Modulation of morphine-induced Fos-immunoreactivity by the cannabinoid receptor antagonist SR 141716

Neuropharmacology 47 (2004) 1157–1169 www.elsevier.com/locate/neuropharm

Modulation of morphine-induced Fos-immunoreactivity by the cannabinoid receptor antagonist SR 141716 M.E. Singh a, A.N.A. Verty a, I. Price a, I.S. McGregor b, P.E. Mallet a,
a

School of Psychology, University of New England, Armidale, NSW 2351, Australia b School of Psychology, University of Sydney, Sydney, NSW 2006, Australia

Received 16 September 2003; received in revised form 19 July 2004; accepted 17 August 2004

Abstract A growing body of evidence suggests the existence of a functional interaction between opioid and cannabinoid systems. The present study further investigated this functional interaction by examining the combined effects of morphine and the cannabinoid receptor antagonist SR 141716 on Fos-immunoreactivity (Fos-IR), a marker for neural activation. Male albino Wistar rats were treated with SR 141716 (3 mg/kg, intraperitoneally), morphine HCl (10 mg/kg, subcutaneously), vehicle, or SR 141716 and morphine combined (n ¼ 6 per group). Rats were injected with morphine or its vehicle 30-min after administration of SR 141716 or its vehicle and perfused 3 h later. Locomotor activity and body temperature were both increased in the morphine-treated group and SR 141716 significantly inhibited these effects. Morphine increased Fos-IR in several brain regions including the caudateputamen (CPu), cortex (cingulate, insular and piriform), nucleus accumbens (NAS) shell, lateral septum (LS), bed nucleus of the stria terminalis (BNST), median preoptic nucleus (MnPO), medial preoptic nucleus (MPO), hypothalamus (paraventricular, dorsomedial and ventromedial), paraventricular thalamic nucleus (PV), amygdala (central and basolateral nuclei), dorsolateral periaqueductal gray, ventral tegmental area (VTA), and Edinger–Westphal nucleus. SR 141716 alone increased Fos-IR in the cortex (cingulate, insular and piriform), NAS (shell), LS, BNST, hypothalamus (paraventricular, dorsomedial and ventromedial), PV, amygdala (central, basolateral and medial nuclei), VTA, and Edinger–Westphal nucleus. SR 141716 attenuated morphine-induced Fos-IR in several regions including the CPu, cortex, NAS (shell), LS, MnPO, MPO, paraventricular and dorsomedial hypothalamus, PV, basolateral amygdala, VTA, and Edinger–Westphal nucleus (EW). These results provide further support for functional interplay between the cannabinoid and opioid systems. Possible behavioural and physiological implications of the interactive effects of SR 141716 on morphine-induced Fos-IR are discussed. # 2004 Elsevier Ltd. All rights reserved. Keywords: Immunochemistry; Opioid; Cannabinoid; Locomotion; Thermoregulation; Rat; Morphine; SR 141716

1. Introduction The brain’s cannabinoid and opioid systems produce many of the same physiological and behavioural effects. For example, administration of opioid or cannabinoid receptor agonists both produce hypothermia, sedation, analgesia and other similar pharmacological effects (Manzanares et al., 1999). These two systems also interactively modulate appetite (Gallate et al., 1999; Verty et al., 2003), analgesia (Tulunay et al., 1981), addiction and reward (Chen et al., 1990;
Corresponding author. Tel.: +61-2-6773-3725; fax: +61-2-67733820. E-mail address: [email protected] (P.E. Mallet).

0028-3908/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2004.08.008

Gardner et al., 1988). At present, not much is known about the neural substrates underlying cannabinoid– opioid interactions and the role of endogenous cannabinoid activation in mediating acute opiate effects. Studies using the expression of the immediate early gene c-fos to map the distribution of CNS neurons activated by stimulation in vivo provide a valuable tool to study the neuroanatomical substrates of many drugs of abuse. An increase in Fos-immunoreactivity (Fos-IR) is found following administration of numerous drugs including cocaine, amphetamine, MDMA (ecstasy), D9-tetrahydrocannabinol (THC), and morphine. These drugs produce characteristic patterns of c-fos expression indicating distinct distribution of drug-induced neuronal activation (Allen et al., 2003;

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Erdtmann-Vourliotis et al., 1999; Frankel et al., 1999; Harlan and Garcia, 1998). Morphine was the first opiate reported to produce Fos in the brain (Chang et al., 1988). Acute administration of morphine produces Fos expression in the ventral tegmental area (VTA) and nucleus accumbens (NAS)—brain areas that have been associated with drug reinforcement and addiction processes (Garcia et al., 1995). Studies also reveal reliable induction of Fos-IR following acute morphine administration in hypothalamic nuclei involved in appetite regulation (Verty et al., 2002). In addition, cannabinoid and opioid induced Fos-IR has been demonstrated in a number of spinal and supraspinal regions known to mediate antinociception (Lichtman et al., 1996; Manzanares et al., 1999). Growing evidence from animal studies suggests that CB1 cannabinoid receptors are involved in the modulation of opiate addiction and reward. SR 141716, a selective antagonist for the CB1 cannabinoid receptor, attenuates heroin self-administration, morphineinduced place preference (Braida et al., 2001; Chaperon et al., 1998; Navarro et al., 2001), alcohol intake (Vacca et al., 2002), and reduces the intensity of opiate withdrawal (Braida et al., 2001; Navarro et al., 2001; Rubino et al., 2000). These findings suggest the possibility that cannabinoid receptor antagonists may have pharmacotherapeutic utility in opiate drug abuse (Navarro et al., 2001). With regard to appetite, functional interactions between the opioid and cannabinoid systems have been well established. For example, the orexigenic effects of THC on feeding induced by electrical stimulation of the lateral hypothalamus can be attenuated by naloxone (Trojniar and Wise, 1991). In addition, normal food intake induced by systemic application of THC is attenuated by naloxone (Williams and Kirkham, 2002). Increased food intake produced by administration of morphine into the paraventricular nucleus of the hypothalamus (PVN) can be attenuated by intraperitoneal application of SR 141716 (Verty et al., 2003) and naloxone and SR 141716 synergistically depress food intake at doses that do not alter feeding on their own (Kirkham and Williams, 2001). However, it is unclear whether the PVN or other hypothalamic sites mediate opioid–cannabinoid interactions in food intake. Hypothalamic and preoptic regions are involved in mediating the thermoregulatory effects of cannabinoids and opioids. Cannabinoid and opioid receptors are located within these regions (Herkenham et al., 1991; Mansour, 1988) and central administration of morphine produces hyperthermia (Adler and Gellar, 1993). Furthermore, THC administration into the preoptic area produces hypothermia (Fitton and Pertwee, 1982; Pertwee et al., 1988). Cannabinoid–opioid interactions in antinociception have been well established. Both cannabinoid and

opioid receptor agonists produce analgesia (Smith et al., 1998) and a combination of these drugs interact synergistically to potentiate analgesia (Welch and Eads, 1999; Welch and Stevens, 1992), which can be blocked by SR 141716 and naloxone (Welch and Eads, 1999). Both opioid and cannabinoid receptors are located within the insular and cingulate cortex (Herkenham et al., 1991; Mansour, 1988); these cortical regions may therefore be involved in cannabinoid–opioid interactions in pain. An interrelationship between cannabinoid and opioid systems was further supported in our recent work examining the interactive effects of THC and the opioid receptor antagonist naloxone on Fos-IR (Allen et al., 2003). THC-induced For-IR was inhibited by naloxone in the central region of the caudate-putamen (CPu), dorsomedial and ventromedial hypothalamus, ventrolateral periaqueductal grey, and VTA. Furthermore, THC and naloxone produced an additive effect on Fos-IR in the bed nucleus of the stria terminalis (BNST, lateral division), insular cortex, central nucleus of the amygdala, and the paraventricular nucleus of the thalamus. The present study sought to build on our previous work with THC and naloxone by examining the effects of SR 141716 administration on morphine-induced Fos-IR. Consistent with previous findings, it was hypothesized that morphine and SR 141716 would produce distinctive patterns of Fos-IR throughout the brain (Alonso et al., 1999; Frankel et al., 1999; Garcia et al., 1995). In order to validate the design and doses utilized, the present study also examined the effects of SR 141716 on two known effects of morphine, namely hyperthermia and locomotor activity changes. Examination of any alterations of morphine-induced Fos-IR by SR 141716 was expected to provide new insights into the mechanisms underlying the functional interactions between these neurotransmitter systems. 2. Materials and methods 2.1. Subjects Male experimentally naive Wistar rats weighing 250  50 g were housed in opaque plastic cages 4–6 per cage on a reverse light–dark cycle (lights off at 08:00 h) with free access to standard lab chow (Barastoc, Ridley AgriProducts, Australia) and tap water. Experimental testing was conducted in the dark cycle between 08:30 and 16:00 h. Animals were treated in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) and the Australian Code of Practice for the care and use of animals for scientific purposes. This study was approved by the University of New England Animal Ethics Committee.

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2.2. Drugs SR 141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4dichlorophenyl)-4-methylpyrazole-3-carboxamide, Sanofi– Synthelabo] was first mixed with a few drops of Tween 80 (polyoxyethylene sorbitan monooleate, ICN Biomedicals). Physiological saline was then added and the solution was stirred and then sonicated. The final vehicle solution contained 15 ll Tween 80 per 2 ml saline (0.0075% Tween 80). SR 141716 was administered intraperitoneally (IP) at a dose of 3.0 mg/kg in a volume of 1 ml/kg. Morphine HCl (API/AMED, Australia) was dissolved in physiological saline (0.9%) and was administered subcutaneously (SC) at a dose of 10 mg/kg in a volume of 1 ml/kg. Vehicle conditions comprised morphine vehicle (0.9% saline) and SR 141716 vehicle. 2.3. Procedure Rats were randomly assigned to one of four treatment groups (n ¼ 6) according to a standard 2  2 design. The groups were: vehicle/saline, SR 141716/ saline, vehicle/morphine, and SR 141716/morphine. All rats were handled for 1 min per day for six consecutive days. Next, all rats were habituated to the testing apparatus, injection, handling and temperature measurement procedures for four consecutive days in order to minimise novelty induced Fos-IR. On each of the habituation days, rats were injected IP with vehicle in their home cages 30 min prior to receiving an SC injection of saline. Rats were then placed into the locomotor activity boxes for 180 min. Treatments were staggered at 10-min intervals to allow sufficient time between consecutive perfusions. Each home cage contained at least one rat from each treatment group. The protocol for the test day was similar to the habituation days with the exception of drug treatments and perfusions. On the test day, rats were injected with either vehicle or 3.0 mg/kg SR 141716 (IP) followed 30 min later by saline or 10 mg/kg morphine (SC). Following the second injection, rats were placed into the locomotor activity boxes for 180 min and then perfused. Body temperature was recorded 90 and 180 min into each session. As we have previously found peak acute locomotor stimulation between 120 and 180 min following morphine administration (Norwood et al., 2003), this delay between injection and perfusion was used in the present study. 2.4. Locomotor activity During testing, individual rats were placed in one of eight dimly lit rectangular Perspex locomotor activity chambers (28 cm wide  23 cm deep  30 cm high) placed inside sound attenuating boxes. The locomotor activity chambers were constructed with aluminium

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plate walls and galvanized wire mesh floors (1 cm2). A computer controlled passive infrared detector (Quantum passive infrared motion sensor, NESS Security Products, Australia, part no. 890-087-2) mounted to the ceiling of each box was used to quantify the locomotor activity of the animals using custom designed software. A 10-lF capacitor located near LK2 of the sensor’s printed circuit board was replaced with a 0.1-lF capacitor serving to alter the sensor alarm period from 5 s to approximately 50 ms. Locomotor activity was defined as time spent in motion and was recorded in 1 min bins. 2.5. Core temperature Core temperature was also indexed by measuring eardrum temperature using a Braun ThermoScan (model IRT 3020) infrared ear thermometer (Morley et al., 2001). 2.6. Immunohistochemistry Rats were deeply anaesthetized with 120 mg/kg sodium pentobarbital (IP) and perfused transcardially with 100 ml of phosphate-buffered saline (PBS, 0.1 M, pH 7.3) followed by 150 ml 4% para-formaldehyde in PBS. Brains were then extracted and placed in 4% v para-formaldehyde at 4 C for 24 h, then transferred to 15% sucrose in a phosphate buffer (PB) solution at v 4 C for 24 h, and finally placed in 30% sucrose solv ution in PB at 4 C for 48 h. Whole brains were then sectioned coronally at 40 lm using a cryostat set at v 19 C and sections were collected in PB. Free-floating sections were given two consecutive 30min washes in PB, a 30-min wash in 0.9% hydrogen peroxide in 50% ethanol, and a 30-min wash in 3% normal horse serum in PB. Sections were then incubated for v 72 h at 4 C in the primary c-fos antibody (s52, Santa Cruz Biotechnology, Santa Cruz, CA; rabbit polyclonal, specific for the amino acid terminus of c-Fos p62, noncross-reactive with FosB, Fra-1 or Fra-2) diluted 1:2000 in phosphate-buffered horse serum (PBH) (0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal horse serum in PB). Next, sections were washed for 30 min in PB and incubated for 60 min in biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:500 in PBH. They were then washed in PB for 30 min, and subsequently incubated for 60 min in extrAvidin-horseradish peroxidase (Sigma-Aldrich, Castle Hill, NSW) diluted 1:1000 in PBH. After three further 30-min washes in PB, horseradish peroxidase activity was visualized with the nickel diaminobenzidine and glucose oxidase reaction with nickel enhancement as described by Shu et al. (1988). The reaction was terminated 10 min later by washing in PB. Sections were mounted onto gelatine-coated slides, dehydrated, histolene-cleared and coverslipped.

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Fig. 1. Schematic diagrams of coronal sections of the rat brain (Paxinos and Watson, 1998). The number of Fos-positive nuclei were counted within the areas numbered and shaded in gray. The areas shown correspond in scale to the exact areas counted. The numbers indicated correspond to the brain regions listed in Table 1.

A total of 26 brain regions or sub regions were examined using light microscopy (Olympus CH-2 microscope) at 200 magnification. The atlas of Paxinos and Watson (1998) was used to identify these

brain regions, which are shown in Fig. 1. A 10  10 square graticule was positioned over each structure and the number of labelled cells within the graticule, which covered a 500  500 lm area, was counted manually.

M.E. Singh et al. / Neuropharmacology 47 (2004) 1157–1169

Because manual counting can be subject to experimenter bias, microscope slides were labelled such that the person counting the labelled cells did not know which group the rats belonged to. Where the structure was larger than the graticule, a standardised region within the structure was counted. Only round and oval cells that were completely black were counted. Images of Fos-IR cells were captured using a Micropublisher FireWire camera (QImaging, Burnaby BC, Canada) attached to an Olympus CH-2 light microscope. Images were acquired using a PowerMac computer running Adobe Photoshop Elements version 2.0. Post-production image processing and layout was conducted using Deneba Canvas 9.0, and included reduction of colour to greyscale, and standardization of brightness and contrast. 2.7. Statistical analysis Temperature data recorded at 90 and 180 min were analysed using a drug treatment by time analysis of variance (ANOVA) with repeated measure on time. Locomotor activity data for the 180 1-min bins were collapsed into two 90-min bins and analysed using a drug treatment by time ANOVA with repeated measures on the second factor. Group differences in temperature and locomotor data were also compared by conducting a one-way ANOVA for the data in each 90-min bin. Groups were then compared using post hoc Tukey-b tests where significant main effects were found. Group comparisons between the numbers of labelled cells were made using one-way ANOVAs. Planned t-test contrasts with Bonferroni correction were used to compare groups when a significant ANOVA main effect was observed. The five contrasts (a¼ 0:01) of interest were: (1) vehicle/saline vs. SR 141716/saline, (2) vehicle/saline vs. vehicle/morphine, (3) vehicle/saline vs. SR 141716/morphine, (4) SR 141716/morphine vs. SR 141716/saline, and (5) SR 141716/morphine vs. vehicle/morphine. A two-way ANOVA was also conducted for each brain region to determine whether the combined effect of the drugs was significantly higher or lower than would be expected by the sum of the individual drug effects. The first factor was the presence or absence of SR 141716 and the second was the presence or absence of morphine. Only the interaction term was reported because the individual main effects in the twoway ANOVAs provided no relevant information. Where ANOVA assumptions were not met, randomization tests of scores were conducted using NPFact version 1.0. In all cases, the randomization tests supported the ANOVA findings so for ease of interpretation only the ANOVA results have been presented. All ANOVAs, t-tests and post hoc tests were conducted using SPSS 11.0.2 for Mac OS X.

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3. Results 3.1. Locomotor activity Locomotor activity data are shown in Fig. 2. ANOVA revealed a main effect of drug treatment [F ð3; 20Þ ¼ 3:41, P < 0:05], main effect of time [F ð1; 20Þ ¼ 30:98, P < 0:001], and drug treatment by time interaction [F ð3; 20Þ ¼ 18:47, P < 0:001]. One-way ANOVAs with Tukey-b post hoc tests revealed that morphine produced a strong stimulation of locomotor activity relative to the control treatment at the 90–180 but not 0–90 min measurement intervals. SR 141716 did not affect locomotor activity when administered alone, but attenuated morphine-induced locomotion significantly at the 90–180 min measurement intervals. 3.2. Temperature Body temperature data are shown in Fig. 3. During the drug phase, time (2) by drug treatment (4) ANOVAs revealed a significant main effect of drug treatment [F ð3; 20Þ ¼ 96:17, P < 0:001] and a drug treatment by time interaction [F ð3; 20Þ ¼ 5:49, P < 0:01], but no main effect of time. A one-way ANOVA with Tukey-b post hoc tests revealed significant morphine-induced hyperthermia compared to vehicle control at the 90- and 180-min measurement intervals. SR 141716 significantly reduced morphineinduced hyperthermia at the 90-min time interval, [F ð3; 20Þ ¼ 63:37, P < 0:001], but not at the 180-min interval. SR 141716 administered alone did not significantly affect body temperature.

Fig. 2. Locomotor activity (mean number of seconds spent in motion) for rats receiving vehicle (VEH), SR 141716 (SR), morphine (MOR), or SR 141716 and morphine combined (SR þ MOR). Values shown represent the cumulative time spent in motion during two 90min measurement intervals.
Significantly different from VEH, P < 0:05; y Significantly different from MOR, P < 0:05.

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3.3.1. Fos-immunoreactivity in control rats Fos-IR in vehicle/saline-treated rats was low in most regions in accordance with the typical low levels of such expression found in well-habituated control animals. Fos-IR was relatively high in the PV. 3.3.2. Fos-immunoreactivity in SR 141716 treated rats The planned contrasts comparing SR 141716 alone to Veh/Sal revealed that SR 141716 increased Fos-IR in the cingulate, insular and piriform cortices, NAS (shell), LS, BNST, PVN, DMH, VMH, PV, CeA, basolateral and medial amygdala, VTA, and EW.

v

Fig. 3. Body temperature ( C) of rats receiving vehicle (VEH), SR 141716 (SR), morphine (MOR), or SR 141716 and morphine combined (SR þ MOR). Temperatures were recorded 90 and 180 min after injection.
Significantly different from VEH, P < 0:05; y Significantly different from SR, P < 0:05; # Significantly different from MOR, P < 0:05.

3.3.3. Fos-immunoreactivity in morphine-treated rats The planned contrasts comparing morphine alone to Veh/Sal revealed that morphine increased Fos-IR in the medial, dorsal and dorsolateral CPu, cingulate, insular and piriform cortices, NAS (shell), LS, BNST, MnPO, MPO, PVN, DMH, VMH, PV, CeA and basolateral amygdala, dorsolateral PAG, VTA, and EW.

3.3. Fos-immunoreactivity The number of labelled cells for the 26 brain regions examined is presented in Table 1. In addition, representative photomicrographs of labelled cells from the NAS (shell) and PVN are presented in Figs. 4 and 5, respectively. The ANOVA comparing groups was significant in 24 regions examined as follows: CPu (central) [F ð3; 20Þ ¼ 4:20, P < 0:01], medial CPu [F ð3; 20Þ ¼ 20:96, P < 0:001], dorsal CPu [F ð3; 20Þ ¼ 61:58, P < 0:001], dorsolateral CPu [F ð3; 20Þ ¼ 4:19, P < 0:05], NAS (shell) [F ð3; 20Þ ¼ 317:54, P < 0:001], lateral septum (LS) [F ð3; 20Þ ¼ 80:11, P < 0:001], BNST [F ð3; 20Þ ¼ 20:34, P < 0:001], cingulate cortex [F ð3; 20Þ ¼ 59:44, P < 0:001], insular cortex [F ð3; 20Þ ¼ 100:18, P < 0:001], piriform cortex [F ð3; 20Þ ¼ 146:97, P < 0:001], medial preoptic nucleus (MPO) [F ð3; 20Þ ¼ 27:68, P < 0:001], median preoptic nucleus (MnPO) [F ð3; 20Þ ¼ 35:09, P < 0:001], paraventricular nucleus of the hypothalamus (PVN) [F ð3; 20Þ ¼ 40:53, P <

0:001], dorsomedial nucleus of the hypothalamus (DMH) [F ð3; 20Þ ¼ 52:51, P < 0:001], ventromedial nucleus of the hypothalamus (VMH) [F ð3; 20Þ ¼ 129:12, P < 0:001], paraventricular nucleus of the thalamus (PV) [F ð3; 20Þ ¼ 16:00, P < 0:001], amygdala (central nucleus, CeA) [F ð3; 20Þ ¼ 90:71, P < 0:001], amygdala (medial) [F ð3; 20Þ ¼ 5:81, P < 0:01], amygdala (basolateral) [F ð3; 20Þ ¼ 67:15, P < 0:001], periaquaductal gray (PAG, dorsolateral) [F ð3; 20Þ ¼ 5:32, P < 0:01], PAG (lateral) [F ð3; 20Þ ¼ 5:77, P < 0:01], VTA [F ð3; 20Þ ¼ 98:11, P < 0:001], and Edinger– Westphal nucleus (EW) [F ð3; 20Þ ¼ 20:30, P < 0:001].

3.3.4. Fos-immunoreactivity in SR 141716/morphinetreated rats Planned contrasts comparing SR/Mor to Veh/Sal were significant for the medial CPu, NAS (shell), BNST, cingulate and piriform cortices, PVN, DMH, VMH, PV, CeA, basolateral and medial amygdala, lateral PAG, and VTA. The planned contrasts comparing SR/Mor to SR 141716 alone and morphine alone revealed that SR/Mor-induced Fos-IR was significantly different from SR 141716 alone in the NAS (shell), LS, BNST, cingulate and insular cortices, PVN, DMH, VMH, PV, CEA, basolateral amygdala, lateral PAG, VTA, and EW. SR/Mor was significantly different from morphine alone in the medial and dorsal CPu, NAS (shell), LS, BNST, cingulate, insular, and piriform cortices, MnPO, MPO, PVN, DMH, VMH, CEA, basolateral amygdala, VTA, and EW. Two two-way interactions were significant in 16 of the 26 structures examined, including the medial [F ð3; 20Þ ¼ 12:89, P < 0:01] and dorsal [F ð3; 20Þ ¼ 73:08, P < 0:001] CPu, cingulate [F ð3; 20Þ ¼ 157:65, P < 0:001], insular [F ð3; 20Þ ¼ 243:28, P < 0:001], and piriform [F ð3; 20Þ ¼ 227:52, P < 0:001] cortices, NAS (shell) [F ð3; 20Þ ¼ 284:97, P < 0:001], LS [F ð3; 20Þ ¼ 232:98, P < 0:001], MnPO [F ð3; 20Þ ¼ 39:29, P < 0:001], MPO [F ð3; 20Þ ¼ 16:35, P < 0:001], PVN [F ð3; 20Þ ¼ 21:81, P < 0:001], DMH [F ð3; 20Þ ¼ 122:98, P < 0:001], VMH [F ð3; 20Þ ¼ 5:89, P < 0:05], PV [F ð3; 20Þ ¼ 7:21, P < 0:05], basolateral amygdala [F ð3; 20Þ ¼ 57:01, P < 0:001], VTA [F ð3; 20Þ ¼ 279:59, P < 0:001], and EW [F ð3; 20Þ ¼ 50:66, P < 0:001]. In a majority of these brain regions, the interaction was due to fewer Fos-labelled cells than can be explained by a simple addition of the individual drug effects. One

M.E. Singh et al. / Neuropharmacology 47 (2004) 1157–1169

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Table 1 Mean number (SEM) of Fos-labelled cells Region

Bregma

Veh/Sal

SR/Sal

Veh/Mor

SR/Mor

+1.00 +1.00 +1.00 +1.00

0:2  0:2 2:0  :07 0:8  0:5 0:2  0:2

0:8  0:4 4:2  2:3 2:2  1:0 1:8  0:7

2:5  0:9 21:0  1:8
14:7  1:2
1:8  0:4

0:5  0:8 9:8  2:1
,

1:7  0:6

0:5  0:5

+1.00 +1.00 +1.00 +1.00 0.26

0:8  0:4 3:2  0:5 1:0  0:5 0:0  0:0 2:7  0:7

1:2  0:5 5:2  0:4
13:5  1:2
7:0  2:3 19:5  3:5


2:2  0:4 29.70.8
16:3  0:9
5:2  1:9 19:8  3:0


2:5  0:8 8:5  0:9
,

,


2:7  0:7

,


6:2  4:4 36.74.1
,

,




Cortex 10. Cingulate 11. Insular 12. Piriform

+1.00 +1.00 +1.00

1:5  0:6 1:2  0:8 2:0  0:6

11:5  0:7
11:8  0:6
6:7  0:7


16:2  1:4
21:2  1:2
19:8  0:8


4:5  0:4
,

,


3:7  0:8

,


4:8  0:5
,



a a a

Hypothalamic regions 13. Median preoptic nucleus 14. Medial preoptic nucleus 15. Paraventricular nucleus 16. Dorsomedial nucleus 17. Lateral hypothalamic area 18. Ventromedial nucleus

0.26 1.30 1.80 2.80 2.80 2.80

8:3  0:9 4:7  0:6 9:7  2:9 2:2  0:9 3:3  0:7 6:0  1:0

9:3  1:1 4:8  0:6 32:8  6:3
15:7  0:8
4:5  1:2 24.31.1


25.72.2
12.30.8
124:5  11:5
14:2  0:9
5:8  1:4 10:0  0:9


8:3  1:0

7:0  0:7

3:7  8:5
,

,


9:0  0:7
,

,


5:3  1:5 33:8  1:5
,

,




a a a a

Thalamus 19. Paraventricular nucleus

2.80

29:7  6:3

52:0  2:7


83:5  8:9


73:7  4:2
,




a

Amygdala 20. Central nucleus 21. Basolateral nucleus 22. Medial nucleus

2.80 2.80 2.80

3:8  1:1 3:5  0:9 4:3  1:2

22:8  3:2
9:2  1:2
11.71.8


56:3  3:6
25:8  1:3
6:2  2:3

77:5  4:9
,

,


14:0  1:2
,

,


15:0  2:6


a

Periaqueductal gray 23. Dorsolateral 24. Lateral

6.72 6.72

4:5  0:6 2:2  0:8

4:8  0:7 2:2  0:8

10:8  1:8
8:2  2:4

7:2  1:5 8:5  1:4
,




Other 25. Ventral tegmental area 26. Edinger–Westphal nucleus

5.30 5.60

1:0  0:5 2:5  1:2

15:7  1:0
6:3  1:1


19:3  1:1
8:0  0:6


4:2  0:9
,

,


1:5  0:7

,




Frontal regions 1. Caudate-putamen, central 2. Caudate-putamen, medial 3. Caudate-putamen, dorsal 4. Caudate-putamen, dorsolateral 5. Nucleus accumbens, core 6. Nucleus accumbens, shell 7. Lateral septum 8. Islands of Calleja, Major 9. BNST lateral division, dorsal

a a

a a

b

a a




P < 0:01 vs.Veh/Sal. P < 0:01 vs. Sal/Mor using planned contrasts assuming unequal variances; a = significantly lower than expected by the sum of individual drug effects; b, significantly higher than expected by the sum of individual drug effects.


P < 0:01 vs. SR/Sal.



exception was the VMH where the interaction was due to a slightly higher (albeit significantly) number of Foslabelled cells than expected by an additive effect of the individual drugs.

4. Discussion The results of the present study provide further evidence for a functional interaction between the cannabinoid and opioid systems. Pre-treatment with SR 141716 prevented morphine-induced Fos expression in several brain regions suggesting that CB1 receptors

have a powerful modulatory effect on opioid-induced neural activation. This provides interesting complementary data to our previous demonstration of region-specific inhibition of THC-induced Fos expression by naloxone (Allen et al., 2003). Morphine induced significant hyperthermia and locomotor activity, consistent with previous reports (Benamar et al., 2001; Norwood et al., 2003), and SR 141716 significantly attenuated these effects. The inhibitory effect of SR 141716 on morphine-stimulated locomotor activity agrees with our recent report (Norwood et al., 2003). To our knowledge, the present study is the

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Fig. 4. Photomicrograph showing Fos-labelled neurons in the nucleus accumbens (shell) in representative sections from rats treated with vehicle (A), SR 141716 (B), morphine (C), or SR 141716 and morphine combined (D). Scale bar ¼ 250 lm. The square indicates the region in which labelled cells were quantified.

first to demonstrate that SR 141716 can attenuate the hyperthermic response to morphine. Consistent with previous studies (Chang et al., 1988; Erdtmann-Vourliotis et al., 1999; Garcia et al., 1995; Liu et al., 1994), morphine increased Fos-IR in numerous brain regions, most notably the PVN, PV, CEA, NAS (shell), and CPu. Morphine also significantly increased Fos-IR throughout the cortex, in preoptic thermoregulatory centres, in various hypothalamic nuclei, the BNST, the basolateral amygdala, the dorsolateral PAG, EW and the VTA. SR 141716, given alone, also stimulated Fos-IR in a large number of brain regions, replicating several findings from a previous study (Alonso et al., 1999). However, in contrast to the study by Alonso et al. (1999), our results revealed significant increases in Fos-IR with SR 141716 in the PVN, DMH, VMH and VTA. The reasons for the discrepant findings are not clear at present. One possibility is our use of Wistar strain rats compared to the Sprague–Dawley rats used by Alonso et al. (1999). Our previous work has documented substantial strain differences in cannabinoid-induced

Fos-IR in Wistar rats compared to Lewis rats (Arnold et al., 2001). There were at least two novel findings with SR 141716 in the present study worth commenting upon. Firstly, SR 141716 significantly increased Fos-IR in several hypothalamic nuclei including the PVN, DMH and VMH. These structures are targets of converging orexigenic and anorexigenic pathways (Elmquist et al., 1998), and are known to regulate food intake by influencing metabolic, hormonal and endocrine responses relating to the nutritional state of the organism (Williams et al., 2001). SR 141716 suppresses food intake when administered alone (Colombo et al., 1998; Verty et al., 2004), and our results suggest that further studies should examine the role of the PVN, DMH and VMH in such effects. Secondly, SR 141716 increased Fos-IR in the Edinger–Westphal nucleus (EW). This small cholinergic structure is the parasympathetic part of the oculomotor nuclear complex and is involved in pupil constriction (Fant et al., 1998). The EW also appears to be involved in nociception (Innis and Aghajanian,

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Fig. 5. Photomicrograph showing Fos-labelled neurons in the paraventricular nucleus of the hypothalamus (PVN) in representative sections from rats treated with vehicle (A), SR 141716 (B), morphine (C), or SR 141716 and morphine combined (D). Scale bar ¼ 250 lm. The square indicates the region in which labelled cells were quantified.

1986), thermoregulation (Bachtell et al., 2003) and the response to alcohol (Bachtell et al., 2002). Our previous work has shown that THC increases Fos-IR expression in the EW (Allen et al., 2003) and the present results further confirm a cannabinoid influence on this structure. It is important to note that in addition to the hypothalamus and the EW, SR 141716 increased Fos-IR in the NAC (shell), LS, BNST, cingulate, insular, and piriform cortices, PVN, VMH, DMH, PV, CEA, basolateral and medial hypothalamus and VTA. Increased Fos-IR in these brain regions was also observed in our previous work using the cannabinoid receptor agonist THC (Allen et al., 2003). It is presently not clear why similar effects on Fos-IR in these regions were observed with both a cannabinoid receptor agonist and a cannabinoid receptor inverse agonist. The most striking finding in the current study was the profound reduction in morphine-induced Fos-IR obtained by co-administration of SR 141716. This was found in several key brain regions and provides valuable clues regarding the mechanisms whereby SR

141716 reverses a variety of functional effects of opioids and other drugs. There is converging evidence that SR 141716 has powerful anti-craving properties and is able to reduce the rewarding effects of many drugs including alcohol, opiates and nicotine (Cohen et al., 2002; Gallate et al., 1999; Navarro et al., 2001). For example, SR 141716 has been found to reduce morphine and heroin self-administration in rodents (Navarro et al., 2001) and morphine-induced conditioned place preference (Chaperon et al., 1998; Singh et al., 2001). Neurochemical studies suggest that SR 141716 inhibition of drug and alcohol-stimulated dopamine release in the mesolimbic pathway may be responsible for realising these anti-craving effects (Cohen et al., 2002; Hungund et al., 2003). The finding that SR 141716 reduces morphine-induced Fos-IR in the NAS (shell) and the VTA is entirely consistent with this hypothesis. Interestingly, naloxone also reduces THC-induced Fos-IR in the VTA (Allen et al., 2003), suggesting that cannabinoid–opioid interaction in drug craving is mediated via a common pathway (Ameri, 1999).

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In addition to the NAS, SR 141716 reduced morphine-induced Fos expression in several other terminal fields of the mesocorticolimbic dopamine system including the LS, basolateral nucleus of the amygdala, and the insular and cingulate cortex. For example, mesolimbic VTA dopamine neurons project to the LS (Jekab and Leranth, 1995) and morphine administered into the VTA stimulates dopamine release in the LS (Cador et al., 1989; Devine et al., 1993). Thus, the local increase in Fos-IR induced by morphine in the LS may at least partly result from a stimulation of VTA dopamine neurons. Thus, the suppression of this by SR 141716 may reflect its action in the VTA as well as a possible action on local l-opioid receptors in the LS (Simantov et al., 1978). Administration of morphine moderately increased Fos-IR in the medial and dorsal regions, but not in central or dorsolateral regions, of the CPu. Pre-treatment with SR 141716 significantly attenuated these morphine effects. Previous studies have indicated that morphine increases c-fos expression in a dorsomedial wedge of the CPu rich in l-opioid receptors (Garcia et al., 1995; Liu et al., 1994). The CPu plays a role in motor integration and the dorsomedial region in particular has been implicated in morphine-induced hyperlocomotion (Stevens et al., 1986). Interestingly, in our previous work, naloxone reduced THC-induced Fos-IR in the CPu (Allen et al., 2003). The ability of SR 141716 to reduce morphine effects in this region may therefore be important in its modulation of morphineinduced locomotor activity. Further behavioural studies using intracranial injections would help to further examine this possibility. Both SR 141716 and morphine administered alone increased Fos-IR in all three cortical regions examined, and the combination of these two drugs produced fewer labelled cells than either drug alone. Examination of several other cortical regions (data not shown) revealed that this pattern of activation was not unique to the cingulate, piriform and insular cortices, but instead was replicated across most of the cortex and is reflective of a general widespread increase of Fos-IR by either SR 141716 or morphine. Interestingly, in our previous work, THC increased Fos-IR in the piriform, but not cingulate or insular cortices (Allen et al., 2003). The hypothalamus proved to be an important site of cannabinoid–opioid interactions in the present study. Morphine-induced Fos-IR in the PVN, DMH and VMH with the highest levels observed in the PVN. Behavioural studies have demonstrated a stimulation of food intake following intra-hypothalamic infusion of morphine (Tepperman and Hirst, 1982; Verty et al., 2003). Like morphine, cannabinoids have been found to stimulate food intake via hypothalamic mechanisms. For example, application of anandamide in the VMH stimulates food intake (Jamshidi and Taylor, 2001)

possibly by stimulation of CB1 receptors within the VMH leading to a trans-synaptic excitation of PVN cells. The PVN receives projections from all hypothalamic regions (for review, see Swanson and Sawchenko, 1983), and appears to be a key region mediating cannabinoid–opioid interactions in food intake. We have recently shown that feeding induced by intra-PVN morphine can be attenuated by the systemic administration of SR 141716 (Verty et al., 2003). The impressive inhibition of morphine-induced Fos-IR by SR 141716 provides an interesting neural corroboration of these findings. It is interesting to note that in our previous work, naloxone reduced THC-induced Fos-IR in the DMH and VMH, but not in the PVN (Allen et al., 2003). This suggests that cannabinoid–opioid interactions in food intake are mediated via different hypothalamic regions. The demonstrations here that morphine increases body temperature agrees with previous studies (Chen et al., 1995). This hypothermic effect was prevented by SR 141716, which also prevented morphine-induced Fos-IR in preoptic thermoregulatory centres. The MnPO and MPO have both been implicated in the control of body temperature with activation occurring in these regions in response to hot environments (Maruyama et al., 2003; Scammell et al., 1993). Further, morphine administered into the MnPO and MPO produces hyperthermia (Trzcinka et al., 1977). A significant increase in morphine-induced Fos-IR was also observed in the EW, and SR 141716 significantly attenuated this effect. Morphine has been shown to affect pain perception (Pakulska et al., 2003), body temperature (Chen et al., 1995) and alcohol consumption (Vacca et al., 2002), all of which are effects that involve the EW (Innis et al., 1986; Bachtell et al., 2003; Bachtell et al., 2002). Interestingly, SR 141716 has been shown to reduce morphine-induced alcohol intake (Vacca et al., 2002), hyperthermia (Fig. 3, present study), and nociception (Reche et al., 1998). Taken together, these data invite the hypothesis that the EW may be an important region mediating the interactive effects of cannabinoid and opioid drugs. Finally, it should be noted that SR 141716 did not reverse morphine activation in all brain regions examined. In two notable cases, the CEA and BNST, the effect of the two drugs were additive with roughly twice as much Fos-IR being produced. This is reminiscent of a similar additive interaction reported by us in the CEA and BNST when THC and naloxone were administered together (Allen et al., 2003) and suggests that SR 141716 and morphine stimulate different CEA and BNST neuronal populations. The exact functional correlate of the additive effect of SR 141716 and morphine within the CEA and BNST is unclear. These regions are anatomically, neurochemically and cytoarchitecturally related (Alheid

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et al., 1995) and have been implicated in cannabinoidand opioid-induced anxiety (Davis, 1998; Onaivi et al., 1990). SR 141716 and morphine have been shown to possess anti-anxiety properties (Haller et al., 2002; Kang et al., 2000; Rodgers et al., 2003). Future studies could assess whether sub-threshold doses of SR 141716 and morphine can produce an additive anxiogenic effect in relevant animal models of anxiety. It is important to note that the CEA and BNST have also been implicated in cannabinoid- and opioid-induced analgesia (Cichewicz, 2004; Crown et al., 2000) and opioidinduced appetite modulation (Ciccocioppo et al., 2003; Park and Carr, 1998; Pomonis et al., 2000) suggesting that the additive effects of SR 141716 and morphine in these regions may have other functional correlates. It is interesting that in several brain regions examined, the combined administration of SR 141716 and morphine reduced Fos-IR below the levels observed with SR 141716 alone. The reasons for this are not clear. However, recent studies have suggested that cannabinoid receptor agonists may stimulate synthesis and release of endogenous opioids (Corchero et al., 1997; Kumar et al., 1990). Hence, the inhibition of morphine-induced Fos-IR by SR 141716 may represent a reduction in an endogenous cannabinoid tone leading to a suppression of opioid release and synthesis. High doses of cannabinoids produce hypothermia and hypolocomotion, and SR 141716 can attenuate both these effects. The present findings that SR 141716 reduced morphine-induced hyperthermia and hyperlocomotion may therefore seem counterintuitive, but it is noteworthy that cannabinoid effects on body temperature and locomotor activity are biphasic with low doses producing hyperthermia and hyperactivity (McGregor et al., 1996; Sulcova et al., 1998; Wenger and Moldrich, 2002). Our results suggest that the effects of opioids on body temperature and locomotor activity are critically dependent on increased endocannabinoid release. In conclusion, results from this study provide an interesting neural portrait of the way in which SR 141716 reverses morphine-induced behavioural and physiological effects. The reversal of morphine effects in the brain were surprisingly extensive and profound, and generate hypotheses as to mechanisms underlying SR 141716-induced prevention of opiate effects with respect to addiction, thermoregulation and appetite. It should, however, be noted that the present results alone are not sufficient to determine causal relationships between neural effects and behaviour. Rather, the present study serves to generate testable hypotheses regarding the interactive effects of morphine and SR 141716 on physiology and behaviour. It remains the goal of future studies to identify causal relationships through the use of other techniques, such as the direct

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microinjection of morphine and SR 141716 into discrete brain regions.

Acknowledgements This study was supported by a grant from the Australian Research Council to ISM and PEM. The authors would like to thank Sanofi–Synthelabo for their supply of SR 141716.

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