P a i n ,67 (1996)79-8801996 International Associationfor the Studyof Pain. All rights reserved0304-3959/96/$15.00

PAIN 3081

Continuous co-administration of dextromethorphan or MK-801with morphine: attenuation of morphine dependence and

naloxone-reversible attenuation of morphine tolerance

Barton H. Manning’*, Jianren Maob, Hanan Frenkc, Donald D. Priceband David J. MayerbDepartments of ‘Anatomy and bAnesthesiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia (USA),

and ‘Department of Psychology, Tel Aviv University, Ramat Aviv (Israel)

(Received28 August 1995,revisionreceived14Deeember1995,accepted30 January 1996)

summary N-Methyl-D-aspartate(NMDA) receptor antagonists have been repeatedly shown to attenuate thedevelopmentof opiate toleranceand dependencein rodents. In the presentexperiments,continuoussubcutaneousin-fusion of either MK-801(0.01mg/kg/hbut not 0.005mg/kg/h)or DM (0.133,0.67 and 1.33mg/kg/h)reliablypro-longedthe antinociceptiveeffectof continuoussubcutaneousinfusionof morphine sulfate (2.0 mg/kg/h),indicatingattenuation of the developmentof morphine tolerance.Furthermore, this prolongedantinociceptionwas completelyreversibleby naloxone(10mgllcg,i.p.). Dosesof MK-801and DM that wereequipotentin attenuatingmorphinetoler-ance(0.01mg/k#h and 1.33mglkgh, respectively)revealeddifferentprofilesof effects,however,on locomotoractivityand naloxone-precipitatedabstinence/withdrawalsymptoms.With regard to locomotoractivity,rats havingreceivedcontinuous(48h) subcutaneousinfusionof morphinesulfateand MK-801,but not rats havingreceivedmorphinesul-fate and DM, displayeda reliableand strikingincreasein locomotoractivityas compared with rats having receivedmorphine alone. With regard to naloxone-precipitatedwithdrawal symptoms,continuous (48 h) subcutaneousco-nfusion of either MK-801 (0.01 mglkglh)or DM (1.33 mg/kg/h)with morphine attenuated naloxone-precipitatedhyperalgesiaas comparedwith rats infusedwith morphinealone.MK-801(0.01mg/k#h) wasmoreeffectivethan DM(0.133,0.67,or 1.33mg/kg/h),however,in reducingother naloxone-precipitatedwithdrawalsymptoms(teeth chatter-ing,jumping and wet dog shakes).The effectsof MK-801on all withdrawalsymptomswere confounded,however,by the appearance of flaccidityfollowingnaloxone administration to rats having receivedMK-801 and morphine.These results extend previous observationsby showingthat the prolonged antinociceptionobserved followingco-adrninistrationof morphineand an NMDA antagonist is completelynaloxone-reversible,supportingthe notion thatthis antinociceptionreflectsprolongationof an opioid receptor-mediatedeffect.The differentprofilesof side effectsassociatedwith MK-801and DM, however,suggestthat (1) attenuation of naloxone-precipitatedwithdrawalsymp-tomsby MK-801may bean artifact of toxicity,and (2)DM may proveclinicallyusefulfor the preventionof morphinetolerance,given its lack of observableside effectswhen administeredconcurrentlywith morphine to rodents.

Key words: Antinociception;Morphine tolerance; Morphine dependence;iV-Methyl-D-aspartatereceptor antago-nists; MK-801;Dextromethorphan

Introduction

N-Methyl-D-aspartate(NMDA) receptor antagonistssuchas MK-801(Marek et al. 1991a,b;Trujilloand Akil

● Corresponding author: Dr. Barton H. Manning, Depart-ment of Anatomy, Medical College of Virginia, Virginia Com-monwealth University, P.O. Box 980709, Richmond, VA23298,USA. Tel.: (804) 828-5336;Fax: (804) 828-4023;E-mail:BMANNING@GEMS.VCU. EDU.Abbreviations: DM: dextromethorphan; NMDA: N-methyl-maspartate; TF: tail-flick; TFL: tail-flick latency.

1991a, 1994;Ben-Eliyahu et al. 1992; Bhargava andMatwyshyn1993;Gutstein and Trujillo 1993;Lutfy etal. 1993;Mao et al. 1994),ketamine (Trujillo and Akil1994),LY274614(Tiseoand Inturrisi 1993;Elliott et al.1994a;Tiseo et al. 1994),phencyclidine(PCP; Trujilloand Akil 1994),ACEA-1328(Lutfyet al. 1995),dextror-phan (Trujillo and Akil 1994)and dextromethorphan(DM; Elliottet al. 1994b)havebeendemonstratedto at-tenuate,at least in part, the developmentof tolerancetothe antinociceptiveeffectsof systemicallyadministeredopiates in rodents. The results of these various studies

suggest that activation of the glutamatergic NMDAreceptorplaysa critical role in cellularprocesseswhichunderliethe developmentof opiate tolerance(Mao et al.1995a).Giventhe generalrole of the NMDA receptorintriggeringintracellularcascadeswhich result in plasticchangesin the centralnervoussystem(Mayerand Miller1990;Sommerand Seeburg 1992),opiate tolerancemayreflectanother exampleof such plasticity.

The experimentalconditionsunder whichNMDA an-tagonistshave been shownto attenuate opiate tolerancehave been quite varied. NMDA antagonists appear tobe effective in attenuating tolerance induced bysustained-releasepreparations of morphine (Gutsteinand Trujillo 1993;Tiseo et al. 1994;Ben-Eliyahuet al.1992),as wellas toleranceinducedby multiple,discreteinjectionsof morphine (Marek et al. 1991a,b;Lutfy etal. 1993;Mao et al. 1994).The eficacy of MK-801treat-ment appears to extend to forms of tolerance whichfollow both acute and chronic exposure to morphine,since MK-801 reportedly attenuates tolerance 24 hfollowinga single injection of morphine (Lutfy et al.1993),as well as the tolerance that developsfollowingadministration of morphine on 3 consecutive days(Lutfy et al. 1993).

Although MK-801treatment appears effectivein at-tenuatingboth ‘acute’and ‘chronic’forms of morphinetolerance, MK-801appears to inhibit the developmentof non-associativemorphine tolerance more so thanassociativetolerance(Trujilloand Akil 1994).This sug-gests that MK-801 disrupts direct neuronal changeswhichresultfrom sustainedexposureto morphinemoreso than it disrupts the learningof associationsbetweendrug effects and environmental cues. These results,coupled with the studiesdescribedabove, tend to sug-gestthat the activationof NMDA receptorscontributesin a direct and generalmanner to the neuronal changeswhichunderliethe developmentof non-associativeopi-ate tolerance in rodents.

In all of the above-mentionedstudies,attenuation ofthe development of morphine tolerance was opera-tionallydefinedas the continuedabilityof morphinetoproduce antinociception despite repeated adtninistra-tion; administrationof an NMDA antagonistappearedto result in prolongationof the antinociceptiveeffectofmorphine. It is necessary to point out, however, thatwhilethese studiesall involvedmeasuringantinocicep-tion following repeated administration of morphine,prolonged antinociceptionwas always observed in thepresence OJ or following prolongedpretreatment with theNMDA antagonist in question. The apparent attenua-tion of morphinetoleranceobservedin thesestudieswasalways assumed to reflect prolongation of an opioidreceptor-mediatedeffect.

MK-801has been reported to result in PCP-likesideeffects (including head-weaving,falling and increasedlocomotor activity; Rasmussen et al. 1991)at doses

similar to those shown to attenuate opiate tolerance.Furthermore, the drug combination of MK-801 andmorphine is more toxic than MK-801 alone (TrujilIoand Akil 1991b;Ben-Eliyahuet al. 1992),especiallyinproducingcatalepsy.Given these potential side effects,it is possiblethat the apparent attenuation of morphinetolerance(i.e., prolongedantinociception)observedac-companying MK-801 treatment reflects toxic effectswhich affect the animal’sability to respond to noxiousstimulation.Although LY274614has been shown to at-tenuate the developmentof morphine tolerance in theabsenceof pronounced toxic sideeffects(Tiseo and In-turnsi 1993), the hypothesis that the prolonged an-tinociception observed following co-treatment withmorphineand an NMDA antagonist reflectsprolonga-tion of an opioid receptor-mediatedeffecthas not beendirectlytested.

The present experimentswere designed to addressseveralmorphine tolerance-relatedquestions. First, wewereinterestedin determiningwhether the antinocicep-tiveeffectof continuoussystemicadministrationof mor-phine in rats can be prolonged by continuous systemicinfusion of either MK-801, a noncompetitiveNMDAantagonist, or DM, a clinically available antitussiveagentwhichhas been shownto possessNMDA antago-nist properties (e.g., Netzer et al. 1993).Second, wequantitatively assessed rats’ capacity for unimpairedlocomotoractivityfollowingcontinuous(48h) adminis-tration of differentdosecombinationsof morphineandDM, or morphine and MK-801. Third, we testedwhetherthe prolongedantinociceptionobservedfoUow-ingcontinuousco-administrationof morphineand DM,or morphineand MK-801,is naloxone-reversible.SinceNMDA antagonistshave low affhity for opioid recep-tors (Lutfyet al. 1993),thiscouldpotentiallyaddressthequestionof whether attenuation of opiate tolerance byNMDA antagonists truly reflects prolongation of anopioid-receptormediated effect.

In addition to attenuating the developmentof mor-phine tolerance in rodents, MK-801 (Trujillo and Akil1991a;Rasmussen et al. 1991;Tanganelli et al. 1991;Ben-Eliyahuet al. 1992;Fundytus and Coderre 1994)and LY274614(Tiseoet al. 1994)have been reported toreduce the severityof the naloxone-precipitatedabsti-nencdwithdrawalsyndromefollowingchronicexposureto morphine. Although several of these studies alsoreported severe side effects associated with MK-801(Rasmussen et al. 1991;Tanganelli et al. 1991) andLY274614(Rasmussenet al. 1991)administration, therelativecontribution of these side-effectsto the reduc-tion in withdrawalsymptomswasnot easilydiscernible.Sincethe evaluationof the effectsof DM and MK-801on withdrawal symptomswas feasible in our presentexperimentaldesign, we also compared the effects ofthese compoundson naloxone-precipitatedwithdrawalbehaviors.

81

Materials and methods

SubjectsExperimentallynaive, male Sprague-Dawleyrats (Hilltop, Scott-

dale, PA)weighing300-325g at the time of surgerywereused. Ratswerehousedindividuallyin cageson a 12:12light/darkschedule(lightson at 07:00h) with food and water availablead Iibitum.All experi-mental procedureswere approved by the Institutional Animal Careand Use Committeeof VirginiaCommonwealthUniversity.

SurgeryAnimals were anesthetized with halothane, and one osmotic

minipump(Alzet,Model2002,Palo Alto, CA)wasimplantedfollow-ingthe instructionsprovidedby the manufacturer.Briefly,a smallin-cisionin the skinoverlyingthe neckwasmade,and the skinoverlyingthe medialportion of the back was detachedfrom the subcutaneousmembranesusing blunt scissors which were inserted caudally andspreadlaterally.A tilled pumpwas then insertedunder the skin withits spout orientedin a caudal direction.The pump was then loweredto the hrmbarregionof the back, and the neck woundclosed.

DrugsOsmoticminipumpswere filled with either morphinesulfate (MS;

0.5or 2.0mgikglh)alone, MS(2.0mgkgh) and MK-801(0.005,0.01or 0.02mgkgih),MS(2.0mgktylr)and dextromethorphanHBr(DM;0.133,0.67,or 1.33mgikg/it),MK-801alone (0.01or 0.02mgikgih),DM alone(1.33mg/kg/h)or physiologicalsaline(the vehicle).Pumpswerefilledto fullcapacity(228PI) 12h prior to surgeryand maintain-ed in 37°Cphysiologicalsaline,therebyensuringimmediatereleaseofthe drugsfromthe momentof pumpimplantation.Themeaninfusionrate of the pumps, as specifiedby the manufacturer, was 0.48plih(SD= 0.02@it).

Behavioral tests(a) Nociceptive threshold: Ambient temperature of the test room

wasmaintainedat 24 + 0.5°Cat all times. Each rat wascomfortablypositionedin a wellventilated,tube-shapedplasticrestrainer(internaldiameter6.5cm; internal length25 cm). The tail-flicklatency(TFL)was defined as the time from the onset of radiant heat to tailwithdrawal(D’Amourand Smith 1941).The radiant heat sourcewasa projectionbrdbsituatedbeneatha 3-mmthick glassplate. The bulbwas aimedat the caudal end (4–IOcm from the tail tip) of the rat’stail and adjustedusinga rheostat to result in baselineIatenciesof approximately3.0sec. An automaticcut-off time of 8 secwas preset tornirtirnizepossibletissuedamagecausedby overstimulationin the ab-senceof a withdrawalresponse.For each time point tested, three TFtrials were made with (1) a l-mitt intertribalinterval (Depauliset al.1987),and(2)changesin the positionof the tail receivingheat stimula-tion oneachtrial. ThemeanTFL fromthe three trials wasusedas thenociceptivescore for that time point.

(b) Locomotor acrivity: Lccomotoractivitywasassessedby placingthe animalin a boxmeasuring44 x 28 x 22cm.The floor of the boxwasdividedinto fourquadrantsof equaldimension.Thetotal numberofepisodesin whichthe animalcrossedcompletely(i.e.,all four paws)into an adjacent quadrant was counted during a 3-reintest interval.

(c) Morphineabstinencelwithdrawal:All animals,includingthose inthe DM alone,MK-801aloneand salinecontrolgroups,wereinjectedwithan intraperitonealdose of 10mgikgof naloxone,whichhas beenshownto yield maximumabstinencdwithdrawalsymptoms(Wei1981)and placedin an open-toppedcylinderwith an upper diameterof 30cm,a lowerdiameterof 22.5cm and a heightof 40cm. The frequencyof occurrenceof the followingwithdrawalsymptomswas recordedduring a lo-mist test interval: jumping, teeth chattering, wet dogshakes, writhing,diarrhea, and seminal plug emission.In addition,post-naloxonenociceptivethresholds(seea above)weremeasuredim-mediatelyfollowingmeasurementof abstinencesymptomsto deter-minethe presenceof naloxone-precipitatedhyperalgesia.

GeneralprocedureAnimalswerehabituatedto the behavioralrestrainersin I h sessions

oneachof the 3daysprior to pumpimplantation.Immediatelyfollow-ing the third habituation session,pre-surgicalnociceptivethresholdsweredeterminedfor each animal.The animalswerethen weighedand

numbered,and randomlyassignedto a treatmentgroup(n= 8/group).The nextday each animalwas implantedwith an osmoticminipump.Thepumpimplantationcaurred between08:30–09:00h, and follow-ingimplantation,nociceptivethresholdsweredeterminedat 2, 4, 6, 9,12, 18,24 and 48 h (three TF trials at each time point, proceduredescribedabove)after surgery.Animalswerereturnedto their colonyroom for all inter-test intervalsto prevent the formation of associa-tions betweendrug effectsand environmentalcues in the test room.Immediatelyfollowingthe 48-hTF session,locomotorscoringof theanimalswasearned out as describedabove.Immediatelyfollowinglo-comotorscoring,each animalwas injectedwith naloxoneand scoredduring a lo-rein interval for the presenm of morphine withdrawalsYmPtoms(SSSBehavioraltests, part c abovefor details).Immediatelyfollowingthis scoringinterval,each rat underwentone final TF ses-sion (three TF trials, prowdure describedabove) to determine thepresenw of naloxone-precipitatedhyperalgesia.

Statistical analysesTFLs,teeth-chattering,and locomotoractivityscoreswereanalyzed

usingseparatetwofactor ANOVAs(Drugtreatment x Time).Muki-ple post-hoccomparisonswere made using Wailer-DuncanK-ratiot-tests.Toeliminateproblemsof heterogeneityof variance,episodesofjumping behavior and wet-dog shakes were analyzed non-parametricallyusingthe Mann-WhitneyU-test.

Results

SummaryContinuoussubcutaneousinfusionof MS (2 mg/kg/h)

alone produced potent antinociception, as comparedwithsaline,at 2, 4 and 6 h followingpump implantation(Fig. 1).After 6 h, however,an antinociceptiveeffectofMS was no longer apparent, indicating tolerance toMS’santinociceptiveeffectshad developedin these rats(Fig. 1). Continuous subcutaneousco-infusion of MSand either MK-801(Fig. 1) or DM (Fig. 2) resulted inprolongedantinociception(P <0.05, as compared with

rats treated with MS alone) which lasted for the entire48-htest session.Furthermore, this effectwascomplete-ly reversedby systemicadministrationof naloxone (10mgjkg;Figs. 1,2). In addition,continuous(48h) subcu-taneousco-infusionof MS with either DM or MK-801prevented,as comparedwith infusionof MS alone, theexpressionof naloxone-precipitatedhyperalgesia(Figs.1,2).Co-infusionof MSand eitherDM or MK-801alsopartially prevented the expression of other naloxone-precipitatedwithdrawalsymptoms(Fig. 3). In the caseof MK-801,however,theseeffectswereaccompaniedbyseveremotor deficits.

Effects of MK-801 on morphine antinociception andtolerance

Followingpump implantation,MS at 2 mglkglh,butnot 0.5 mg/kg/h,produced reliable antinociceptionascomparedwith saline(Fig. 1;P < 0.05).As a result, the2 mglkglhdose of MS was co-administeredwith MK-801or DM in subsequentexperiments.Antinociceptionat thisdosewasobservedat the 2, 4 and 6 h time pointsfollowingpump implantation, with the maximal effectoccurringat the 4-h timepoint. No antinociceptionwasobservedafter 6 h (Fig. 1; P > 0.05).

Although three different doses of MK-801 (0.005,

82

- 0- Saline

- Morphine Sulfate (MS, 0.5 mg/kg/hr)*

~ MS(2.0mg/kg/hr)

- v- MK-801 (0.01 mg/kg/hr)

+ MS (2.0 mg/kg/hr) +

MK-801 (0.01 mg/kg/hr)

~0248 912 18 24 48 Naloxone

Time After Pump Implant (hrs.)

Fig. 1. Mean tail-flickIatencies(TFLs) of rats receivingcontinuoussubcutaneousinfusion of saline alone, morphine alone or MK-801aloneare comparedwithTFLs of rats receivingcontinuoussubcutan-eousco-infusionof morphinesulfate(MS)and MK-801.Continuoussubcutaneousinfusion of MK-801alone did not result in reliablyhigherTFLs than saline at any time point. when MK-801was con-tinuouslyco-infusedwith MS,however,the antinociceptiveeffectsofMSwereboth potentiated(at timepoints wheninfusionof MSalonewas still antinociceptive)and prolonged.This prolongedantinociception was completely reversed by naloxone. Furthermore, thehyperalgesiawhichwurred followingadministrationof nafoxonetorats treated with MS alone (i.e., morphine-tolerant rats) was notapparent followingnaloxoneadministrationto rats having receivedco-infusionof MK-801with MS. *P < 0.05,Wailer-DuncanK-ratior-test(WD),ascomparedwithrats receivingcontinuoussubcutaneousinfusionof saline alone or rats receivingcontinuous subcutaneousinfusionof MS alone. Comparisonsare across the same time point.Xp < 0.05~) as compar~ with rats receivingcontinuousSubcu-taneousinfusionofsalinealone.Comparisonsareacrossthe sametime pint. #pc 0.05,Wailer-DuncanK-ratio r-test (WD) u com-

paredwithrats treatedwithnaloxonefollowingcontinuous(48h) srrb-cutaneous infusionof saline alone.

0.01,and 0.02mg.kgih)were co-infusedwith MS, onlythe intermediatedose was effective(Fig. 1). The 0.005mgk~ dose had no effecton the developmentof mor-phine tolerance (results not shown). The 0.02 mg/lcg/hdose of MK-801 proved to be toxic in the presenceofMS — this combinationresultedin the developmentofa profound catatonic state culminatingin the deaths ofthree rats 4-5 h after pump implantation, followedbythe deaths of three more rats 48 h after pump implanta-tion (followingnaloxoneinjection).Onlytwo rats out ofeight in this group survived.

MK-801(0.01mg/kg/h)reliablypotentiatedmorphineantinociceptionat the 2 h time point followingpumpimplantation (Fig. 1; P < 0.05). Furthermore, ratsreceivingthe combination of MK-801 (0.01 mg/kgih)and MS remainedreliablyantinociceptivefor the entire48-htest period (Fig. 1;P < 0.05),in contrast with ratsinfused with MS alone. The same dose of MK-801alone,however,was ineffectivein elevatingTFLs above

- 0- Saline

-9- Morphine Sulfate (MS; 2.0 mg/kg/hr)**

* - v- Dextromethorphan (DM; 133 mg/kg/hr)

*< 4 MS 2.0 + DM (0. !33 mg/kg/hr)

~ MS 20 + DM (0.67 mg/kg/hr)

● --- MS 2.0+ DM (1 33 mg/kg/hr)

y ~

1~0246 912 18 24 48 Naloxone

Time After Pump Implant (hrs.)

Fig. 2. Mean tail-flickIatencies(TFLs) of rats receivingcontinuoussubcutaneous isditsion of safinealone, morphinesrrffate(MS)alone ordextromethorphsn(DM) afone are compared with TFLs of ratsreceivingcontinuoussubcutaneousco-infusionof MSand DM. Con-tinuoussubcutaneousinfusionof DM alone did not resuft in reliablyhigherTFLsthan salineat anytimepoint. WhenDM wascontinuous-ly co-infusedwith MS, however,the antinociceptiveeffects of MSwereboth potentiated(at timepointswheninfusionof MS alonewasstill antinociceptive)and prolonged(comparewith Fig. 1).This pro-longedsntinociceptionwascompletelyreversedbynafoxone.Further-more, the hyperafgesiawhich occurred followingadministration ofnaloxoneto rats treated with MS alone (i.e., morphine-tolerantrats)was not apparent followingnatoxoneadministration to rats havingreceivedco-infusionof the highest dose of DM with MS (comparewithFig. 1).*P < 0.05,Wailer-DmrcanK-ratio &test(WD),as com-pared with rats receivingcontinuoussubcutaneousinfusionof salineatoneor rats receivingcontinuoussubcutaneousinfusionof MSafone.Comparisonsare across the same time point. ‘P < 0.05 (WD) ascompared with rata receivingcontinuous subcutaneous infusion ofssdinesfone.Comparismsare across the sametimepoint. #P <0.05,Wailer-DurrcanK-ratior-teat(WD)aa comparedwithrats treated withnaloxone following continuous (48 h) subcutaneous infirsion of

safinealone.

levelsdisplayedby rats treated with salinealone (Fig. I;P > 0.05).It shouldbe pointed out that whilethe com-binationof MK-801and MS resulted in a longer dura-tion of reliableantinociceptionthan that obtained withMS alone, the antinociceptiveeffectof the MS/MK-801combination gradually, and reliably, decreased overtime (Fig. 1; P < 0.05).

Finally, systemic administration of naloxone (10mg/kgi.p.)had no effecton nociceptivereactivityin ratshaving receivedcontinuous (48 h) subcutaneous infu-sion of saline or MK-801 alone (Fig. 1; P > 0.05).Naloxonecompletelyreversed,however, the prolongedantinociceptionobservedin rats co-infusedwith MS andMK-801,returning the nociceptivethresholds of theserats to levelsdisplayedby rats havingreceivedsalineorMK-801alone (Fig. 1; P > 0.05).

Effects of DM on morphineantinociceptionand toleranceContinuoussubcutaneousinfusionof the highestdose

83

of DM used in the present study(1.33mgkgh) failedtoproduce reliable antinociception as compared withsaline(Fig. 2; P > 0.05).Continuoussubcutaneousco-nfusion of MS and DM (0.133,0.67, or 1.33mg/kg/h),however,potentiatedantinociception,as comparedwithMSalone,during the first6 h followingpump implanta-tion (Fig.2; P < 0.05).Furthermore,continuoussubcu-taneousco-infusionof MS and DM (0.133,0.67,or 1.33mg/kg/h)continued to result in reliableantinociceptionat timeswhen MS alonewas no longereffective(Fig. 2).

Continuousco-infusionof MS and the lowestdose ofDM (O.133mg/kg/h)resultedin reliableantinociceptionup to 12 h followingpump implantation (Fig. 2; P <0.05).Co-infusionof MS and either of the two higherdosesof DM (0.67or 1.33mgkgh) resulted in reliableantinociceptionfor the entire duration (48h) of the testsession(Fig. 2; P < 0.05).The effectsof DM werecon-sistent with a dose-effectrelationship, in that the twohigherdoseswere reliablymore effectivein prolongingantinociceptionthan the lowestdose 18-48 h followingpump implantation(Fig.2; P < 0.05).The combinationof MS and DM (1.33mgkgh) did not, however,resultin a reliably higher level of antinociception than thecombinationof MS and DM (0.67mgkgh) at any timepoint (Fig. 2; P > 0.05). It should be pointed out thatwhile the combination of MS and either of the threedosesof DM resultedin a longerduration of reliablean-tinociceptionthan that obtainedwith MS alone, the an-tinociceptiveeffectsof all three MWDM combinationsgradually,and reliably,decreasedovertime(Fig. 2; P <0.05).

Finally, systemic administration of naloxone (10mgikgi.p.)had no effecton nociceptivereactivityin ratshaving receivedcontinuous (48 h) subcutaneous infu-sion of saline or MK-801 alone (Fig. 2; P > 0.05).Naloxonecompletelyreversed,however, the prolongedantinociceptionobservedin rats co-infusedwith MSandeither of the two higher doses of DM (0.67 or 1.33mg/kg/h),returning nociceptivethresholdsof these ratsto levelsdisplayedby rats havingreceivedsalineor MK-801 alone (Fig. 2; P < 0.05).

Effects of IUK-801 and DM on naloxone-precipitatedhyperalgesia

TFLs of saline-treatedrats followingsystemicadmin-istration of naloxone were not different from thosedisplayed by saline-treated rats immediately prior tonaloxone injection (Fig. 2; P > 0.05).Similar resultswere obtained followingadministration of naloxone torats havingreceivedcontinuous(48h) subcutaneousin-fusionof MK-801(0.01mgkgh) or DM (1.33mg/kg/h)alone (Figs. 1,2; P > 0.05).In rats havingreceivedMSalone,however,reliablehyperalgesia,as compared withsaline-treatedrats, was observedfollowingnaloxonead-ministration(Fig. 1;P < 0.05).Similarhyperalgesia,ascompared with saline-treatedrats, was observedin rats

co-infusedwith MS and eitherof the two lowerdosesofDM (0.133or 0.67 mg/kg/h;Fig. 2; P < 0.05).In ratsco-infusedwith MS and MK-801(0.01mg&/h) or MSand DM (1.33mg/k#h), however,systemicadministra-tion of naloxone did not elicit reliable hyperalgesia(Figs. 1, 2; P > 0.05).

Effects of A4K-801 and DM on other naloxone-precipitated withdrawal symptoms

Systemicadministration of naloxone (10 mg/kg i.p.)produced a robust abstinence/withdrawalsyndrome inrats havingreceivedcontinuous(48h) subcutaneousin-fusionof either0.5or 2 mglkgfhMS.This syndromewascharacterizedpredominantlyby jumping, teeth chatter-ing,and wet-dogshakes,To a lesserextent,diarrhea andseminalplugemission(lessthan 50’?/oof the animals)andsporadic writhing (less than 20?/o of the animals) wereobserved. Given the low incidence of diarrhea andseminalplug emissionepisodes,only episodesof teethchattering,jumping,and wet dog shakeswere subjectedto statisticalanalyses.Rats receivingcontinuoussubcu-taneous infusionof salinedisplayedincidentalwet-dogshakes and teeth chattering, but no other withdrawalbehaviorswere observed(Fig. 3).

Rats havingreceivedcontinuous(48 h) subcutaneousinfusionof MS (2 mg/kg/h)and MK-801(0.01mg/kg/h)displayedreliablylessjumping,teeth chattering,and wetdog shakesfollowingnaloxoneadministrationthan ratshavingreceivedMS alone (Fig. 3; P > 0.05). Some oftheserats also displayedincidentaldiarrhea and seminalplug emissionepisodes(data not shown in Fig. 3), buttheseepisodeswerefar lessfrequentthan thoseobservedin rats treated with MS alone.

None of the quantifiablewithdrawal behaviorswerereduced, as compared with rats having received MSalone,in rats havingreceivedMS and the lowestdose ofDM (0,133mg/kg/h).In rats treated with MS and eitherof the two higher doses of DM (0.67 or 1.33mg/kg/h),however,episodesof both jumpingand teeth chattering,but not wet-dogshakes,were reliablyreduced(Fig. 3; P> 0.05).

Toxic effects of the combination of morphine, A4K-801,and naloxone

Naloxone(10mg/kg)did not appear to affectthe gen-eral behaviorof rats which had receivedcontinuous (48h) subcutaneous infusion of MK-801 (0.01 mg/kg/h)alone.Naloxonedid, however,dramaticallyalter the be-haviorof rats havingreceivedcontinuous (48 h) subcu-taneous co-infusion of MS (2 mg/kg/h) and MK-801(0.01 mgkgh). Followinga brief period in which therats displayedthe withdrawalsymptomssummarizedinthe previoussection,each subjectturned on its sideandremained in this position until the end of the 10-mintestingsession.Rats did not right when touched, and incontrastwith rats in all other groupsthey did not active-

84

m Teeth Chattering Episodes

_ Jumps

= Wet Dog Shakes

,2 M2 M2,++.133 D 0.67 D 1.33

Drug Treatment

Fig. 3. Frequencyof naloxone-precipitatedwithdrawalbehaviorsinvariousgroupsof rats havingreceivedcontinuous(48h) subcutaneousinfusionof salinealone, morphinesulfate (MS)alone, MK-801aloneor DMalone,or variouscombinationsof MSand MK-801or MSandDM. The frequency of occurrence of various withdrawal-relatedbehaviors(teeth-chattering,jumpingand wet-dogshakes)werescoredduringa lo-tin interval immediatelyfollowinginjectionof naloxone(IOmg’lcgi.p.). In the figure, the relativecontributionsof teeth chat-tering,jumpingand wetdog shakesto the overallwithdrawalbehaviorscore is indicatedfor each group. Note that co-infusionof MS withMK-801resulted in a marked reduction in episodes of naloxone-precipitated withdrawal behaviors as compared with rats havingraived MS rdone.Co-infusionof MS with the higherdoses of dex-tromethorphanrdsoreducedwithdrawalbehaviorsas comparedwithrats treated with MSalone,but the reductionwas not as great as thatseen in rats co-infusedwith MS and MK-801(see text for details).DrugTreatmentsare abbreviatedin the figure in the followingman-ner: D = dextromethorphan;M = morphine sulfate; MK = MK-801;Sal= Saline.The numbersfollowingthe letter abbreviationsfor eachdrug indicate drug dosage in mg@lt (e.g., M 2 = 2.0 mg@ih

morphinesulfate).

ly walk into the TF restrainers (in preparation for thefinal TF trials under naloxone) but remained passivewhen inserted by the experimenter.When returned totheirhomecage, theserats failed to right themselvesandmaintained their flaccid position.

No DM-treated rats displayedevidenceof flaccidityfollowingnaloxoneadministration.

Locomotor activityFollowingcontinuous(48h) subcutaneousinfusionof

MS(2mgkgh) alone, rats displayeda slightbut reliableelevation,as comparedwith salinecontrol rats, in loco-motor activity during the second minute of the 3-rninlocomotor scoring interval (Fig. 4A; P < 0.05). Con-tinuous(48h) subcutaneousinfusionof the highestdoseof DM (1.33mg/kg/h)alone, however,did not reliablyaffectlocomotoractivityscoresas comparedwith salinecontrol rats (Fig. 4; P > 0.05), and did not produce

other observable side effects in behavior. Continuoussubcutaneousadministration of MK-801 (0.01 or 0.02mg/kg/h) alone, on the other hand, produced (par-ticularlyat the higherdose)pronounced effectson loco-motor activity.Within6 h followingimplantationof thepumps, rats receiving MK-801 (0.02 mg/kg/h) alonedisplayedsustained agitation both in their cages, andwhen restrained for TF testing. Following continuous(48 h) infusionof MK-801 (0.01 mg/kg/h)alone, loco-motor activitywas reliablyenhanced as compared witheither saline control rats or rats treated with MS (2mg/kg/h)alone (Fig. 4A; P < 0.05). Following con-tinuous (48 h) subcutaneousinfusion of MK-801 (0.02mgkgih) alone, locomotor activity scores were nearlytripled as compared with salinecontrol rats (Fig. 4A).

Continuous(48h) subcutaneousco-infusionof MS (2mg/kg/h)and MK-801(0.01mgkgih) reliablyincreasedlocomotoractivityas compared with continuous (48 h)subcutaneousinfusion of either compound alone (Fig.4A; P < 0.05).In fact, rats having receivedcontinuous(48h) subcutaneousco-infusionof MS (2 mg/kg/h)andMK-801 (0.01 mg/kg/h)displayed levels of locomotoractivitycomparable to those displayed by rats havingreceivedMK-801 (0.02 mg/kg/h)alone (Fig. 4A; P >0.05).In addition,homecageand restraineragitation ofrats treated with MS (2 mg/kg/h) and MK-801 (0.01mgkgh) appearedto be as pronouncedas that observedin rats receivingcontinuous subcutaneous administra-tion of MK-801(0.02mg/kg/ml)alone.

In contrast with MK-801- or MS-treated rats, con-tinuous (48 h) subcutaneous infusion of DM (1.33mgllcgih)alone did not reliablyalter locomotor activityscoresas compared with saline-treatedrats (Fig. 4B; P> 0.05).Furthermore, locomotoractivityscoresof ratshavingreceivedcontinuous(48 h) co-infusionof MS (2mgkglh) and DM (0.133 or 1.33 mg/kg/h) were notsignificantlydifferent from rats having received MSalone(Fig.4B;P > 0.05).Continuous(48h) co-infusionof MS (2 mg/kg/h)and the intermediate dose of DM(0.67 mg/kg/h),however, resulted in slightly increasedlocomotoractivityscores,as comparedwith rats treatedwith MS alone, during the secondminute of the 3-reinlocomotor scoring interval (Fig. 4B; P < 0.05).

No DM-treatedrats showedevidencsof MK-801-likeagitation during TF testing.

Discussion

The results of the present studies support earlierreports suggesting that NMDA antagonists such asMK-801(Trujilloand Akil 1991a;Marek et al. 1991a,b;Ben-Eliyahu et al. 1992; Bhargava and Matwyshyn1993;Gutsteinand Trujillo 1993;Lutfy et al. 1993;Maoet al. 1994)and DM (Elliott et al. 1994b)attenuate thedevelopmentof morphine tolerance in rodents. Usingosmoticminipumpsfor continuous(48 h) subcutaneous

85

A- 0- Saline - A- MK-801

-9- Morphine Sulfate (O01 mglkglhr)

(MS: 2.0 mg/kg/hr) -0- MK-801(0.02mg/kg/hr)

Tf -A- MS 2,0+ MK.801 0.01w**..----**.-.-..*

1 2 3

B- 0- Saline A MS 2.0 + DM 0.133

+ Morphine Sulfate ~ MS 2.0 + DM 0.67(MS; 2.0 mg/kg/hr)

-0- MS 2.0+ DM 1.33- v- Dextromethorphan

(DM; 1,33 mg/kg/hr)

.I I I

1 2 3

Time (rein)

Fig.4. Profileof locomotoractivitycorrespondingto varioustreatment groupsof rats. Locomotoractivity(auadrant crossings.seeMethodssec-tion)wasscoredduringa 3-reinobservationperiodfollowing48h of continuoussubcutaneousdrug treatment(beforeadministrationof naloxone).

. . -.

(A)Averagenumberof quadrant crossingsof rats havingreceivedcontinuoussubcutaneousinfusionof salinealone, morphinesulfate(MS)aloneor MK-801alone, or continuousco-infusionof MS and MK-801.Verticalbars indicatestandard error of the mean. MS produceda slight, butreliable,increasein locomotoractivityat the 2-nrirrtime point, as comparedwith saline.Bothdosesof MK-801producedelevationsin locomotoractivityas did the combinationof MS and MK-801.(B)Averagenumberof quadrant crossingsof rats havingreceivedcontinuoussubcutaneousinfusionof salinealone, MSalone or DM alone, or one of the variouscombinationsof MS and DM. Verticalbars indicatestandard error of themean.MSproduceda slight,but reliable,increasein hxomotor activityat the 2-reintime point as comparedwith saline.The combinationof MSand DM faded to reliablyincreaselocomotoractivityabove that of rats treated with MS alone, exceptwhen MS was combinedwith DM (0.67mfiti) — the combinationof MSand DM (0.67m~fi) produ~d a slight,but reliableelevationin quadrant crossingsat the 2-reintime pointascomparedwithrats havingreceivedcontinuoussubcutaneousinfusionof MSalone.*P <0.05, Wailer-DuncanK-ratiot-test (WD),as comparedwithrats havingreceivedcontinuous(48h) subcutaneousinfusionof salinealoneor rats havingreceivedcontinuous(48h) subcutaneousinfusionof MSalone.Comparisonsare acrossthe sametimepoint. xp <0.05 @JD)ascomparedwithrats havingrezeivedcontinuous(48h) subcutaneous

infusionof saline alone. Comparisonsare across the same time point.

deliveryof drugs,wefound that MS(2mg/kg/h)produc-ed antinociceptionfor a period of 6 h, with its maximaleffectoccurring4 h followingpump implantation (Fig.1).Continuoussubcutaneousinfusionof MK-801(0.01mg/kg/h)or DM (1.33 mg/kg/h)did not produce an-tinociceptionin the absence of MS (Figs. 1, 2). WhenMS (2 mgkglh) was co-infused with MK-801 (0.01m@#h) or DM (0.133,0.67or 1.33mg/kg/h),however,antinociceptionwas observed for a longer period thanthat observedwith MS alone (Figs. 1,2).WhileMK-801and DM both appear to have attenuated the develop-ment of morphinetolerance in the present experiments,it is necessaryto point out that neithercompoundcom-pletelyblockedthe developmentof tolerance.Therewasa gradual reduction over time in the magnitudeof an-tinoceptionobservedin rats treated with MS and MK-801, or MS and DM (Figs. 1, 2).

Followingcontinuous(48h) subcutaneousinfusionofMK-801 (either alone or in combination with MS (2mgikgh)) rats displayed enhanced locomotor activity(Fig. 4A) and general activation, confirming previousobservationsin mice(Kuribara et al. 1992).In contrast,continuous(48 h) subcutaneousco-infusionof MS andDM did not result in any strong increasesin locomotor

activity scores as compared with rats having receivedcontinuous subcutaneous(48 h) infusion of MS alone(Fig. 4B).

SinceDM and MK-801 either reliably enhanced, orhad no effecton, locomotor activity as compared withsaline-treatedcontrol rats, it is unlikelythat an NMDAantagonist-inducedmotor deficit accounts for the in-creased TFLs (and apparent attenuation of morphinetolerance)observedin the presentexperiments.Further-more, in rats displayingprolonged antinociception(in-dicating attenuation of morphine tolerance), theantinociceptionwhich remained after the 48 h test ses-sion was completelyreversed by naloxone to baselinelevelsor below(Figs. 1, 2). This suggeststhat the pro-longed antinociceptionproduced by co-treatment withMS and an NMDA antagonist reflects an opioidreceptor-mediatedeffect. Taken together, these datasupportthe hypothesis(Trujilloand Akil 1991a;Mao etal. 1995a)that pharmacologicalblockade of NMDAreceptorsspecificallyinterfereswith the developmentoftolerance to the antinociceptiveeffectsof morphine.

Mao et al. (1995a)recentlyproposed that an increasein responsivenessof excitatory amino acid receptorscontributes to the developmentof opiate tolerance, as

86

wellas the developmentof hypera]ge8iaassociatedwithsuch pathologies as peripheral neuropathy and peri-pheral inflammation.This hypothesis is supported byevidencesuggestingthat MK-801preventsthe develop-ment of the hyperalgesicstate which developsconcur-rentlywith morphinetolerancein rats (Mao et al. 1994).Furthermore,rats whichdevelophyperalgesiafollowingligation of the common sciaticnerve appear to be lesssensitiveto the antinociceptiveeffects of systemicallyadministeredmorphine (Mao et al. 1995b). In the pres-ent study, the effectsof continuous subcutaneousinfu-sion of MK-801 or DM on naloxone-precipitatedthermalhyperalgesiawereassessedin rats havingreceiv-ed continuous subcutaneous infusion of morphine.Reliable hyperalgesia,as compared with saline-treatedcontrol rats, was observedfollowingnaloxoneadminist-ration (10 mg/kg i.p.) to rats having received con-tinuous(48h) subcutaneousinfusionof MS (2 m#lc#h;Fig. 1). Hyperalgesiawas also seen followingnaloxoneadministrationto rats havingreceivedcontinuous(48h)subcutaneousco-infusionof MS and DM (0.133or 0.67mg/kg/h;Fig. 2). Continuous (48 h) subcutaneousco-nfusion of MS (2 mg/kg/h)and either MK-801 (0.01mg/lcgih)or DM (1.33 mg/kg/h),however, resulted inpreventionof naloxone-precipitatedhyperalgesia.Theseresults,coupledwith the observationthat DM and MK-801 prevent the development of morphine tolerance(Figs. 1, 2), support the proposed role of the NMDAreceptor in the developmentof morphine toleranceandconcurrent hyperalgesia(Mao et al. 1994,1995a).

An important issuepertainingto the presentresultsisthe question of whether administration of an NMDAantagonistincreasesthe antinociceptiveporerzcyof mor-phine. This issue is particularly relevant to the presentexperimentssince the antinociceptionobservedfollow-ing 2, 4 or 6 h of continuous subcutaneousinfusion ofMS(2 mg/kgih)alonewasreliablypotentiatedif the MSwas co-infusedwith MK-801 (Fig. 1) or DM (Fig. 2).Although MK-801and DM were ineffectivein elicitingantinociception when continuously infused withoutmorphine(Figs. 1,2), the potentiationand prolongationof antinociceptionobservedin rats co-infusedwith mor-phine and either MK-801 or DM could conceivablyhave resulted from an antinociceptivesynergybetweenthese dregs, and not a blockade in the developmentofmorphine tolerance per se. Several lines of evidencecount against this possibility, however. Pretreatingopioid-naiverats witha singlesystemicinjectionof MK-801 has no effect on the antinociceptionproduced bysystemicallyadministeredmorphine (Trujillo and Akil1991a;Marek et al. 1991a;Elliott et al. 1994a),whileinmice, similar pretreatment results in attenuation of theantinociceptiveeffect of morphine (Lipa and Kavaliers1990;Lutfy et al. 1993).Furthermore, a singlesystemicinjectionof MK-801fails to restore the ability of mor-phine to produce reliableantinociceptionin morphine-

tolerant rats (Trujillo and Akil 1991a). Pretreatingopioid-naivemicewitha singlesystemicinjectionof DMalso fails to alter the antinociceptiveeffectof morphine(Elliottet al. 1994b),and a similarpattern of resultshasbeen obtained for the competitiveNMDA receptor an-tagonistLY274614(Tiseo and Inturrisi 1993;Elliott etal. 1994a;Tiseo et al. 1994).Sincesinglesystemicinjec-tions of MK-801 or DM do not potentate theantinociceptiveeffectof morphine in opioid-naiverats,it is unlikelythat the potentiation and prolongation ofmorphineantinociceptionobservedin the presentexper-iments(Figs. 1, 2) was due to a synergisticeffect of thecombinationof MS and MK-801,or MS and DM.

Although it is unlikelythat the potentiation of mor-phine antinociceptionproduced by MK-801 or DM atearlytimepoints(2,4 and 6h followingpump implanta-tion; Figs. 1, 2) reflectsan antinociceptivesynergybe-tween the NMDA antagonists and morphine, thispotentiation may reflect attenuation of the early, or‘acute’(Kissinet al. 1991)developmentof morphinetol-erance. Although continuous subcutaneousinfusion ofmorphine alone still elicits reliable antinociception atthese early time points, the NMDA receptor-mediatedintracellularchangeswhich contribute to the develop-ment of morphine tolerance may be ongoing at theseearly time points. Consequently, blockade of thesechangesby MK-801or DM could result in an apparentpotentiationof the antinociceptiveeffectof morphineatthese times.

It is interestingthat the doses of DM and MK-801which were equipotent in potentiating morphine an-tinociceptionand attenuating morphine tolerance didnot producs equipotent effectson locomotor behavior— only MK-801 strongly increased locomotor activityscoresabove those seen in rats treated with MS alone(Fig. 4A). Rasmussenet al. (1991)observedsimilardif-ferences in locomotor effects between LY274614andMK-801and attributed thesedifferencesto the fact thatMK-801is a non-competitiveantagonist at the NMDAreceptor,whereasLY274614isa competitiveantagonist.SinceDM and MK-801showdifferentpatterns of bind-ing sites throughout the CNS (compare Canon et al.1989withSakuraiet al. 1991and Boweryet al. 1988)andDM has beenshownhaveeffectson CNS neuronsin ad-dition to its effectson NMDA receptor-mediatedevents(Netzer et al. 1993),these factors may account for thedifferencesin side effectsassociatedwith MK-801 andDM. SinceDM appears to be less toxic than MK-801when combinedwith morphine, DM may prove usefulas an opiate tolerance blockingagent in humans.

Theeffectsof chronicsystemicadministrationof MK-801 on morphine dependencehave been tested in onlytwo studiesprior to the present study (Tlujilloand Akil1991a;Ben-Eliyahuet al. 1992).In one of these studiesMK-801 diminished abstinence-relatedjumping (Tru-jillo and Akil 1991a). Although the second study

87

reporteda reductionin teeth chatteringand ptosis(Ben-Eliyahuet al. 1992),this studydid not report a reductionin episodesofjumpingand diarrhea. The presentstudiesconfirm that some morphine abstinenc.dwithdrawalsymptoms(jumping, wet dog shakes, teeth chatteringand hyperalgesia)can be diminishedbychronicMK-801treatment (Figs. 1, 3). These observations were con-founded, however, by severe motor deficitswhich ap-peared immediately following the administration ofnaloxone. These motor deficits were characterized byflaccidity(i.e., the animalsdid not spontaneouslyrightthemselves)but not completeparalysis— the TF reflexof these rats was unimpaired as compared with rats inboth the saline-and MK-801-treatedcontrol groups.

In the present study, the motor deficits observedfollowing naloxone administration in rats co-infusedwith morphineand MK-801did not appear to be medi-ated by MK-801 alone. Instead, the observed motordeficitsappeared to reflectan interaction betweenmor-phineand MK-801.Evidenceto support this conclusionincludesthe observationsthat (1) decreasedlocomotoractivity appeared only after opioid receptors in theserats were blocked; prior to naloxone administrationtheseanimalsdisplayedincreased locomotoractivity,aneffectsimilarto one reported by Rasmussenet al. (1991)and (2)continuous(48h) subcutaneousinfusionof MK-801 or MS alone did not result in similar flaccidityfollowingnaloxoneadministration.

In contrast with those animals treated with MS andMK-801,rats havingreceivedcontinuous(48 h) subcu-taneous co-infusion of MS and DM did not displaydetectable motor deficits followingnaloxone adminis-tration. AlthoughDM (0.133mg/kg/h)potentiatedteethchatteringduring naloxone-precipitatedabstinence,thetwo higher doses of DM (0.67 and 1.33 mg/kg/h)decreasedthis behavior, without significantlyaffectingother withdrawal behaviors. It should be pointed outthat while the highestdose of DM (1.33mg/k@) wasequipotentwith MK-801in attenuatingmorphinetoler-ance, DM was less effectivethan MK-801 in reducingthe severityof withdrawalsymptoms(Fig. 3). If NMDAreceptorsare involvedin both the developmentof mor-phine tolerance and the developmentof morphine de-pendence, it might be expected that doses of DM andMK-801which are equipotent in attenuating morphinetolerancewould be equipotent in suppressingbehaviorsindicativeof morphinedependence.The present resultssuggest the distinct possibility that the reduction ofnaloxone-precipitatedwithdrawal symptoms observedin rats co-treated with MS and MK-801in the present,and previous, studies reflectsmotor deficitsand not atrue reduction in morphine dependence.

In summary, the present results suggest that con-tinuoussubcutaneousadministrationof MK-801or DM(1) attenuates the developmentof toleranceto the anti-nociceptiveeffectsof continuoussubcutaneousadminis-tration of morphine and (2) diminishes naloxone-

precipitated withdrawal behaviors observed followingcontinuous(48 h) subcutaneousadministrationof mor-phine. In addition, the present results indicate that theprolonged antinociceptionobserved in rats co-treatedwith morphineand an NMDA antagonist is completelyreversibleby systemicadministrationof naloxone.Pro-longedexposureto MK-801,but not DM, was accom-paniedby noticeablesideeffects.It is unlikely,however,that these side effectscontributed to the apparent at-tenuation of morphine tolerance by MK-801 since (1)continuous (48 h) subcutaneous co-infusion of mor-phineand MK-801producedincreasedlocomotoractiv-ity rather than sedation and (2) the prolongedantinociceptionobserved following continuous (48 h)subcutaneousco-infusionof morphine and either MK-801or DM was completelyreversedby administrationof the opioid antagonist naloxone. The possibilityremains, however, that the pronounced side effectsinduced by naloxone administration following con-tinuous (48 h) subcutaneousco-infusion of morphineand MK-801 contributed to the reduced withdrawalsymptomsobservedin these rats.

Acknowledgements

Supportedby PHS Grants NS-24009and DA-08835.We thank LeslieKeniston and Juan Lu for assistancewith testingof animals.

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