THE OFFSET OF MORPHINE TOLERANCE IN RATS AND MICE

Br. J. Pharmac. (1975), 53, 383-391

THE OFFSET OF MORPHINETOLERANCE IN RATS AND MICE

B.M. COX, M. GINSBURG & JULIA WILLIS,with an Appendix by J.K. DAVIESBasic Medical Sciences Group, Department of Pharmacology, Chelsea College, Manresa Road, London SW3 6LX

I In rats and mice made tolerant to morphine by pretreatment with the drug, the shift to theright of the log dose/analgesic response line for morphine from its position in naive animalsoccurs without significant change in slope provided that sufficient time is allowed forelimination of pretreatment drug.2 Responsiveness to the analgesic effects of morphine, given together with cycloheximide toprevent reinforcement of tolerance, was measured in rats (paw pressure method) and mice (hotplate method) at intervals during 1-23 days following cessation of a variety of regimens oftolerance-inducing drug treatments.3 A biphasic pattern of recovery of responsiveness was observed, which was independent ofthe regimen or the drug (morphine, methadone or diamorphine) used to induce tolerance.Estimates of the rates of the first, fast phase are imprecise but the rate of the second phase ofoffset, from 4th day after cessation of pretreatment had, in rats, a mean half-time of13.2 ± 0.53 days-for all pretreatments combined, there being no significant differencesbetween the various pretreatment regimens employed. In mice, similarly, a biphasic recovery ofanalgesic responsiveness was seen after morphine pretreatment, the mean half-time of theslower phase being 17.4 days.4 Precipitation of an acute withdrawal syndrome in rats by naloxone HCI given 6 h after thefinal injection of a tolerance-inducing treatment with morphine did not affect the subsequentrate of recovery from tolerance.5 During the period following a tolerance-inducing pretreatment with morphine in mice, therate of attenuation of the naloxone-evoked jumping response was faster than the rate of offsetof tolerance.

Introduction

Opioid narcotic drugs produce numerous effectson neuronal function in the central nervoussystem, and it has proved difficult to identifyseparately those processes concerned in themediation of particular opioid agonist actions orthose involved in the genesis of tolerance anddependence. Identification of the mechanismsresponsible for the phenomenon of tolerance maybe assisted by knowledge of the rate of recoveryfrom the tolerant state, because it seems likelythat the rate of reversion towards the level ofresponsiveness to morphine seen in naive animalsreflects the recovery from the underlying meta-bolic perturbation.

Reports on the rate of offset of opioidtolerance are sparse and conflicting; Cochin &Kornetsky (1964) report that in rats significanttolerance to morphine was retained for more thanone year following single or multiple doses ofmorphine, whereas Goldstein & Sheehan (1969)

estimated the time of recovery from levorphanoltolerance in mice to be 'essentially the same,within the limits of experimental error, as the rateof onset of tolerance,' (that is, in theirexperiments, a half-time of 1648 hours).We have, therefore, reassessed the rate of

recovery from tolerance to the analgesic effects ofmorphine in both rats and mice. A difficulty inmeasuring the level of morphine tolerance arisesfrom the fact that the test procedure necessarilyinvolves the administration of an opioid analgesicwhich then reinforces the phenomenon beingmeasured. This problem has been circumvented inthe present experiments by the simultaneousadministration of a protein synthesis inhibitorwith the test dose of opioid drug, a procedurewhich prevents the further development oftolerance whilst not affecting previouslyestablished tolerance (Cox, Ginsburg & Osman,1968; Cox & Osman, 1970). A preliminary

384 B.M. COX, M. GINSBURG & JULIA WILLIS

account of this work has been presented to ameeting of the British Pharmacological Society(Cox, Ginsburg & Willis, 1973).

Methods

Induction of opioid drug tolerance

All drugs were dissolved in 0.9% w/v NaCl solution(saline), and were given s.c. to both rats and mice,except for methadone which was given i.p. becausewhen given s.c. it causes, in some animals, skinlesions at the site of injection. Injections weregiven twice daily, at 09 h 00 min and 17 h 00 min;on the starting day the first injection was given at17 h 00 min and on the concluding day the finalinjection was given at 09 h 00 min. Controlanimals were given twice daily injections of salinesimulating the regimens of drug administrationwhich were as indicated in Table 1.

Assessment of analgesic responsiveness

Analgesic responsiveness was tested in rats by apressure method during continuous intravenousinfusion of morphine HCI with cycloheximide viaan exteriorized cannula inserted under etheranaesthesia in an external jugular vein at least 18 hbefore the beginning of the infusion (Cox, et al.,1968). The flow rate of the infusion was 0.5 ml/hand the infusate (in saline) contained cyclo-heximide such that each rat received 200,ug kg-'h-'. In most experiments the rate of concomitantmorphine HCI infusion was 5 mg/kg-' h-1.

Analgesia was assessed in rats by estimation, atintervals during an infusion, of responsiveness to apainful stimulus using an Analgesimeter (UgoBasile, Milano) to measure the minimum load thatmust be applied to a rat's hind paw to cause theanimal to react, see Randall & Selitto (1957).

In mice, analgesia was measured by amodification of the hot-plate method described byWoolfe & McDonald (1944); the surface tempera-ture of the plate was 550 C. The response time toelicit the reaction (licking or blowing on the frontpaws, kicking the hind feet or hopping on all feetor jumping) was noted before and 30 min after as.c. injection of morphine HCI (12 mg/kg) pluscycloheximide (20 mg/kg). Mice that did not reactwithin 30 s were removed from the hot-plate toavoid damaging their paws. In fact, all miceresponded within that time.

Assessment of morphine dependence in mice

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MORPHINE TOLERANCE 385

with naloxone (10 mg/kg i.p.) following pretreat-ment with morphine and is often taken as ameasure of dependence (Way, Loh & Shen, 1969;Cheney & Goldstein, 1971). The jumping rate wascounted using the apparatus (Figure 8) describedin the Appendix.

Results

Relationship of morphine dosage to analgesicresponse in naive and tolerant animals

Groups of 6 rats were given an intravenousinfusion of morphine at 1,2,3,4 and 5 mg kg-' h-1.Cycloheximide was infused concurrently with themorphine HCl at a rate of 200p,g kg-' h'1 toprevent the development of tolerance to themorphine during the course of infusion (Cox &Osman, 1970), and the nociceptive responsivenessfor each rat was assessed by determination of thepressure threshold at 60 or 90 min intervals afterthe start of the infusion. At each infusion rate, themean analgesic index rose during the first 2-3 h toreach a plateau level which was maintained to theend of the infusion (Figure 1). Similar experimentswere carried out in groups of rats which had beenrendered tolerant to the analgesic effects ofmorphine by pretreatment with the drug; thesetest infusions were administered 2 days or 7 daysafter cessation of pretreatment. The meananalgesic indices again reached a plateau afterabout 3 h of the infusion as with the naive animalsbut, naturally, greater amounts of morphine wererequired to produce analgesic effects of com-parable magnitude. The steady state analgesicindices were plotted against the morphine infusionrates (on a logarithmic scale). The straight linefitting the points obtained in these experiments,with animals 7 days after cessation of thetolerance-inducing treatment, paralleled that fornaive animals and was shifted to the right(Figure 2a). However, 2 days after the end of thepretreatment period the more marked shift to theright was accompanied by significant flattening ofthe log-dose response line.

Log-dose response curves were also determinedin naive mice and mice 2 days after a period of 7days treatment with morphine HCI (dosageschedule D, Table 1). Analgesic responsiveness wastested by the hot plate method. Single sub-cutaneous doses of morphine HCI were given andthe analgesic response was measured 30 min later.There was no significant deviation from parallelismof the log-dose response curves for naive andtolerant animals (Figure 2b).

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Figure 1 Time course of analgesic responses todifferent doses of morphine plus cycloheximide(200Mg kg-' h-'), infused intravenously for 6 h intoconscious rats. Mean analgesic index was estimatedfrom 4-6 rats. In this and subsequent figures s.e. meanare not shown where they are < symbol signs. (A)1 mg kg-' h-1 morphine HCI; (o)2 mg kg-' h-'morphine HCI; (.) 3 mg kg-' h-' morphine HCI; (o)4 mg kg-' h-' morphine HCI; (A) 5 mg kg-' h-'morphine HCI.

Recovery of the analgesic response to morphineafter the development of tolerance

Recovery following morphine pretreatment inrats. The offset of morphine tolerance wasstudied by measuring the analgesic response to astandard infusion of morphine, at various timeintervals after cessation of the tolerance-inducingtreatments. The choice of this approach, ratherthan the more expensive, arduous, and timeconsuming procedure of determining for eachstage the dose of morphine that would elicit astandard analgesic effect, was justified in the lightof the results described above, showing that thereduced responsiveness of tolerant animals couldbe described by a parallel shift to the right fromthe dose-response curve for morphine in naiveanimals.On selected days after the termination of a

period of morphine pretreatment, rats in separategroups of between 4 and 6 were given intravenous


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Period of infusion (h)Figure 3 Antinocioceptive responses during intra-venous infusion of morphine HCI (5 mg/kg ' h-1) pluscycloheximide (200 lg/kg-l h-') in rats duringrecovery from morphine tolerance (pretreatment C).Numbers on curves refer to the number of daysafter cessation of the pretreatment. Mean analgesicindex was estimated from 4-6 rats. Vertical barsindicate s.e. mean. (o) results from naive rats whichreceived saline only in the pretreatment period.

infusions of morphine HCl (5 mg kg-' h-1)together with cycloheximide (200 jig kg-' h-');pressure thresholds were determined at intervalsduring the infusion (Figure 3).

During each infusion the mean analgesic indexvalue increased for the first 2-3 h when a steadystate level was reached which was maintained forthe rest of the infusion period. The steady stateanalgesic index increased progressively as theinterval between the test infusion and the finaldose of the pretreatment. A plot of the steadystate analgesic index value (on a logarithmic scale)against the days after termination of morphinepretreatment showed that the recovery ofsensitivity to morphine occurred in two phases; an

initial rapid phase of recovery followed by a

slower phase (Figure 4a). Similar results were

obtained for the three different regimens ofmorphine pretreatment, which were chosen toinduce initial tolerances of varying intensity.

From the point of inflection on the curve (atabout the fourth day) until offset of tolerance was

virtually complete (up to 23 days) the points were

well-fitted by straight lines, the slopes of whichwere similar for all tolerance-inducing treatments.The mean slopes during this phase correspond to a

half-time for recovery of morphine sensitivity of13.2 + 0.53 days. No estimates of the rate ofrecovery during the initial rapid phase have beenmade because of low precision in the determina-tion of the analgesic indices at the low valuesobtained in highly tolerant animals and becausethe testing procedure itself occupies a period of

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time that is significant in relation to the recoveryrate.

Recovery following methadone or diamorphinepretreatment in rats. Measurements of therecovery of sensitivity to morphine after pretreat-ment of rats with methadone or diamorphineshowed that the analgesic response returned in asimilar biphasic manner following toleranceinduction with these drugs (Figure 4b). From thefourth day onwards the points are fitted bystraight lines whose slopes correspond to half-times for recovery of 14.2 and 11.4 days formethadone and diamorphine respectively.

Recovery following morphine pretreatment inmice. At predetermined time intervals after thecompletion of the tolerance induction in mice, theresponse, to a s.c. injection of the standard dose ofmorphine HCI (12 mg/kg) plus cycloheximide(20 mg/kg), was tested by measuring the increasein reaction time on the hot plate; different groupsof animals were used at each time of testing. Whenthe responses were plotted on a logarithmic scaleagainst the time after the final morphine dose, itwas seen that once again recovery occurred in twophases. In mice the half-time for the second phasewas estimated to be 17.4 days (Figure 5). Thepossibility that the protein synthesis inhibitor

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Figure 5 Rate of recovery of responsiveness tomorphine in tolerant mice. Reaction time wasestimated 30 min after a s.c. injection of morphineHCI (12 mg/kg) plus cycloheximide (20 mg/kg) intolerant mice (observations in 16-25 animals). Thehorizontal broken line indicates the mean increase inreaction time in naive mice.

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alone might affect recovery was tested by givingone group of eight mice, cycloheximide (20 mg/kgs.c.) on the ninth day of recovery. The increase inreaction time produced by morphine and cyclo-heximide in these animals on the following daywas not different from that in a control group ofmice.

Effects of naloxone on recovery from morphinepretreatment in rats. It has been suggested thatmorphine antagonists such as nalorphine ornaloxone interact competitively with morphine ata common receptor site (Cox & Weinstock, 1964;Grumbach & Chernov, 1965; Pert & Snyder, 1973)and that displacement of morphine from bindingsites might be responsible for the rapid onset ofwithdrawal symptoms induced by these drugs inmorphine-dependent animals (Goldstein, Aronow& Kalman, 1969). It was therefore of interest toinvestigate the effects of naloxone treatment onthe recovery of sensitivity to morphine. A groupof rats tolerant to morphine (pretreatment C,Table 1) received a dose of naloxone HCI (1 mg/kgs.c.) 6 h after the last morphine dose. This dosewas sufficient to precipitate withdrawal symptomsnamely diarrhoea, irritability, ptosis, headshakes,

Figure 7 Comparison of offset of morphinetolerance and naloxone-elicited jumping in mice.

Increase in hot plate reaction time was estimated30 min after s.c. injection of morphine HCI(12 mg/kg) plus cycloheximide (20 mg/kg). Meannumber of jumps per mouse was determined during a

20 min period after i.p. injection of naloxone HCI(10 mg/kg; observations in 16-25 mice). The inter-section of the abscissa and the left-hand ordinate givesthe mean increase in hot plate reaction time in naivemice.

body trembling, etc., symptoms which were more

intense and more acute than those observedfollowing simple withdrawal of the drug. Differentsubgroups of these animals were then given thestandard morphine and cycloheximide infusion on

subsequent days and the steady state analgesicindices determined. Animals not tested formorphine responsiveness before day 9 of recovery

received an additional dose of naloxone HCI(10 mg/kg s.c.); the second naloxone dose did notelicit any observable withdrawal symptoms.

The responses of the naloxone-treated rats were

compared with those of a control group whichreceived an identical morphine pretreatment butdid not receive naloxone. The results (Figure 6)showed that naloxone administration did notaffect the recovery of the analgesic response tomorphine.

Rate of loss of naloxone sensitivity in mice

Of the techniques available for assessing the degreeof physical dependence in opioid-treated small

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laboratory animals, the procedure which mostreadily yields quantitative data is the measurementof the incidence of the characteristic jumping(escape) response elicited by naloxone in depen-dent mice (Marshall & Weinstock, 1969; Cheney &Goldstein, 1971).

Experiments to compare the rate of loss ofnaloxone sensitivity with the rate of recovery ofthe analgesic response to morphine were carriedout at various times after the cessation ofpretreatment D (Table 1). On each day the testsfor responsiveness to the antinociceptive effects ofmorphine HCI (12 mg/kg) given subcutaneouslywith cycloheximide (20 mg/kg) were followed4-5 h later by an injection of naloxone HCI(10 mg/kg i.p.) and determination of the jumpingactivity over the ensuring 20 minutes.

Figure 7 shows, plotted on arithmetic scale,the loss of the naloxone response and the loss oftolerance to the antinociceptive effect ofmorphine as time elapses following the final doseof the morphine pretreatment, the former beingexpressed as the mean number of naloxone-elicitedjumps per mouse and the latter in the increase inhot plate reaction time induced by morphine.Naloxone sensitivity declined much more rapidlythan tolerance. Thus on day 8, no jumps could beelicited by the naloxone treatment although themice were still clearly tolerant to the analgesiceffect of morphine.

Discussion

The rate of offset of morphine tolerance may beobtained by estimating at time intervals after

cessation of pretreatment with morphine either,the recovery of the response to a fixed dose ofmorphine or the dose of drug required to elicit anarbitrarily defined standard response. For thepresent experiments the former strategy wasadopted because it is more economical in time andin resources.

In general a pattern of change in one of theseparameters will be reflected in the other providedthat alteration in the position of the log dose-response curve is not accompanied by alteration ofslope. This has been shown to be the case in thecomparison of naive mice with mice two days aftermorphine withdrawal and in the contrast of naiverats with rats 7 days after morphine withdrawal.However, slight flattening of the log dose-responsecurve was observed after 2 days abstinence in rats,an effect which could be predicted on pharmaco-kinetic grounds from the persistence of some ofthe morphine from the tolerance-inducing treat-ment. Thus any discrepancy arising from thissource would affect estimates of the recovery rateonly in the period immediately following cessationof pretreatment, a period in which, for otherreasons given in Results, no attempt was made toreach a precise estimate of recovery rate.

It should be remembered that owing to arestriction imposed by the experimental design(arising from avoidance of injurious nocioceptivestimuli), all the analgesic responses probably lie inthe lower half of a complete log dose-responsecurve. Nevertheless, the smallest analgesic responseconsidered by us to be significant was representedby points on the straight line portion of the curve.

In both rats and mice the return of theanalgesic response to the standard morphine

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treatment occurred in two phases. There was aninitial rapid recovery which was complete in 3-4days, followed by a slower phase. The half-timefor this slower recovery phase (T½l 13.2 days inrats, 17.4 days in mice) was independent of theintensity or means of the tolerance induction.These results differ from those reported byGoldstein & Sheehan (1969) on recovery fromtolerance to the 'running-fit' caused bylevorphanol in mice. They found that recoveryoccurred as a single phase, with a half-time of 16 hwhen tolerance-inducing doses of levorphanol weregiven at 8 h intervals, or with half-time of 48 hwhen the tolerance-inducing doses were given at1 6 h intervals. Goldstein & Sheehan suggest thattheir results are consistent with the propositionthat the opioid drug induces a change in thesynthesis rate of an enzyme of functional proteinmaintained at steady state level by end-productcontrol of the synthesis rate and an exponentialrate of breakdown, although it is not clear why therecovery half-time should vary with the frequencyof administration of the tolerance-inducing drug.

Our results on the recovery of the analgesicresponse are not consistent with this explanationsince the half-time of slower recovery phase isconsiderably longer than the half-time fortolerance development (e.g. consider morphinepretreatment A which lasted for 3 days only).Recovery times greatly in excess of these havebeen described by Cochin & Kornetsky (1964)who reported that the analgesic response tomorphine in rats still had not returned to normal14 months after a single prior application ofmorphine (20 mg/kg). We cannot explain thediscrepancy between their results and the progres-sive return over a period of 20-30 days to thesensitivity of naive animals, as described in thispaper.

The contrast between the single phase rapidrecovery of the 'running fit' response (Goldstein &Sheehan, 1969) and the more prolonged biphasicrecovery of the analgesic response suggests thatdifferent processes are concerned in tolerancedevelopment with regard to these two opioid drugeffects, or that a similar mechanism with a rapidrecovery time is involved in tolerance to botheffects but that an additional process with a slowerrecovery time is also involved in tolerance to theanalgesic response.

The close agreement of the half-time values ofthe second recovery phase after morphine,diamorphine or methadone treatment, despite thedifferences in the manner in which these drugs aredistributed within and eliminated from the body(Misra, 1 972) and in their physical properties(Herz & Taschemacher, 1972) argues against theconclusion that the second phase reflects the time

course ot elimination of the opioid drug from thebody and strongly reinforces the view that thenature of the persistent functional changeunderlying the morphine tolerant state is indepen-dent of the opioid drug used to induce that state.Furthermore, the failure of naloxone treatment toinfluence either the rapid or slow phases ofrecovery of the analgesic response to morphinealso suggests that neither of these phases reflectthe rate of dissociation of morphine-receptorcomplexes, since opioid antagonists are thought tocompete with morphine for a common receptorsite (Cox & Weinstock, 1964; Grumbach &Chernov, 1965; Pert & Snyder, 1973).

The rate at which naloxone-induced jumpingactivity in mice declined after the termination ofthe initial morphine treatment was considerablyfaster than the rate of loss of tolerance to theanalgesic effects of inorphine; Cheney & Goldstein(1971) also have reported a rapid dissipation ofnaloxone sensitivity in the mouse.

If there is a common mechanism associatedwith physical dependence and tolerance then it ischaracterized by a rapid relaxation (Tl2 3-6 h)whilst there is associated separately with toleranceto the analgesic action or morphine, an additionalprocess with a half-time of about 1 7 days in miceand 1 3 days in rats. It is probable that the latterreflects the reversal of an opioid-inducedmetabolic disturbance, and knowledge of itsrecovery rate may assist in identification of theprocesses involved.

Appendix

Counter for recording mouse jumping activity

The jumping counter (Figure 8) operates on acounter-balance principle. The lever, 1, is a flatperspex strip 3 cm wide which pivots about a rod,2, and supports the floor and rear wall of thejumping compartment, 3, which are made of1.5 mm white perspex sheet. The excursion of thelever assembly is limited by a gate, 4, and adashpot, 5, is provided to dampen oscillations. Theposition of the counter balance weight, 6, is soadjusted that the weight of a mouse on the floorof the jumping compartment is just sufficient todepress that end of the lever assembly. When themouse jumps the position of equilibrium of thelever shifts and the tip of the adjustment screw, 7,closes the microswitch, 8 (Honeywell B2-2RW84N27-D14/68) and activates a digitalcounter (Type E 350:ITT Electronic Services).

The dimensions of each jumping compartmentare 20 x 15 x 30 cm high; the counter used in theexperiments described above has five of these,


mounted side-by-side. The three walls whichenclose the moving parts of each compartment arealso made of white perspex and are fixed to thechassis of the counter assembly. The front wall, 9,is hinged to facilitate cleaning the apparatus afteruse.

This work was supported by a grant from the MedicalResearch Council. We should like to thank Dr M.J. Fersterof Endo Laboratories, Inc., Brussels, for a gift ofnaloxone.

References

CHENEY, D.L. & GOLDSTEIN, A. (1971). Tolerance toopioid narcotics: time course and reversibility ofphysical dependence in mice. Nature, Lond., 232,477-478.

COCHIN, J. & KORNETSKY, C. (1964). Developmentand loss of tolerance to morphine in the rat aftersingle and multiple injections. J. Pharnac. exp. Ther.,145, 1-10.

COX, B.M., GINSBURG, M. & OSMAN, O.H. (1968).Acute tolerance to narcotic analgesic drugs in rats. Br.J. Pharmac. Chemother., 33, 245-256.

COX, B.M., GINSBURG, M. & JULIA WILLIS (1973).The offset of morphine tolerance in rats and mice. Br.J. Pharmac., 49, 159P.

COX, B.M. & OSMAN, O.H. (1970). Inhibition of thedevelopment of tolerance to morphine in rats by drugswhich inhibit ribonucleic acid or protein synthesis. Br.J. Pharmac., 38,157-170.

COX, B.M. & WEINSTOCK, M. (1964). Quantitativestudies of the antagonism by nalorphine of some ofthe actions of morphine-like analgesic drugs. Br. J.Pharmac. Chemother., 22, 289-300.

GOLDSTEIN, A., ARONOW, A. & KALMAN, S.M.(1969). Principles of drug action. New York, London:Harper & Row.

GOLDSTEIN, A. & SHEEHAN, P. (1969). Tolerance toopioid narcotics: I. Tolerance to the "running-fit"caused by levorphanol in the mouse. J. Pharmac. exp.Ther., 169, 175-184.

GRUMBACH, L. & CHERNOV, H.I. (1965). The

analgesic effect of opiate-opiate antagonist combina-tions in the rat. J. Pharnac. exp. Ther., 149, 385-396.

HERZ, A. & TASCHEMACHER, H.J. (1972). Activitiesand sites of antinocioceptive action of morphine-likeanalgesics and kinetics of distribution followingintravenous, intracerebral and intraventricular applica-tion.Adv. inDrugRes., 79-119.

MISRA, A.L. (1972). Disposition and metabolism ofdrugs of dependence. In: Chemical and biologicalaspects of drug dependence, pp. 219-277, ed. Mule,S.J. & Binn, H. Cleveland: CRC Press.

MARSHALL, I. & WEINSTOCK, M. (1969). Aquantitative method for the assessment of physicaldependence of narcotic analgesics in mice. Br. J.Pharmac., 37, 505-506P.

PERT, C.B. & SNYDER, S.H. (1973). Opiate receptors:demonstration in nervous tissue. Science, 179,1011-1014.

RANDALL, L.O. & SELLITO, J.J. (1957). A method formeasurement of analgesic acitivity on inflamed tissue.Arch, Int. Pharmacodynam, 111, 409-419.

WAY, E.L., LOH, H.H. & SHEN, F.H. (1969).Simultaneous quantitative assessment of morphinetolerance and physical dependence. J. Pharmac. exp.Ther., 167, 1-8.

WOOLFE, G. & MacDONALD, A.D. (1944). Theevaluation of the analgesic action of pethidine HCI(Demerol). J. Pharmac. exp. Ther., 80, 300-307.

(Received May 22, 19 74)

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THE OFFSET OF MORPHINE TOLERANCE IN RATS AND MICE