I.G. (1982) Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects. Psychopharmacology (Berl.), 76, 245± 250. Zuardi, A.W., Teixeira, N.A. & Karniol, I.C. (1984) Pharmacological interaction of the effects of delta 9-trans-tetrahydrocannabinol and cannabidiol on serum corticosterone levels in rats. Arch. Int. Pharmacodyn. Ther., 269, 12±19. ß 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 19, 678±686