Psychopharmacology (2003) 170:80–88 DOI 10.1007/s00213-003-1504-0
Takeshi Watanabe · Takayuki Nakagawa · Rie Yamamoto · Akifumi Maeda · Masabumi Minami · Masamichi Satoh
Involvement of noradrenergic system within the central nucleus of the amygdala in naloxone-precipitated morphine withdrawal-induced conditioned place aversion in rats Received: 3 November 2002 / Accepted: 2 April 2003 / Published online: 27 May 2003 Springer-Verlag 2003
Abstract Rationale: We previously reported that the central nucleus of the amygdala (CeA) plays a crucial role in the negative affective, rather than somatic, component of morphine withdrawal. However, numerous studies have reported that the central ascending noradrenergic system is implicated in morphine withdrawal syndrome, although the roles of the noradrenergic system within the CeA in the negative affective component remains less clear. Objectives: The possible role of the noradrenergic system within the CeA in the negative affective component of morphine withdrawal was investigated. Methods: The extracellular noradrenaline level within the CeA during naloxone-precipitated morphine withdrawal was measured using an in vivo microdialysis experiment on unanesthetized and freely moving rats. The effects of microinjection of b-adrenoceptor antagonists into the bilateral CeA on the naloxone-precipitated morphine withdrawal-induced conditioned place aversion (CPA) and somatic signs were examined. Results: The extracellular noradrenaline level within the CeA was transiently elevated during morphine withdrawal. Intra-CeA injections of b-adrenoceptor antagonists propranolol (30 nmol per side) and timolol (10 nmol per side) significantly attenuated the morphine withdrawal-induced CPA. Similarly, b1-antagonist atenolol (30 nmol per side) or b2antagonist butoxamine (30 nmol per side) significantly attenuated the CPA. In contrast, they did not affect morphine withdrawal-induced somatic signs, except for propranolol. Conclusion: These results suggest that the activation of the noradrenergic system within the CeA contributes to naloxone-precipitated morphine withdrawal-induced CPA, rather than somatic signs, through b1and b2-adrenoceptors. T. Watanabe · T. Nakagawa · R. Yamamoto · A. Maeda · M. Minami · M. Satoh ()) Department of Molecular Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606–8501, Japan e-mail: [email protected]
Keywords Morphine withdrawal · Conditioned place aversion · Central nucleus of amygdala · Noradrenaline · b-Adrenoceptor
Introduction Chronic use of opiates such as morphine is well known to lead to physical and psychological dependence, characterized by the expression of a withdrawal syndrome upon cessation of drug administration. The withdrawal syndrome includes both somatic and affective components. In animals, morphine withdrawal produces various characteristic somatic signs, as well as disruption of schedulecontrolled operant responses for food (Koob et al. 1989), elevation of intracranial self-stimulation thresholds (Schaefer and Michael 1986) and aversively motivated avoidance behavior from the environment previously associated with morphine withdrawal (conditioned place aversion; CPA) (Mucha 1987; Stinus et al. 1990). These behavioral changes are thought to reflect the negative affective component of morphine withdrawal, which might contribute to aversively motivated drug seeking. A body of evidence provides support for the involvement of the central noradrenergic (NA) system in the expression of morphine withdrawal syndrome (for review, see Maldonado 1997). It has been reported that systemic administration of b-adrenoceptor antagonists or a2-agonists attenuated the expression of morphine withdrawal somatic signs (Tseng et al. 1975; van der Laan 1985), as well as conditioned aversions (Harris and Astone-Jones 1993; Kosten 1994; Nader and van der Kooy 1996). Recently, it has been reported that NA inputs to the bed nucleus of the stria terminalis (BNST) via the ventral NA bundle from the A1/A2 cell groups of the caudal medulla are involved in morphine withdrawal-induced CPA (Delfs et al. 2000). These findings suggest that activation of the central ascending NA system could contribute to the negative affective component of morphine withdrawal.
The amygdala is a forebrain structure composed of several distinct subnuclei including the central (CeA), lateral and basolateral nuclei, and is thought to be a key neural substrate underlying emotional responses such as anxiety and fear (Gallagher and Chiba 1996). It has been reported that microinjection of opioid antagonists into the amygdala produced morphine withdrawal-induced CPA, as well as several somatic signs (Stinus et al. 1990; Maldonado et al. 1992). Furthermore, we recently reported that the CeA contributes to the morphine withdrawal-induced CPA (Watanabe et al. 2002a, 2002b). The CeA is known to be highly innervated by NA afferents (Asan 1998), and the ascending NA systems to the CeA are reported to be involved in conditioned fear and modulating affective responses to stress (Williams et al. 1998; Clayton and Williams 2000; Khoshbouei et al. 2002). However, the involvement of the NA systems within the CeA in the negative affective component of morphine withdrawal remains unclear. Therefore, in this study, we examined (i) the extracellular NA level within the CeA and (ii) the effects of microinjection of badrenoceptor antagonists into the bilateral CeA on the morphine withdrawal-induced CPA, as well as somatic signs. Our data suggest a critical role of the NA system within the CeA in the negative affective, rather than somatic, component of morphine withdrawal.
Materials and methods
microdialysis guide cannula (o.d., 0.5 mm, AG-8; Eicom, Kyoto, Japan) above the CeA at coordinates of 1.8 mm posterior to bregma, 4.0 mm lateral to the midline, 8.0 mm below the surface of the skull, according to the atlas of Paxinos and Watson (1998). The guide cannulae were held firmly in place by dental acrylic cement and secured with a dummy cannula. After the surgery, the rats were individually returned to the cages. One day after the implantation of the guide cannula (day 4), microdialysis experiments were performed in unanesthetized and freely moving rats. Ringer-primed microdialysis probes (dialysis membrane: length 1.0 mm, o.d. 0.22 mm, A-I-8-01; Eicom) were inserted through the guide cannula and continuously perfused with normal Ringer’s solution (Na+ 147 mM, K+ 4 mM, Ca2+ 2.3 mM, Cl 155.6 mM) at a constant flow rate of 1 ml/min. The rats were then placed in a Plexiglas chamber (303035 cm: width length height). For a stabilization period, the dialysates obtained over the first 1 h were discarded, and collected for 60 min before and for 80 min after i.p. injection of naloxone (0.3 mg/kg). Perfused dialysates of 20 ml were collected on ice every 20 min, and injected into the HPLC-ECD system. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.0) containing 400 mg/l hexadecyltrimethylammonium bromide, 50 mg/l ethylene diamine tetraacetic acid (EDTA) acid and 5% methanol delivered at a constant flow rate of 0.23 ml/min using a high-performance liquid chromatography (HPLC) pump (EP300). NA was separated on a reverse-phase column (Eicompak CA5ODS, 2.1 mm i.d. 150 mm), which was detected by an electrochemical detector (ECD-300) with a working electrode set at +450 mV versus a Ag/AgCl reference electrode. The temperature of the column was maintained at 25C by oven (ATC-300). Under these experimental conditions, the retention time for NA was 8.5– 9.0 min. Chromatogram peaks were analyzed by means of the PowerChrom data recording system (EPC-300) with a computer. NA was quantified by reference to a calibration curve ranging from 1 fmol to 100 fmol. Detection limit for NA was approximately 1 fmol per sample.
Microinjection of drugs into the CeA
A total of 242 male Sprague-Dawley rats initially weighing 200– 230 g (6 weeks old) was used. They were kept at a constant ambient temperature of 24€1C under a 12-h/12-h light/dark cycle with free access to food and water. After arrival, the rats were individually housed in plastic cages with wood-chip bedding for at least 1 day until surgery. The experiments were conducted in accordance with the ethical guidelines of Kyoto University animal experimentation committee, and the guidelines of the Japanese Pharmacological Society.
Under sodium pentobarbital anesthesia (50 mg/kg, i.p.), each rat was bilaterally implanted with guide cannulas (o.d., 0.5 mm; i.d., 0.22 mm) above the CeA at coordinates of 1.8 mm posterior to bregma, 4.0 mm lateral to the midline, and 3.0 mm below the surface of the skull. The guide cannulae were held firmly in place by dental acrylic cement. After surgery, the rats were individually returned to their cages and left to recover for at least 1 week before the experiments. Drugs were microinjected into the CeA 10 min before i.p. injection of naloxone. Stainless-steel injection cannulae (33 Gauge) fitted to a 100-ml Hamilton syringe by PE-8 tubing were inserted just 8.0 mm below the surface of the skull when attached to the guide cannula. Drugs or vehicle (PBS) were bilaterally administered in a volume of 1 ml per side over 2 min by a microinfusion pump. The injection cannula was left in place for an additional 2 min to prevent backflow of drugs.
Materials Morphine hydrochloride was purchased from Takeda Chemical Industries (Osaka, Japan). Morphine pellets each containing 75 mg morphine base were prepared according to the method of Gibson and Tingstad (1970). Timolol, butoxamine, and naloxone hydrochloride were purchased from Sigma (St. Louis, USA). (€)Atenolol was purchased from RBI (Berkeley, USA). dl-Propranolol was purchased from Nacalai Tesque (Kyoto, Japan). Naloxone hydrochloride was dissolved in saline and other drugs were dissolved in phosphate-buffered saline (PBS). These drug solutions were freshly made each day. All other reagents were of the best available quality from commercial sources. In vivo microdialysis procedure and measurement of the NA level In vivo microdialysis experiment was performed in 15 rats. On the first day (day 1), under light ether anesthesia, the rats had a morphine pellet or a placebo pellet implanted in the back of the neck. After 48 h (day 3), under sodium pentobarbital anesthesia (50 mg/kg, i.p.), each rat was unilaterally implanted with a
CPA paradigm Apparatus CPA was conducted in 173 rats as previously described (Watanabe et al. 2002a, 2002b). A place conditioning apparatus, consisting of a shuttle box (306030 cm: width length height), divided into two equal-sized compartments with distinctive visual color and floor texture, was used to measure naloxone-precipitated morphine withdrawal-induced aversive behavior. One compartment was black with a smooth floor; the other was white with a textured floor. The infrared beam sensors were positioned on each cover, and the time spent in each compartment during a period of 15 min (900 s) was measured automatically in a blind fashion using a computer system (KN-80; Natsume Seisakusho, Tokyo, Japan). The apparatus was enclosed by a sound- and light-attenuated box
82 under conditions of dim illumination (40 lux) and masking white noise. Preconditioning session The experimental process consisted of three distinct sessions: preconditioning session, conditioning session, and test session. On the first day (day 1), under light ether anesthesia, the rats had either a morphine or placebo pellet implanted in the back of the neck. The implanted pellet was left in place until the test session. On day 2, the partition separating the two compartments was raised 12 cm above the floor, and a neutral platform (5212 cm) was inserted along the seam separating the compartments. The rats were individually placed on the platform and stepped down to the horizontal floor. The rats were allowed to freely explore the two compartments for 900 s and acclimatized to the apparatus. On day 3 (preconditioning session), the same trial was performed, and the time spent in each compartment for 900 s was measured. There were no significant differences between time spent in the black compartment with a smooth floor [441€13.8 s, n=110 (final numbers used in the test)] and the white compartment with a textured floor [458€13.8 s, n=110 (final numbers used in the test)], indicating that there was no preference bias before conditioning in the apparatus itself. Nevertheless, in this study, we selected the bias-like protocol in order to nullify each rat’s initial preference, as previously discussed (for review, see Tzschentke 1998). Thus, we determined the preferred compartment for each rat by establishing the compartment in which the rats spent more than 50% of the total time (i.e., 450 s). Rats that spent more than 80% of the time (i.e., 720 s) in one side on day 3 (19 animals) or showed a difference of more than 200 s between the time spent on day 2 and that on day 3 in one side (19 animals) were eliminated. Conditioning session On day 4, place conditioning was performed as follows: in the morning, each rat was intraperitoneally (i.p.) injected with saline (1 ml/kg) and then confined to its non-preferred compartment for 1 h. After at least 3 h, in the afternoon, the rat was i.p. injected with naloxone (0.3 mg/kg) and then confined to its preferred compartment for 1 h. The dose of naloxone is reported to be sufficient to induce CPA by morphine withdrawal (Frenois et al. 2002). Drugs were microinjected into the CeA 10 min before i.p. injection of naloxone. Test session On day 5, the partition separating the two compartments was raised, and the rats were individually placed on the neutral platform and allowed to freely explore the two compartments. The time spent in each compartment for 900 s was then measured. The CPA scores represent the time spent in the naloxone-paired compartment in the test session minus the time spent in the same compartment in the preconditioning session.
signs (Frenois et al. 2002). Then, the rats were immediately returned to the cylinder and behavior was observed for 1 h. Body weight was measured just before and 1 h after naloxone injection, and is presented as the means €SEM of percentage for body weight loss. The numbers of occurrences of rearing, stretching, wet-dog shaking, teeth chattering, paw shaking, head shaking, and backwards walking were counted, and are presented as means €SEM of total numbers. The occurrence of diarrhea, ptosis and rhinorrhea was checked and are presented as the number of rats showing positive signs over the total number of rats tested. Histology After all tests, histological analyses were performed. The rats were sacrificed by decapitation and the brain was rapidly removed and frozen in powdered dry ice. In the case of the microinjection experiments, to verify the placement of each injection cannula, 1 ml of a solution of cresyl violet including thionine in saline was infused through a similar injection cannula before the rats were sacrificed. Then, coronal sections (50 mm) including the amygdala (anterior posterior 1.8 to 2.8 mm from bregma) were prepared on a cryostat, thaw-mounted onto gelatin-coated slides and stored at 80C until use. The slices were stained with cresyl violet and each section was examined under the microscope (400). Statistical analyses In the case of the in vivo microdialysis experiment, the differences in the NA levels between naive and morphine-dependent rats were analyzed using two-way analysis of variance (ANOVA) followed by the Bonferroni’s post-hoc test. In the case of CPA experiments, statistical significance was calculated using one-way ANOVA followed by the Student-Newman-Keuls post-hoc test. In the case of morphine withdrawal somatic signs, because of considerable variability in the data of body weight loss and counted signs, the non-parametric statistic (Mann-Whitney U-test) was used. The data of checked signs were compared using the Chi-square test. Differences with P