Ž .Brain Research 854 2000 85–92www.elsevier.comrlocaterbres

Research report

Microinjections of an opiate receptor antagonist into the bed nucleus of thestria terminalis suppress heroin self-administration in dependent rats

John R. Walker ) ,1, Serge H. Ahmed 1, K. Noelle Gracy, George F. KoobDiÕision of Psychopharmacology, Department of Neuropharmacology, The Scripps Research Institute, 10550 North Torrey Pines Road, CVN-7 La Jolla,

CA 92037 USA

Accepted 26 October 1999

Abstract

Recent anatomical evidence suggests that the shell of the nucleus accumbens, the bed nucleus of the stria terminalis, and the centralnucleus of the amygdala, together referred to as the extended amygdala, may play a role in opiate dependence. The bed nucleus of thestria terminalis and the shell of the nucleus accumbens have a moderately high density of opiate receptors, which allows for manipulationof opiate neurotransmission with receptor antagonists. The goal of this study was to determine the role these regions play in opiatereinforcement, and whether dependence alters the reinforcing effects of opiates by examining the effect of local administration of theopiate receptor antagonist methylnaloxonium on heroin self-administration in dependent and nondependent rats. Previous studies revealedthat blockade of the reinforcing effects of opiates with systemic administration of opiate receptor antagonists results in an increase inheroin self-administration in nondependent rats, and a greater increase in dependent rats. In the present study, methylnaloxoniumdose-dependently suppressed heroin intake when injected into the bed nucleus of the stria terminalis and shell of the nucleus accumbensof dependent rats, and had no effect in nondependent rats. These results demonstrate that opiate receptors in parts of the extendedamygdala may be responsible for the reinforcing effects of opiates in dependent animals and suggest that activity in this system may berecruited during the development of dependence. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Bed nucleus of the stria terminalis; Nucleus accumbens; Methylnaloxonium; Self-administration; Dependence; Heroin

1. Introduction

In order to understand the biological basis of drugaddiction, an understanding of how the brain is altered in

w xaddicted versus nonaddicted states is needed 20,27 . Drugaddiction or substance dependence is characterized by acompulsion to take drugs, a loss of control of drug intake,and emergence of a negative affective state upon drug

w xabstinence 18,24 . It is clear that there is a differencebetween occasional use and abuse of drugs, yet the respon-sible neurobiological mechanisms are still unknown.

Of recent interest in drug dependence is the possibleinvolvement of the extended amygdala which intercon-nects limbic brain structures and structures involved in

w xmotor function 3,23,25 . The extended amygdala is com-posed of several basal forebrain regions that share similar

) Corresponding author. Fax: q1-858-812-1584; e-mail:john_r.walker@nifg.novartis.com

1 Both authors contributed equally to this work.

morphology, immunoreactivity, and connectivity. It is es-Ž .sentially continuous rostral-caudally from the medial shell

Ž .portion of the nucleus accumbens AcbSh , through theŽ .bed nucleus of the stria terminalis BST , to the central

Ž .nucleus of the amygdala ACE . Afferent inputs to theseregions include various cortical structures, the hippocam-pal formation, basolateral amygdala, the ventral tegmental

Ž .area VTA , thalamic nuclei, lateral hypothalamus, andlateral septum. Efferent connections include the sublenticu-lar ventral pallidum, medial VTA, reticular formation,

w xcentral grey, and the lateral hypothalamus 2,9,14,22 . Theextended amygdala therefore interconnects various brainstructures hypothesized to be involved in the reinforcing

w xeffects of abused drugs 6,19,37 .Very low systemic doses of the opiate receptor antago-

nist naloxone block heroin reinforcement to a greaterdegree in dependent than nondependent subjects, suggest-ing that the reinforcing effects of heroin become sensitized

w xduring dependence 7 . Of interest would be to determinewhich brain regions are responsible for this differentialsensitivity to opiates brought about by dependence.

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02288-X

( )J.R. Walker et al.rBrain Research 854 2000 85–9286

The two goals of this study were to determine ifportions of the extended amygdala are involved in thereinforcing effects of opiates in nondependent rats, and ifthese brain regions are involved in the increased sensitivityto opiate receptor antagonists in dependent rats. The BSTand the AcbSh have a moderate to high density of opiatereceptors, but it is not known if these discrete brain regions

w xare involved in the reinforcing effects of heroin 5,15,26 .Local injection of the quaternary derivative of the opiate

Ž .receptor antagonist naloxone, methylnaloxonium MN , isan effective means to map brain sites involved in the

w xreinforcing effects of drugs of abuse 8,35 . Therefore, MNwas microinjected into the BST and AcbSh of nondepen-dent and dependent rats self-administering heroin to deter-mine if heroin reinforcement can be altered and if opiatedependence alters sensitivity to opiate receptor blockade.

2. Materials and methods

2.1. Animals

ŽSixty male Wistar rats 220–250 g at the start of the.experiments, up to 500 g at the end were housed in groups

of two per cage. Wistar rats were bred at the BeckmanLaboratories of The Scripps Research Institute from a

Žstock originally derived from Charles River Kingston,.NY . Rats were bred using a circular pair random system

of breeding in order to maintain genetic heterogeneity, andnew breeders were obtained from Charles River as deter-mined by our internal Genetics Advisory Board. All exper-iments were performed in accordance with the NationalInstitutes of Health Guide for the Care and Use of Labora-tory Animals. In addition, all efforts were made to mini-mize animal suffering, and a within-subjects design forMN doses was utilized to reduce the number of subjects.

ŽRats were maintained on a 12:12 light:dark cycle lights.on at 1000 h , and all experiments were performed duringŽ .their active dark phase. Rats were trained to lever press

for food on a FR 1 TO 20 s schedule prior to implantationof jugular catheters and cannulae.

2.2. Surgery

Catheter surgeries were performed according to meth-w xods described previously 1 . During intravenous catheter

surgeries, rats were anesthetized with an oxygenrhalothaneŽ .mixture 1.5% halothane , then allowed to recover for 7

days. During recovery they were given daily intravenousŽ .0.1 ml of a 100 mgrml solution administrations of the

w Žantibiotic Timentin SmithKline Beecham Pharmaceuti-.cals, Philadelphia, PA . After recovery, baseline heroin

self-administration was assessed. When a catheter becamenon-functional during the course of the experiment, it wasassumed that the tubing was broken and a new catheterwas implanted into the opposite jugular vein.

For cannula implantation surgeries, rats were anes-Žthetized with an oxygenrhalothane mixture 1.5%

.halothane . Bilateral 23 G guide cannulae were positionedŽ . Ž .3.0 mm AcbSh or 2.5 mm BST above the intended

injection site using a Kopf stereotaxic instrument. Aftersurgery, rats were allowed to recover for 7 days. Stainless

Ž .steel stylets o.d. 0.011 in. were inserted into the guidecannulae to maintain patency, and extended 2.5 mm be-yond the end of the cannulae for BST implantations, or 3.0

Žmm for the AcbSh implantations. For the BST dorso-.lateral portion , the intended target was 0.35 mm posterior

to Bregma, 1.5 mm lateral to the mid-sagittal suture, and6.5 mm ventral from the skull surface according to a

w xprevious study 11 . For AcbSh implantations, coordinatesfor the intended target site were: 1.4 mm anterior toBregma, 0.7 mm lateral to the mid-sagittal suture, and 8.0mm ventral to the skull surface according to the atlas of

w xPaxinos and Watson 28 .

2.3. Self-administration

Self-administration sessions consisted of a FR 1 TO 20s schedule, and a heroin training dose of 10 mgrinfusionwas used in all of the experiments. Drug solutions weredelivered in a volume of 0.1 ml over a period of 4 s. Therewere no priming injections given during self-administra-tion sessions. After each infusion, a time-out period of 20 sbegan during which the lever was inactive and a lightabove the lever remained lit. Sessions lasted 1 h per day, 5days per week.

ŽOnce responding was stable "10% of the mean for.three consecutive sessions , rats with approximately equal

mean numbers of infusions per session were divided intomorphine and placebo groups. Rats were subcutaneouslyimplanted under halothane anesthesia with two 75 mg

w xmorphine or placebo pellets, wrapped in nylon 21 . Thisprocedure of morphine pellet implantation results in toler-ance and dependence, which lasts for at least 12 days after

w xpellet implantation 12 . Heroin self-administration ses-sions were continued the day following pellet implanta-

Ž .tions. Initial stability once pellets were implanted inŽresponding "10% of the mean for two consecutive ses-

.sions was achieved usually after three sessions. Intracra-nial injections were administered only when respondingreturned to the baseline value before the previous injection.This usually occurred two days after each injection period.Old pellets were removed and replaced with new pelletsevery 12 days.

2.4. Drug microinjections

ŽMethylnaloxonium hydrochloride Organon, Amster-.dam, The Netherlands was dissolved in sterile saline, and

injected based on the molecular weight of the free base.Vehicle injections were 0.9% saline. Intracranial injectorsconsisted of 30 Ga stainless steel tubing connected to 23

( )J.R. Walker et al.rBrain Research 854 2000 85–92 87

Ga stainless steel tubing which allowed the 30 Ga tubingŽ . Ž .to extend 2.5 mm for BST or 3.0 mm AcbSh beyond

the end of the guide cannulae. Intracranial injections weregiven in a volume of 0.5 ml per side over a period of 90 susing a Harvard pump apparatus, and the injectors wereleft in place for an additional 90 s to allow for diffusion ofthe fluid from the injector tip. Stylets were reinserted, ratswere immediately placed into the operant chamber, andsessions were started. The same MN dose was given to allrats on a single day. Saline was given on the first injectionday, followed by MN doses in descending order, thenanother saline injection. A final injection of the mosteffective dose then was given to determine if there is achange in sensitivity with repeated MN injections. MNdoses tested varied depending on the injection site. For theBST, doses tested were 0, 2, 8, 32, and 128 ng. For theAcbSh, doses tested were 0, 8, 32, and 128 ng.

The rationale for choosing the MN dose to be injectedinto each brain region was based on findings from a

w xprevious self-administration study 35 . The minimum ef-Ž .fective dose injected into the nucleus accumbens Acb

which blocked heroin self-administration in the Vaccarinostudy was 125 ng. Since an increase in sensitivity to MNwas expected with dependence, a similar dose was chosenas the highest dose for this study. The MN doses chosen

Fig. 1. MN microinjections into the bed nucleus of the stria terminalisŽ . ŽBST suppress heroin self-administration in dependent morphine-pel-

. Ž . Ž .leted but not nondependent placebo-pelleted rats. A The number ofŽ .infusions of heroin 10 mgrinfusion is represented for the entire 1-h

self-administration session. U P -0.05, UU P -0.01, different from vehi-Ž . Žcle. B Reinforcer delivery record for a single morphine-pelleted rat rat

.aAV21 injected with different doses of MN into the BST. The horizon-tal axis denotes time over the 1 h heroin self-administration session. Eachvertical line represents a self-infusion of heroin.

Fig. 2. MN microinjections into the shell of the nucleus accumbensŽ . ŽAcbSh suppress heroin self-administration in dependent morphine-pel-

. Ž . Ž .leted but not nondependent placebo-pelleted rats. A The number ofŽ .infusions of heroin 10 mgrinfusion is represented for the entire 1-h

U Ž .heroin self-administration session. P -0.05 different from vehicle. BŽ .Reinforcer delivery record for a morphine-pelleted rat rat a337 injected

with different doses of MN into the AcbSh. The horizontal axis denotestime over the 1 h heroin self-administration session. Each vertical linerepresents a self-infusion of heroin.

for injection into the BST were the same as for the Acb,except for the inclusion of a 2 ng MN dose injected intothe BST.

2.5. Histology

At the end of the experiment, rats were deeply anes-thetized with 100 mgrkg sodium pentobarbitalŽ w .Nembutal , Abbott Laboratories, Chicago, IL , and per-fused with phosphate-buffered formalin. Brains were ex-tracted, post-fixed in buffered formalin, placed into abuffered 30% sucrose solution for at least 2 days, and cuton a cryostat in 40 mm coronal sections. Sections wereadhered to glass slides, stained with 0.5% Cresyl violet,cover-slipped, and examined under a microscope to deter-mine location of injector tips.

2.6. Statistical analyses

Twenty-nine rats were used for injections of MN intothe AcbSh. Three rats were used exclusively as a guide forcannulae placement. Six rats chewed excessively on thespring-housed tubing connecting the drug syringe to thecatheter, and were eliminated from the study. Two rats

( )J.R. Walker et al.rBrain Research 854 2000 85–9288

( )J.R. Walker et al.rBrain Research 854 2000 85–92 89

. .Fig. 3. Injector tip locations for MN microinjections into the A bed nucleus of the stria terminalis and B shell portion of the nucleus accumbens. NumbersŽ . w xrepresent anterior–posterior levels from Bregma mm according to the atlas of Paxinos and Watson 28 . Relevant abbreviations: AcbC, core portion of

the nucleus accumbens; AcbSh, shell portion of the nucleus accumbens; BSTLD, BST lateral division, dorsal part; BSTLJ, BST lateral division, juxtacaps;BSTLP, BST lateral division, posterior; BSTLV, BST lateral division, ventral; BSTMA, BST medial division, anterior; BSTMV, BST medial division,ventral; mfba, medial forebrain bundle, ‘a’ component; VDB, vertical limb diagonal band. Ovals represent location of injector tips in intended region.Triangles represent location of injector tips outside of the intended region and were not included in statistical analyses.

became ill and were eliminated from the study. One ratlost both its original catheter and a second replacementcatheter before all MN doses could be completed, and its

data was not included in the analysis. Seventeen rats wereinjected with MN in the BST. The cannulae for one ratwere found to be too ventral upon histological analysis,

( )J.R. Walker et al.rBrain Research 854 2000 85–9290

and the data for this rat was eliminated from statisticalanalyses.

The effect of central injections of MN in rats implantedwith morphine or placebo pellets was analyzed by two-way

Ž .analysis of variance ANOVA with one between-subjectsŽfactor experimental groups: placebo vs. morphine-pel-

.leted and with repeated measures on the second factorŽ .dose of MN in ng . Individual means comparisons were

w xconducted with the Newman–Keuls a posteriori test 36 .

2.7. Materials

Ž .The National Institute on Drug Abuse Bethesda, MDŽ .provided heroin 3,6-diacetylmorphine hydrochloride .

Methylnaloxonium hydrochloride was kindly provided byŽ .Organon Amsterdam, The Netherlands .

3. Results

3.1. Bed nucleus of the stria terminalis microinjections

Morphine- and placebo-pelleted rats with cannulaeaimed at the BST did not differ in their baseline heroin

Ž .intake. Before the first MN injection, mean "S.E.M.Ž .values were 15 "1.7 infusions per hour for the mor-

Ž .phine-pelleted rats and 16.4 "1.6 for the placebo-pel-leted rats. There was still no difference in baseline heroin

Ž .intake at the end of the experiment: 15.3 "1.8 for mor-Ž .phine-pelleted and 15.8 "2.2 for placebo-pelleted rats.

MN doses tested were 0, 2, 8, 32, and 128 ng. MNdepressed heroin intake at low doses. A group X dose

Ž w x .interaction occurred F 4,60 s3.1, P-0.05 , with a sig-Ž w x .nificant effect of MN dose F 4,60 s7.9, P-0.001 for

the morphine-pelleted group. Individual mean comparisonsrevealed significant differences between vehicle and MN

Ž . Ž .doses of 8 ng P-0.05 , 32 ng, and 128 ng P ’s-0.01Ž .for the morphine-pelleted rats Fig. 1 . MN injections had

no effect on responding in the placebo-pelleted ratsŽ w x .F 4,60 s1.0, NS . Injections given to either the placeboor morphine-pelleted groups had no obvious effect on therats’ behavior observed while handling.

3.2. Nucleus accumbens shell microinjections

Morphine-pelleted and placebo-pelleted rats implantedwith cannulae aimed at AcbSh did not differ in theirbaseline heroin intake. Before the first vehicle injection,

Ž . Ž .mean "S.E.M. values were 15.1 "2.9 infusions perŽ .hour for the morphine-pelleted rats and 12.0 "1.6 for

the placebo-pelleted rats. There was still no difference inbaseline heroin intake at the end of the experiment: 17.3Ž . Ž ."2.5 for morphine-pelleted and 17.2 "1.3 for

Ž .placebo-pelleted rats. A significant group pellet conditionŽ w xX MN dose interaction was observed F 3,45 s4.12,

. Ž w xP-0.05 , with a significant effect of dose F 3,45 s10.6,

.P-0.001 for the morphine-pelleted group. Individualmean comparisons yielded a significant difference betweenthe 128 ng dose and the vehicle dose for the morphine-pel-

Ž . Ž .leted rats P-0.01 Fig. 2 . MN injections had no effectŽ w xon responding in the placebo-pelleted rats F 3,45 s0.65,

.NS . Injections given to either the placebo or morphine-pelleted groups had no obvious effect on the rats’ behaviorobserved while handling.

3.3. Histological controls

Histological examination of brains from rats which hadcannulae aimed at the BST indicated that all rats except

Žone had at least one injector tip located in the BST Fig.. Ž .3A . This rat placebo-pelleted which had ventrally placed

injector tips was eliminated from statistical analysis. Allrats, whether morphine or placebo-pelleted, had expandedventricles. However, there was no relationship between the

Ž .size of the ventricle by visual inspection and the magni-tude of the MN effect in dependent rats.

Histological examination of brains from rats which hadcannulae aimed at the AcbSh indicated that most injector

Ž .tips were located in the AcbSh Fig. 3B . All of theplacebo-pelleted rats had at least one injector tip located inthe AcbSh; most had both in the shell, and for this reasonall were included in the statistical analysis. Eight out of tenof the morphine-pelleted rats had both injector tips in theshell. The two animals that had excessively ventral place-ments with both injector tips outside of the shell portionwere the only two that did not increase their heroinconsumption with the 128 ng dose of MN, and wereeliminated from statistical analysis.

4. Discussion

MN injections into different components of the ex-tended amygdala dramatically affected heroin self-adminis-tration in opiate-dependent rats, without producing anyobvious physical signs of withdrawal. Specifically, MNinjections into the BST strongly suppressed heroin intake;injections into the AcbSh also suppressed intake, but in aless potent manner. In sharp contrast, MN injections intoeither the AcbSh or the BST had no effect on heroinself-administration in nondependent rats. These observa-tions suggest that the transition to opiate dependence isassociated with a qualitative ‘‘switch’’ in the functionalstatus of opiate transmission in critical parts of the ex-tended amygdala.

One surprising finding of this study is that MN injec-tions into the AcbSh did not affect heroin intake in nonde-pendent rats. This finding contrasts with earlier reports ofincreases in heroin self-administration following local MN

w xinjections into the nucleus accumbens 8,35 . There aretwo obvious explanations for this apparent discrepancy.First, the MN doses used here were too low compared to

( )J.R. Walker et al.rBrain Research 854 2000 85–92 91

w xearlier studies 8,35 . Indeed, the highest MN dose testedŽ .i.e., 128 ng was equivalent to the lowest dose used in

Ž .previous studies i.e., 125 ng which did not produce anysignificant effect on heroin self-administration. Low dosesof MN were chosen in the present study because anincrease in sensitivity to MN with dependence was ex-

w xpected 7 . Another important difference between the cur-rent study and previous studies was the injection site. TheAcbSh was targeted in this study while the Acb core wastargeted in earlier studies. Since MN does not spread far

w xfrom injection sites 31 , it is possible that with the MNdoses used here, more lateral Acb opiate receptors in thecore were not blocked to a sufficient degree to alter heroinself-administration. MN injections into the BST also didnot affect heroin self-administration in nondependent rats.Again the lack of effects of intra-BST injections in nonde-pendent rats could be explained by the relatively low MNdoses tested. It is also possible, however, that the BSTdoes not contribute to the acute reinforcing effects ofheroin, a question that remains to be addressed empiri-cally. Clearly, further studies using higher MN doses innondependent animals will be required to settle theseimportant issues.

The major finding of the present study is that MNinjections into both the BST and the AcbSh suppressedheroin intake in dependent rats, without producing obviousphysical signs of withdrawal. Suppression of operant re-sponding following systemic or intracranial injections ofopiate antagonists has been generally interpreted as evi-dence for the negative affective state associated with opi-

w xate withdrawal 22 . This conclusion is reinforced by ob-servations showing that opiate-dependent rats avoid a placewhich has been repeatedly paired with opiate withdrawalw x13,32 . Different parts of the brain have been previouslyshown to contribute to the genesis of affective withdrawal,including the nucleus accumbens, the central amygdala and

w xthe periaqueductal gray 21,34 . The present study extendsthese previous studies by showing that both the shelldivision of the nucleus accumbens and the BST are alsoinvolved in affective opiate withdrawal. Interestingly, theBST appeared to be the most sensitive component of theextended amygdala. A dose as low as 8 ng injected into theBST was effective in suppressing heroin-taking behaviorin dependent rats; much higher MN doses in the AcbShŽ .present study and the central amygdala were required to

w xproduce significant affective withdrawal 21,34 . The ori-gin of this differential sensitivity is not known at presentbut it may result from differences either in opiate receptordensity andror in the functional specialization of differentparts of the extended amygdala.

A puzzling aspect of the present data is that MNinjections into the extended amygdala decreased ratherthan increased heroin consumption. At first glance, onemight have expected that the negative affective state pro-duced by intracranial MN injections would increase theneed for taking heroin. There are two possible explanations

for why this was not the case in the present study. First, itis possible that before opiate affective withdrawal acquiresany motivational effect, rats must first learn to repeatedlyassociate the alleviation of withdrawal with heroin self-ad-ministration. This phenomenon, called cathexis in the liter-

w xature on animal learning 10 , has been recently observedin rats self-administering ethanol. Ethanol-dependent ratshaving had a chance to drink ethanol while withdrawnshow a long-lasting increase in consumption compared to

w xethanol-dependent rats without such an experience 30,33 .It seems that this effect increased by repeating the experi-

w xence of drinking while ethanol withdrawn 16,29 . In thiscontext, the conditions of the present study were perhapsnot optimal for dependent rats to learn to alleviate affec-tive withdrawal by taking heroin. The second reason whyantagonist administration may not have any motivationaleffects under these conditions is that withdrawal producedby antagonist administration to dependent rats may notadequately mimic the nature of spontaneous withdrawalobserved following abrupt termination of opiate adminis-tration, which more closely resembles the experience of ahuman addict. Indeed, behavioral changes produced byantagonist-precipitated opiate withdrawal have a faster on-set and shorter duration than behavioral changes produced

w xby spontaneous withdrawal 4,17 . It may be that thoughuseful for mapping the brain regions involved in affectivewithdrawal, opiate antagonists are not appropriate for gen-erating a motivational state that will drive continued druguse in dependent animals.

In summary, opiate receptor blockade in the bed nu-cleus of the stria terminalis and the shell of the nucleusaccumbens dramatically alters heroin self-administration inopiate-dependent rats. These effects were observed withoutevidence of physical signs of withdrawal suggesting theiraffective mediation. It seems therefore that the transition toopiate dependence is associated with a neurobiological‘‘switch’’ in the functional status of opioid transmission incritical components of the extended amygdala. Furtherstudies will determine whether such neuroadaptations areassociated with a change in the motivational impact ofopiates and whether different components of the extendedamygdala, though having similar anatomical connectionsand histochemical makeup, mediate somewhat differentaspects of opiate withdrawal.

Acknowledgements

This is publication number 12205-NP from The ScrippsResearch Institute. This work was supported by NIDAgrant DA04043 to George F. Koob. The authors wish tothank Robert Lintz and Elena Battenberg for excellenttechnical assistance, Dr. Kyle Frantz and Mike Arends forcritical reading of the manuscript, and Drs. A. van Delftand Ril Broekkamp from Organon for generously supply-ing the methylnaloxonium hydrochloride.

( )J.R. Walker et al.rBrain Research 854 2000 85–9292

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