Destruction of the locus coeruleus decreases physical signs of opiate withdrawal

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1 128 Brain Research, 605 (1993) Elsevier Science Publishers B.V. BRES Destruction of the locus coeruleus decreases physical signs of opiate withdrawal R. Maldonado * and G.F. Koob Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA (USA) (Accepted 22 September 1992) Key words: Morphine dependence; Locus ceruleus; Electrolytical lesion; Methylnaloxonium; Intracerebroventricular The purpose of the present study was to investigate the role of the locus coeruleus in the development of opiate dependence. Two groups of rats each were subjected to either a electrolytic lesion of the locus coeruleus or a sham lesion. All animals were implanted with an intracerebroventricular (i.c.v.) cannula, and made physically dependent by subcutaneous insertion of two 75-mg morphine (base) pellets. Abstinence was precipitated by i.c.v, administration of methylnaloxonium (31-1,000 ng) 72 h after pellet implantation. Methylnaloxonium administered intracerebroventricularly induced a withdrawal syndrome characterized by the appearance of teeth chattering, mastication, rearing, wet dog shakes, jumping, piloerection, hyperactivity, ptosis and eye twitch. Withdrawal observed in the electrolytic lesion groups was less severe than in sham group. The presence of mastication, rearing, piloerection, hyperactivity, ptosis and eye twitch was significantly lower. These results support the hypothesis that the locus coeruleus has an important role in the expression of the physical signs of opiate dependence. INTRODUCTION Physical dependence on opiate drugs is characterized by a withdrawal syndrome following either the abrupt termination of morphine intake or precipitated by the administration of a narcotic antagonist. The neuroanatomical substrates implicated in the expression of the physical dependence have been explored using both intracerebral injections of opiate antagonists and central nervous system lesions in dependent animals. These techniques revealed the participation of multiple brain sites in the morphine physical withdrawal syndrome, including the periaqueductal gray matter 24'28, medial thalamus 38'4 '41, hypothalamus 6,16,2, amygdala 6'3s, substantia nigra 3, globus pallidus 38 and several mesencephalic and pontine structures as the nucleus raphe magnus and the locus coeruleus 4A1'24'28. The locus coeruleus represents the largest cluster of noradrenergic neurons in the brain 1 '14. This nucleus possesses a high density of opioid receptors, particularly of the mu and kappa subtype 37. Experimental evidence from neuroanatomical, neurophysiological and biochemical studies suggests functional interactions in the locus coeruleus between opioid and noradrenergic systems during opiate withdrawal. A noradrenergic hyperactivity in the locus coeruleus has been hypothesized to mediate the expression of some components of the physical morphine withdrawal ~'26. Supporting this hypothesis, an increase in the noradrenergic neuron firing rate in the locus coeruleus ~, an increase in the turnover of norepinephrine 25 and an increase in the levels of 3-methoxy-4-hydroxyphenyetyleneglycol 9 has been reported during naloxone-precipitated morphine withdrawal. Moreover, electrical stimulation of this nucleus simulates behavior induced by the opiate withdrawal state 17'33'35. Further, administration of clonidine, a drug that decreases noradrenergic activity, blocks behavior induced by both opiate withdrawal 5'13'33'36'39 and electrical stimulation of the locus coeruleus 33,35. Accordingly, a morphine withdrawal syndrome, associated with increased concentrations of 3-methoxy-4-hydroxyphenylethylene-glycol in the cerebral cortex, is precipitated by injecting naloxone in the locus coeruleus 1~. Correspondence: G.F. Koob, Neuropharmacology Department, Scripps Research Institute, North Torrey Pines Road, La Jolla, CA 92037, USA. Fax: (1) (619) * Present address: Unite de Pharmacochimie Mol6culaire, INSERM U 266, Facult6 de Pharmacie, Paris, France.

2 129 Recent work has established that the region of the locus coeruleus appears to play a critical role in the expression of morphine physical dependence. Indeed, the administration of the hydrophilic opiate antagonist methylnaloxonium (MN) in several brain structures (classically related to the development of the dependence) in chronically morphine-treated rats revealed that the locus coeruleus is the most sensitive site to precipitate withdrawal syndrome 28. However, in contrast to all these data, other reports appeared to indicate that hyperactivity in the noradrenergic pathway from the locus coeruleus to the forebrain played a limited role in the expression of the opiate abstinence. This was suggested by the fact that the 6-hydroxydopamine lesion of the dorsal noradrenergic bundle originating into the locus coeruleus failed to alter both the severity of morphine withdrawal and the clonidine is attenuating effect on withdrawal 5. However, dorsal noradrenergic lesions destroy only the noradrenergic fibers projecting to the forebrain and fail to eliminate projections to the cerebellum and other and other parts of the brainstem. The purpose of this study was therefore to investigate the effects induced by the electrolytic lesion of the locus coeruleus in the development of physical opiate dependence in order to clarify the role played by this nucleus. The morphine withdrawal syndrome evoked by acute i.c.v, injection of MN in dependent rats sham-operated or with a electrolytic lesion of the locus coeruleus was compared by measurements of the characteristics behavioral signs of physical abstinence. MATERIALS AND METHODS Animals and surgery Male Wistar rats (Charles River, Kingston, NY) ranging in weight from 260 to 280 g at the beginning of the experiment were used in this study. Rats were maintained in a light- and temperature-controlled environment. For surgery, animals were anesthetized with halothane and secured in a Kopf stereotaxic instrument. Surgery consisted of a two-stage procedure. Bilateral electrolytic lesions of the locus coeruleus were made by passing 1.0 ma DC current for 12 s between the tip of a 0.25-ram-diameter electrode (insulated to 0.5 mm of the tip) and a rectal anode. The coordinates, according to Paxinos and Watson 29, were: -0.8 mm from interaural bar _ mm lateral to midline, -7.3 mm from skull surface, incisor bar 3.3 mm below the ear bar. lesions were produced by lowering the electrode to within 1 mm of the lesion site (-6.3 mm). In a second stage, a unilateral stainless steel guide cannula (10 mm long, 23 gauge) was implanted 1 mm above the left lateral ventricle. The cannula was secured to the skull with stainless-steel screws and dental cement. The coordinates, for the final site of injection, were: -0.6 mm from bregma; -2.0 mm lateral to midline, -4.2 mm from skull surface, incisor bar 5.0 mm above ear bar 3. Behavioral experiments began 4 weeks after the surgery. Injection schedule Cannula guides were permanently kept open with wire stylets (10 mm long, 30 gauge) which were removed for intraventricular injections and then immediately replaced. Two ~1 of solution was injected over 70 s through a 30-gauge cannula which was removed after one additional minute (diffusion time). Rats were then immediately placed in the testing chambers. Induction of morphine dependence and withdrawal Rats were implanted subcutaneously (lower back) with two 75 mg morphine (base) pellets under anesthesia. Pellets were obtained from the National Institute on Drug Abuse (NIDA, USA). Methylnaloxonium (MN) hydrobromide (Boehringer-lngelheim) was dissolved in isotonic saline. The following doses were injected in order to precipitate the withdrawal syndrome: 32, 64, 125, 250, 500 and 1,000 ng (base) by rat. Animals were placed in test cages for a 30-min habituation period prior to injection of MN. One min after injection the withdrawal syndrome was observed for a period of 10 min. This procedure was repeated at 12-h intervals until a total of 6 injections were made. The injection doses of MN were randomly assigned across animals and days (latin square). Vehicle injections were performed 12 h before the first MN administration and 12 h after the last injection, in order to detect the appearance of any conditioned withdrawal. Two classes of signs were observed. The number of bouts of teeth chattering, mastication, rearing, wet dog shakes and jumping were simply counted. Ptosis, rhinorrhea, lacrimation, salivation, diarrhea and eye twitch were evaluated over 2-min periods with one point being given for the presence of each sign during each period. The TABLE I Two-way analysis of variance (ANOVA). The factors of variation were lesion (between subjects) and dose (within subjects). Simple main effects were analyzed when the main effect of lesion and/or interaction between lesion and dose were significant. Doses of methylnaloxonium (MN) are expressed in ng (base). ANOVA Simple main effects (interaction between lesion and dose) Lesion Dose Interaction Saline MN MN MN MN MN MN F2.I 7 p F7,4! p F p Teeth chattering 1.33 N.S < N.S Mastication 7.17 < < N.S. N.S. N.S. N.S. < 0.01 < 0.05 < 0.05 Rearing 7.74 < < < 0.01 N.S. N.S. < 0.05 N.S. < 0.05 N.S. Wet dog shakes 5.72 < < N.S. N.S. N.S. N.S. N.S. N.S. < Piloerection 2.32 N.S < < N.S. N.S. < 0.05 < 0.02 N.S. N.S. Locomotor activity 5.13 < < N.S. N.S. N.S. < 0.01 N.S. < 0.05 N.S. Ptosis 8.01 < < < 0.05 N.S. < 0.01 N.S. < N.S. N.S. Eye twitch 5.95 < < N.S. N.S. N.S. N.S. N.S. N.S. < Saline <0.001 N.S. <0.001 <0.05 <0.001 N.S. < 0.01 N.S. N.S. N.S. < N.S. < N.S.

3 130 number of periods showing the sign were then counted (maximum score: 5). Piloerection and locomotor activity were also evaluated over 2-min periods giving a value between 0 and 2 for each period. Here the values were added for the whole 10-min period (maximum score 10). Body weight was determined before and 60 min after MN administration. Statistical analysis Individual comparisons between the different group of animals were made using a two-way analysis of variance (ANOVA) with repeated measures. The factors of variation were lesion (between subjects) and dose (within subjects). Individual effects of each dose were analyzed using a Newman-Keuls comparisons following significant simple main effects when the main effect of lesion and/or the interaction between lesion and treatment were significant (Table I and Figs. 3 and 4). In each group of animals (sham, partial lesion and full lesion) individual dose effects were also analyzed using a Newman-Keuls comparisons following significant main effects of dose by ANOVA within subjects (Table II). The level of significance was P < Histological verification and biochemical analysis The cannula localization in the ventricle was verified by the ability of saline in the catheter to flow rapidly when the extremity of catheter wa: placed higher, as a consequence of negative pressure in the ventricle. After behavioral testing all animals were sacrificed by decapitation. The brain was removed and the cortex, hippocampus, hypothalamus, dorsal half of the cerebellum and dorsal medulla were dissected and stored at -70 C. Levels of noradrenaline were determined using high-pressure liquid chromatography with electrochemical detection 12. The hindbrain was dissected free from the rest of the brain and preserved in 10% formalin for verification of lesion placement. Coronal sections (50 ~m thick) of the brain were cut on a cryostat at -17 C and stained with Cresyl violet. Representative sections showing the electrolytic lesion of the locus coeruleus were then compared to standard stereotaxic plates 29. Lesion rats were divided in two groups, partial lesion and full lesion, based on the histological visualization (Fig. 1). Six animals were included in the full lesion group and seven animals in the partial lesion group. This classification was performed without knowledge of the individual behavioral response of each animal. RESULTS Biochemical depletion The electrolytical lesion of the locus coeruleus produced a significant decrease in brain norepinephrine in cortex, hippocampus and cerebellum. Decreases ob- TABLE II Summary table of one-way ANOVA within subjects and post-hoc comparisons performed in each group of animals (sham, partial lesion and full lesion). Significant differences from both of the two saline controls performed in each group are indicated, Newman - Keuls a posteriori test. Doses of methylnaloxonium (MN) are expressed in ng (base). Teeth chattering Mastication Rearing Wet dog shakes Piloerection Locomotor activity Ptosis Eye twitch ANOVA Newman - Keuls F P MN MN MN MN MN MN (7,41) = 5.78 < N.S. < 0.05 < 0.05 < 0.05 < 0.01 < 0.01 (7,49) = 4.97 < N.S. < 0.05 < 0.05 N.S. N.S. < 0.01 (7,41) = 3.14 < 0.02 N.S. N.S. N.S. N.S. N.S. < 0.05 (7,41) = < < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 (7,49) = 8.93 < N.S. < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 (7,41) = 6.56 < < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 (7,41) = 6.25 < N.S. < 0.05 N.S. N.S. N.S. < 0.01 (7,49)= 1.81 N.S. N.S. N.S. N.S. N.S. N.S. N.S. (7,41)= 1.73 N.S. N.S. N.S. N.S. N.S. N.S. N.S. (7,41) = 8.06 < N.S. N.S. N.S. N.S. < 0.05 < 0.01 (7,49) = 2.67 < 0.05 N.S. N.S. N.S. N.S. N.S. < 0.05 (7,41) = 6.62 < N.S. N.S. N.S. N.S. N.S. < 0.01 (7,41) = < N.S. < 0.01 < 0.01 < 0.05 < 0.01 < 0.01 (7,49) = 6.70 < N.S. N.S. N.S. N.S. < 0.05 < 0.01 (7,41) = 6.62 < N.S. N.S. N.S. N.S. N.S. < 0.01 (7,41) = < < 0.05 < 0.01 N.S. < 0.05 < 0.01 < 0.01 (7,49)= 3.37 < 0.01 N.S. N.S. N.S. N.S. N.S. N.S. (7,41) = 4.83 < N.S. N.S. N.S. N.S. N.S. < 0.01 (7,41) = < < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 (7,49)= 3.37 < 0.01 N.S. N.S. N.S. N.S. N.S. N.S. (7,41)= 2.13 N.S. N.S. N.S. N.S. N.S. N.S. N.S. (7,41) = < N.S. N.S. N.S. < 0.05 < 0.01 < 0.01 (7,49) = 6.60 < N.S. N.S. N.S. N.S. < 0.05 < 0.01 (7,41) = 3.49 < 0.01 N.S. N.S. N.S. N.S. N.S. < 0.01

4 131 " Fig. 1. Photomicrographsof coronal sections through the dorsal pons at the level of the maximum development of the locus coeruleus. Cresyl violet staining. A: sham lesioned brain. B: full lesion of the locus coeruleus. served in hypothalamus and spinal cord were not significant. Animals in the full lesion group showed a 59, 52, 14, 43 and 13% depletion in norepinephrine in the cortex, hippocampus, hypothalamus, cerebellum and spinal cord, respectively, relative to the control (shamoperated) rats. The partial lesion group exhibited a lower depletion in cortex (54%), hypothalamus (8%), cerebellum (23%) and spinal cord (5%), and a higher depletion in hippocampus (57%) (Fig. 2). Withdrawal syndrome evaluation MN administration into the lateral ventricle precipitated a withdrawal syndrome characterized by the presence of teeth chattering, mastication, rearing, wet dog shakes, jumping, piloerection, hyperactivity, ptosis and eye twitch. As it has been reported in previous studies 28, other signs of withdrawal (salivation, lacrimation, rhinorrhea and diarrhea) were not observed after central injection of MN in morphine dependent rats. Weight

5 132 loss observed was very slight and has also been reported in previous studies. Teeth chattering (Fig. 3) Two-way analysis of variance revealed a significant treatment effect for teeth chattering. There was no significant effect of lesion nor interaction between dose and lesion (Table I). One-way ANOVA within subjects revealed a significant dose effect in the sham, partial lesion and full lesion groups. In the sham group the incidence of teeth chattering was dose-dependent with a significant effect, revealed by post-hoc comparisons within subjects, starting at the dose of 62 ng. Partial lesion animals exhibited a more variable response with a significant effect at the doses of 62, 125 and 1,000 ng. Teeth chattering was significant in the full lesion group only after the administration of the highest dose (1,000 ng) of MN (Table II). Mastication (Fig. 3) Two-way ANOVA revealed a significant dose-effect and lesion-effect for mastication. There was no interac- tion between treatment and lesion. Simple main effect analysis revealed a significant overall difference between both lesion groups from sham group when MN was administered at the doses of 125, 250, 500 and 1,000 ng (Table I). Post-hoc comparisons between subjects (Newman-Keuls) indicated that these differences were significant in both partial and full lesion groups in the four cases (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect on the sham, partial lesion and full lesion groups. These effects were significant at all the doses used of MN in the three groups, with the exception of the dose of 31 ng in the partial lesion group (Newman-Keuls within subjects) (Table II). Rearing (Fig. 3) Two-way ANOVA revealed a significant dose-effect, lesion-effect and interaction between treatment and lesion in the case of mastication. Simple main effect analysis showed a significant overall difference between both lesion groups from sham group when MN was administered at the doses of 62, 250 and 1,000 ng G) 300 O 300 -~ 200 W = 200 O) OD I= loo C loo CORTEX HIPPOCAMPUS 4) (n 2000 Q :S M M 200,~ Q O) 0 t- O HYPOTHALAMUS CEREBELUM 3O0 O m I SHAM 200 1o0 D PART, A" LES,ON,- ~ 0 [~ FULL LESION SPINAL CORD Fig. 2. Norepinephrine levels (ng/g wet tissue) in several structures (cortex, hippocampus, hypothalamus, cerebellum and spinal cord) after electrolytic lesion of the locus coeruleus. Values are means _+ S.E.M. * P < 0.05; * * P < 0.01 vs. sham group (Newman-Keuls test).

6 133 30] A 40- [3 30 * "~ 20..Ic **. o o 10 ~ ~ " --,d METHYLNALOXONIUM.a,d METHYLNALOXONIUM ~ <.< < (ng) < r~ (ng) r~ 1 sham 1 sham [~ LC partial lesion [~ LC partial lesion 1-'] LC full lesion 1"] LC full lesion C D 20- [3 ~tt 30 ~ 2O o 10. tam < : METHYLNALOXONIUM.a < (ng) r~ METHYLNALOXONIUM.a < (ng) ~3 < 1 sham 1 sham [~ LC partial lesion [~ LC partial lesion ["-] LC full lesion [_7 LC full lesion Fig. 3. Effects of locus coeruleus electrolytic lesions on the different signs of morphine withdrawal syndrome (a: teeth chattering; b: mastication; c: rearing; d: wet dog shakes) after i.c.v, administration of methylnaloxonium. Abscissa represents the different doses: saline group placed at the left shows the control vehicle injection performed before the injections of methylnaloxonium; saline group placed at the right shows the control vehicle injection performed after methylnaloxonium injections. Ordinate expresses the values of the means_+ S.E.M. for each group. * P < 0.05, * P < 0.01 main overall lesion effect ANOVA with significant overall difference between both lesion groups and the sham group (Newman- Keuls test).

7 A 10- [3 8 ~ 6 ~ 6. r.r k.1 k.l "-1 METI-IYLNALOXONIUM.t METHYLNALOXONIUM.1 "< (ng) "< ~ < (ng) < 1 sham 1 sham [~ LC partial lesion [~ LC partial lesion rl LC full lesion [~] LC full lesion 4 D ~ o g~ METHYLNALOXONIUM "-1.< < (ng) r.r "500 i000-1 METHYLNALOXONIUM.<.< (ng) l sham l sham [~ LC partial lesion [] LC partial lesion 1--'] LC full lesion ~] LC full lesion Fig. 4. Effects of locus coeruleus electrolytic lesions on the different signs of morphine withdrawal syndrome (a: piloerection; b: locomotor activity; c: ptosis; d: eye twitch) after i.c.v, administration of methylnaloxonium. Abscissa represents the different doses: saline group placed at the left shows the control vehicle injection performed before the injections of methylnaloxonium; saline group placed at the right shows the control vehicle injection performed after methylnaloxonium injections. Ordinate expresses the values of the means+ S.E.M. for each group. * P < 0.05, ** P < 0.01 main overall lesion effect ANOVA with significant overall difference between both lesion groups and the sham group (Newman-Keuls test).

8 135 (Table I). Newman-Keuls (between subjects) comparisons indicated that these differences were significant in the full lesion group when MN was administered at the dose of 250 ng, and in both the partial lesion and full lesion groups after MN administration at the dose of 1,000 ng (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect on the sham group but not on the partial lesion or full lesion groups. This effect was significant only after the administration of MN at the doses of 62 and 1,000 ng (Newman-Keuls within subjects) (Table II). Wet dog shakes (Fig. 3) Two-way ANOVA revealed a significant dose-effect and lesion-effect for wet dog shakes. There was no interaction between dose and lesion. Simple main effect analysis revealed a significant overall difference between both lesion groups and the sham group when MN was administered at the doses of 500 and 1,000 ng (Table I). Post-hoc comparisons between subjects (Newman-Keuls) indicated that these differences were significant in both cases (500 and 1,000 ng of MN) in both partial and full lesion groups (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect in the sham, partial lesion and full lesion groups. Newman-Keuls within-subjects comparisons showed that these effects were significant in sham group after the administration of 500 and 1,000 ng of MN and in the partial and full lesion groups after the dose of 1,000 ng (Table II). Jumping (data not shown) Two-way ANOVA did not reveal any significant effect of dose, lesion nor interaction between dose and lesion in the case of jumping. Jumping behavior was only observed after the injection of the highest dose of MN (number of jumps in sham group= 1.83_ partial lesion group = 0.57 _+ 0.39, full lesion group = ). Piloerection (Fig. 4) Two-way ANOVA revealed a significant dose-effect, lesion-effect and interaction between dose and lesion in the case of piloerection. Simple main effect analysis showed a significant overall difference between both lesion groups and the sham group when MN was administered at the doses of 62, 125 and 1,000 ng (Table I). However, Newman-Keuls comparisons (between subjects) did not reveal any significant difference between these three groups of animals (Fig. 3). One-way ANOVA within subjects revealed a significant treatment effect in the sham, partial lesion and full E lesion groups. Post-hoc comparisons exhibited a significant effect in sham group at all the doses used of MN, except at the dose of 31 ng. Piloerection was significant in partial lesion group after the administration of MN at the doses of 500 and 1,000 ng, and in full lesion group only after the dose of 1,000 ng (Newman-Keuls within subjects) (Table II). Locomotor activity (Fig. 4) Two-way ANOVA revealed a significant dose-effect and lesion-effect for locomotor activity. There was no interaction between dose and lesion. Simple main effect analysis revealed a significant overall difference between both lesion groups and the sham group when MN was administered at the doses of 62 and 250 ng (Table I). Post-hoc comparisons between subjects (Newman-Keuls) indicated that these differences were significant (at both 62 and 250 ng of MN) only in full lesion groups (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect on the sham, partial lesion and full lesion groups. These effects were significant in sham group at all the doses of MN, with the exception of 125 ng, and in both the partial and full lesion groups after the administration of the highest dose of MN (1,000 ng) (Newman-Keuls within subjects) (Table II). Ptosis (Fig. 4) Two-way ANOVA revealed a significant treatmenteffect, lesion-effect and interaction between dose and lesion in the case of ptosis. Simple main effect analysis showed a significant overall difference between both lesion groups and the sham group when MN was administered at the doses of 31, 125 and 1,000 ng (Table I). Newman-Keuls (between-subject) comparisons indicated that these differences were significant in both partial lesion and full lesion groups when MN was administered at the doses of 125 and 1,000 ng (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect on the sham and partial lesion groups but not full lesion group. Post-hoc comparisons (Newman-Keuls within subjects) revealed a significant effect in the sham group at all the doses of MN, but did not reveal any effect in partial lesion group (Table II). Eye twitch (Fig. 4) Two-way ANOVA revealed a significant dose-effect and lesion-effect for eye twitch. There was no interaction between dose and lesion. Simple maineffect analysis revealed a significant overall difference between both lesion groups and the sham group when MN was administered at the doses of 500 and 1,000 ng (Table I). Post-hoc comparisons (Newman-Keuls between subjects) indicated that these differences were signifi-

9 136 cant in both cases (500 and 1,000 ng of MN) in both partial lesion and full lesion groups (Fig. 3). One-way ANOVA within subjects revealed a significant dose effect in the sham, partial lesion and full lesion groups. These effects were significant in sham (at the doses of 250, 500 and 1,000 ng) partial lesion (at the doses of 500 and 1,000 ng) and full lesion (at the dose of 1,000) groups (Newman-Keuls within subjects) (Table II). Weight loss (data not shown) Two-way ANOVA did not reveal any significant effect of treatment, lesion nor interaction between treatment and lesion in the case of weight loss. Weight losses observed were very small, and were similar in the three groups of animals (values for weight loss following the highest dose of MN: sham group = % _+ 0.22, partial lesion group = % , full lesion group = % ). DISCUSSION The present study provides evidence that the integrity of the locus coeruleus is important for the development and the expression of the central nervous system component of physical opiate dependence in rats. The withdrawal syndrome induced by i.c.v, administration of MN in animals with a partial or a full lesion of the locus coeruleus was less severe than the one induced in sham-operated animals. The presence of mastication, rearing, wet dog shakes, piloerection, hyperactivity, ptosis and eye twitch was significantly lower in the lesion animals. This lesion produced a significant decrease in brain norepinephrine levels in cortex, hippocampus and cerebellum and a slight decrease in hypothalamus and spinal cord. The variability in the expression of the morphine withdrawal syndrome induced with the present experimental protocol has been tested in a previous study 28. The administration of MN (500 rig) in morphine dependent rats was repeated each 12 h until a total of 6 injections was made in the same animal. The severity of the different signs of withdrawal observed after these 6 injections was similar. These data indicate that the protocol used in the present study to precipitate the withdrawal syndrome several times in the same animal shows a reliable response in the expression of the withdrawal over repeated testing. The present results support recent studies showing that the region of the locus coeruleus plays an important role in the expression of morphine physical dependence 28. Consistent with this hypothesis, previous reports implicated noradrenergic hyperactivity in the locus coeruleus in the expression of the morphine absti- nence 1'9'17'25'26. Furthermore, others 32 have observed a striking parallel between the time course of behavioral abstinence symptoms, the increase in the firing rates of the locus coeruleus noradrenergic neurons, and aspects of the biochemical alterations (increased level of G- proteins and up-regulation of camp system) that occur in the locus coeruleus during antagonist-precipitated morphine withdrawal. In a previous study, the 6-hydroxydopamine lesion of the dorsal noradrenergic bundle originating into the locus coeruleus failed to alter the severity of morphine withdrawal 5. The noradrenergic depletion obtained after these lesions was severe, but it was less widespread that the one observed after electrolytic lesion of the locus coeruleus. Indeed, the norepinephrine levels in the cerebellum were not modified after these 6-hydroxydopamine lesions 5'23 whereas in the present study, as well as in previous studies 23, electrolytic lesions of the locus coeruleus produced a significant depletion. Consequently, it is possible that some noradrenergic fibers, such as the pontine or descending projections of the locus coeruleus, are critically involved in the expression of the withdrawal syndrome and remain untouched after the 6-hydroxydopamine lesion of the dorsal noradrenergic bundle. Consistent with this hypothesis, other reports suggested that the spinal sympathetic preganglionic neurons may contribute to the expression of morphine abstinence ~s. However, in the present study only a very slight, non-significant, depletion of norepinephrine in the spinal cord was observed. The activation of the locus coeruleus during morphine withdrawal syndrome appears to be mediated in part by afferent inputs to the locus coeruleus. Recent data showed that the administration into the locus coeruleus of kynurenate, a general antagonist of excitatory amino acids, significantly reduced the activation of locus coeruleus cells induced by naloxone precipitated withdrawal, and suggests that an important part of locus coeruleus hyperactivity during opiate withdrawal is mediated by an excitatory amino acid input to the locus coeruleus. This input may derive from the nucleus paragigantocellularis, since lesions of this area greatly attenuated withdrawal-induced activation of the locus coeruleus 31. However induction of c-los is increased in the locus coeruleus during opiate withdrawal, but no increase is observed in the nucleus paragigantocellularis, suggesting that the withdrawal activation of the locus coeruleus is due largely to cell bodies and/or nerve terminals in the locus coeruleus that are supersensitive to a relatively constant input from the paragigantocellularis ~8. While earlier work showed that locus coeruleus neurons themselves do not show withdrawal-induced acti-

10 137 vation in isolated brain slices preparations taken from morphine-dependent rats 2'8, more recent studies have shown that some intrinsic withdrawal effects can be observed in the locus coeruleus itself 43. These results suggest that both within-system and between-system changes 22 may be involved in the development of the opiate-dependent state at the level of the locus coeruleus. Multiple brain regions, other than the locus coeruleus, have been implicated in the development and expression of the opiate dependence 4. The injection of opiate antagonists in various brain areas of morphine dependent animals results in a withdrawal syndrome 6,24,2s'38'4 '4t, and the metabolic activity of many central structures is altered during morphine abstinence 21'43. However, many of the brain structures which have been suggested to be important for the expression of morphine withdrawal behaviors receive a prominent noradrenergic input 34, e.g., periaqueductal grey matter 24'28, amygdala 6, hippocampus 19, serotonin neurons 7 and hypothalamus 2. The important role of locus coeruleus in the expression of the physical abstinence may explain the mild withdrawal syndrome observed after the chronic administration of inhibitors of the enkephalin catabolism 27, because there is an absence of endogenous opioid tone in the locus coeruleus. Indeed, the administration of inhibitors of the enkephalin degrading enzymes have no effect of their own on the properties of locus coeruleus neurons, suggesting that there is little tonic nor synaptically evoked release of endogenous opioid in the locus coeruleus 42. In conclusion, the decrease in the expression of opiate abstinence induced by the electrolytic lesion of the locus coeruleus represents further evidence supporting an important role for the locus coeruleus in the opiate dependence. Further studies will be necessary to identify the precise afferent and efferent projections of the locus coeruleus implicated in the expression of the opiate withdrawal syndrome. Acknowledgements. We would like to thank Diana Smith and Richard Schroeder for their excellent technical assistance. This work was supported by NIDA Grant DA We thank Molecular and Experimental Medicine's Word processing Center for their help in preparing the manuscript. REFERENCES 1 Aghajanian, G.K., Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal response by clonidine, Nature, 276 (1978) Andrade, R., Vandermaelen, C.P. and Aghajanian, G.K., Morphine tolerance and dependence in the locus coeruleus: single-cell studies in brain slices, Eur. J. 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