Voluntary consumption of morphine in 15 inbred mouse strains

To determine genetic differences in voluntary morphine consumption, 15 commonly used inbred strains of mice were given ad libitum two-bottle choice between saccharin alone or saccharin/morphine in one bottle and water in the other bottle. Subsequently, the saccharin was gradually reduced to zero, leaving only morphine. Independent groups of mice of the same strains were exposed to quinine in a parallel manner to control for the bitter alkaloid taste of morphine. Of the 15 strains, the C57BL/6J strain showed the highest consumption of morphine, both with or without saccharin and greatest consumption of morphine relative to quinine; it also showed only a slight decline in fluid consumption when morphine was added to the saccharin bottle. In marked contrast, the SWR/J strain showed the least consumption of morphine by the same criteria, followed closely by the AKR/J, CE/J, DBA/2J and SJL/J strains. The strain differences for all the morphine drinking measures exceeded an order of magnitude. Strain-specific voluntary morphine/saccharin consumption was not significantly correlated with saccharin consumption alone, but was highly correlated with morphine consumption alone. The results show that these behaviors are under an unusually large degree of genetic determination, and some of the largest strain differences remained essentially the same regardless of whether saccharin was present, or whether quinine was used as a control tastant.     

5-HT(3A) receptor subunit is required for 5-HT3 antagonist-induced reductions in alcohol drinking.

The ionotropic serotonin subtype-3 (5-HT3) receptor has emerged as a potential therapeutic target in the treatment of alcohol abuse and alcoholism because selective pharmacological antagonists reduce alcohol consumption in preclinical and clinical models. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit but receptor activation is also enhanced by distinct allosteric sites, which indicates the presence of other receptor subunits. It is not known if specific molecular subunits of the 5-HT3 receptor modulate alcohol drinking. To address this issue, we characterized acute locomotor response to alcohol and alcohol consumption in a two-bottle home-cage procedure by congenic C57BL/6J mice with a targeted deletion of the 5-HT(3A) receptor subunit gene. 5-HT(3A)-null mice did not differ from wild-type littermate controls on measures of spontaneous locomotor activity, habituation to a novel environment, or locomotor response to ethanol (0, 0.5, 1, or 2 g/kg). Moreover, null mice did not differ from controls on measures of ethanol (2-10%) intake and preference during or after a two-bottle home-cage sucrose fading procedure. Systemic administration of the 5-HT3 antagonist LY-278,584 (0-10 mg/kg) decreased intake of both sweetened (2% sucrose+10% ethanol) and unsweetened (10% ethanol) ethanol in wild-type mice only. These findings indicate that reduction of alcohol drinking produced by 5-HT3 antagonism is dependent on the presence of 5-HT(3A)-containing receptors.     

Efficacy of choice testing to predict chronic ingestion of drinking solutions adulterated with chemicals

The feasibility of using a measure of palatability in a 2-bottle choice paradigm to determine detriments in fluid intake when unpalatable solutions containing drugs or chemicals were provided as a sole source of fluid was examined. Palatability measures obtained from testing various concentrations of quinine with water in a two-bottle choice paradigm were compared with intake of these same solutions when they were the sole fluid source for 20 consecutive days. Mice were observed to significantly avoid quinine solutions at concentrations as low as 0.0001 mg/ml in a choice situation while fluid intake was reduced in a forced drinking situation only at a concentration of 0.1 mg/ml. Palatability altered forced fluid intake only when quinine solutions comprised 20% or less of total intake in a choice situation. This approach was successfully employed to predict whether various concentrations of halogenated hydrocarbons could be administered in a repetitive forced drinking situation without reducing total fluid intake of mice.     

Influence of genetic background on alcohol drinking and behavioral phenotypes of 5-HT3 receptor over-expressing mice

Behavioral effects of genetic manipulations are influenced by the background genetics of mouse strains used for the creation of transgenic mice. One strategy to address whether background genes may compromise interpretation of phenotype is the production of congenics. 5-HT3 receptor over-expressing mice have been behaviorally characterized on a B6SJL/F2 background (B6SJL/F2-OE mice), and were found to consume less ethanol failed to develop conditioned place preference to moderate doses of cocaine and demonstrate improved hippocampal-dependent learning. To assess the contribution of parental strain genetics to these behaviors, we bred the transgene onto two well-defined backgrounds that differ in ethanol consumption and contextual fear conditioning, C57Bl/6J (B6) and DBA/2J (D2) strains. The behavioral phenotype of B6SJL/F2-OE was recapitulated in C57Bl/6J-OE mice. However, the effect of transgene over-expression on behavior was only apparent for one aspect of the novelty test using DBA/2J-OE mice. Results underscore the need to consider the genetic environment conferred by strain selection on the effects of genetic manipulation in mice.     

Ethanol preexposure increases ethanol self-administration in C57BL/6J and DBA/2J mice

Genetic variables are thought to interact with environmental factors, such as alcohol exposure history, to produce individual differences in alcohol abuse and alcoholism. The objective of this study was to test the potential interaction between genetic predisposition to consume alcohol and alcohol pretreatment on subsequent self-administration. To accomplish this goal, four groups of mice from the ethanol-avoiding DBA/2J (D2) and ethanol-preferring C57BL/6J (B6) inbred strains were exposed to saline, acute ethanol (2 g/kg), or chronic intermittent ethanol (1 or 2 g/kg) intraperitoneal (i.p.) injections. Locomotor activity was monitored after each injection. After preexposure, animals were given a two-bottle choice test with various concentrations of ethanol/sucrose vs. sucrose or ethanol vs. water for 4 days at each concentration. Then, all animals were challenged with a 2.0 g/kg ethanol i.p. injection and locomotor activity was assessed. Acute and chronic ethanol pretreatment increased locomotor activity in response to a challenge dose of ethanol (2 g/kg) in D2 mice but had no effect on B6 mice. Prior exposure to ethanol altered the amount of ethanol consumed in a mouse strain-dependent manner. D2 mice showed a positive relationship between ethanol intake and dose or duration of ethanol preexposure. B6 mice preexposed to ethanol consumed more ethanol than naive animals, independent of dose or duration of exposure. During the last phase of self-administration testing, D2 mice exposed to chronic ethanol (2 g/kg) consumed as much ethanol as B6 from the same pretreatment condition. After a history of ethanol self-administration, saline control mice from the D2 strain showed equal locomotor activation as compared to D2 mice that were pretreated with ethanol injections. B6 mice showed no change in locomotor activity after ethanol self-administration or injection. These results demonstrate that genetic predisposition to avoid alcohol (D2 mice) can be modified by a history of preexposure and that a predisposition to prefer alcohol (B6 mice) may be also amenable to influence by drug history. In general, the results of this study suggest that genetic factors may interact with previous exposure to ethanol to modify ethanol self-administration.     

Contributions of taste factors and gender to opioid preference in C57BL and DBA mice

C57BL/6J and DBA/2J mouse strains have been characterized as morphine preferrers and avoiders, respectively (Horowitz et al. 1977). Previously, sweetened morphine solutions were presented with a water alternative, primarily with male subjects. Because sweetness may affect the endogenous opioid system and rodents have shown strain and sex differences in taste preferences, this study looked for strain- and gender-related taste preferences that might have affected opiate consumption. Preference for sweetened and unsweetened morphine and etonitazene was compared across gender and strain. In all choice tests, the control was a similar tasting quinine sulphate solution. Under these conditions, C57BL/6J mice continued to show strong preference for morphine. However, DBA/2J mice drank approximately equal amounts of morphine and quinine solutions, rather than avoiding morphine as when water was the alternative. Both strains appeared surprisingly indifferent to the synthetic opioid etonitazene, compared because it is potent at concentrations having barely perceptible bitterness. This raises the possibility of unexpected differences in post-ingestional effects between morphine and etonitazene. Contrary to reports of gender differences in sweet preference in rats, none were found in either strain of mouse. Neither were there any significant sex differences in opiate preference in either strain. C57 mice preferred sweetness more than did DBA mice.     

Age-dependent and strain-dependent influences of morphine on mouse social investigation behavior

Opioid-coded neural circuits play a substantial role in how individuals respond to drugs of abuse. Most individuals begin using such drugs during adolescence and within a social context. Several studies indicate that adolescent mice exhibit a heightened sensitivity to the effects of morphine, a prototypical opiate drug, but it is unclear whether these developmental differences are related to aspects of motivated behavior. Moreover, exposure to opioids within the rodent brain can alter the expression of social behavior, yet little is known about whether this relationship changes as a function of development or genetic variation. In this study, we conducted a series of experiments to characterize the relationship between genetic background, adolescent development and morphine-induced changes in mouse social investigation (SI). At two time points during adolescent development [postnatal days (PD) 25 and 45], social interactions of test mice of the gregarious C57BL/6J (B6) strain were more tolerant to the suppressive effects of morphine [effective dose 50 (ED50)=0.97 mg/kg and 2.17 mg/kg morphine, respectively] than test mice from the less social BALB/cJ (BALB) strain (ED50=0.61 mg/kg and 0.91 mg/kg morphine, respectively). By contrast, this strain-dependent difference was not evident among adult mice on PD 90 (ED50=1.07 mg/kg and 1.41 mg/kg morphine for BALB and B6 mice, respectively). An additional experiment showed that the ability of morphine to alter social responsiveness was not directly related to drug-induced changes in locomotor behavior. Finally, administration of morphine to stimulus mice on PD 25 reduced social investigation of test mice only when individuals were from the B6 genetic background. Overall, these results indicate that alterations in endogenous opioid systems are related to changes in SI that occur during adolescence, and that morphine administration may mimic rewarding aspects of social encounter.     

Metabolic, molecular genetic and toxicological aspects of the acetylation polymorphism in inbred mice.

Over the past 10 years, much fascinating information has been obtained concerning the biochemistry, genetics, toxicological implications and molecular genetics of the N-acetylation polymorphism in mice. Using C57BL/6J (B6) mice as representative of rapid acetylation and A/J (A) mice as representing slow acetylation, it has been shown that the polymorphism observed in N-acetyltransferase (NAT) activity in liver also occurs in kidney, bladder, blood, and other tissues. The development of congenic acetylator mouse lines derived from B6 and A, have provided the necessary tools to study the role of the acetylation polymorphism, on either the B6 or A genetic background, free of nearly all other genetic differences between these strains. Eliminating genes which modify and complicate the differences due to the acetylator genes make the congenic lines very useful in toxicology studies, particularly those involving carcinogenesis. The molecular genetic basis of the acetylator polymorphism in B6 and A mice involves two Nat genes. Nat-1 encodes a protein termed NAT1 which is identical in rapid and slow acetylator strains. Nat-2, however, differs between rapid and slow strains by a single nucleotide change in the coding region. The corresponding NAT2 proteins differ by a single change at amino acid 99: an hydrophilic asparagine in rapid acetylator NAT2 to an hydrophobic isoleucine in NAT2 from slow acetylators. The mechanistic basis for the differences between rapid and slow acetylation in mice appears to be that NAT2 from the rapid B6 strain is 15-fold more stable at 37 degrees C and is transcribed/translated with a maximal efficiency twice that of the enzyme from slow acetylator A mice. Results discussed in this review indicate that mice provide an excellent system for studying the N-acetyltransferase polymorphism and also are useful for modelling several aspects of the human N-acetyltransferase polymorphism.     

Genetic Control of Drug-induced Recovery from Murine Visceral Leishmaniasis

Abstract— The influence of host genetic background on the response of Leishmania donovani-infected mice to chemotherapy was studied using the H-2^d and H-2^b haplotypes on a BALB or a B10 genetic background. Animals were treated with free or liposomal sodium stibogluconate and parasite burdens in the liver, spleen and bone marrow were assessed. In all the mouse strains and their congenic derivatives examined, the liver responded best to therapy regardless of drug formulation, whilst the spleen and the bone marrow respectively were increasingly less responsive to chemotherapy. Treatment with free drug was more effective in congenic mice carrying the H-2^b haplotype than in those carrying the H-2^d haplotype and in mice carrying the same H-2 haplotype, animals from a BALB background were better responders than those from a B10 genetic background. Liposomal drug was more effective than free drug treatment in all four mouse strains and produced a similar significant suppression (> 99%, P < 0·001) in liver parasite burdens to that obtained using a six times greater free drug dose. This liposomal drug dose was more effective than free drug in reducing bone marrow parasite burdens in all four mouse strains and equally (BALB/c mice) or more effective (P < 0·01, BALB/B, B10 and B10.D2 strains) in reducing spleen parasite numbers. Liposomal drug, particularly in the B10 genetic background, effectively negated the H-2 dependent influences apparent using free sodium stibogluconate. These results are discussed in relation to the genetic factors which are known to control the course of L. donovani infection in mice.     

Congenic strains developed for alcohol- and drug-related phenotypes

Quantitative trait loci (QTLs) for many alcohol- and drug-related traits have been mapped using well-accepted mapping techniques. The ultimate goal of gene identification necessitates confirmation of the QTL and reduction of the interval surrounding the QTL; both can be accomplished in congenic strains. These strains carry a chromosomal region introgressed from a donor strain onto the genetic background of a second, recipient strain. Multiple generations of backcrossing reduce the unlinked donor genome to less than 0.1%. Then, phenotypic comparisons between mice congenic for the donor region and controls from the recipient strain allow confirmation of the QTL effect. Animals with recombinations in the donor region can be used to generate interval-specific congenic recombinant lines. Numerous congenic strains are currently being developed in which chromosomal regions carrying QTLs for alcohol- and drug-related traits have been transferred from one strain onto a second strain. The purpose of this review is to summarize the chromosomal regions, donor and recipient strains, and results obtained from these congenics. Most researchers developing such strains are willing to share these resources to facilitate localization of the genetic bases of other phenotypes.     

Mapping murine loci for physical dependence on ethanol

RATIONALE: Alcoholism is associated with withdrawal (physical dependence), tolerance, or a maladaptive pattern of alcohol (ethanol) use. The well-documented difference in susceptibility to withdrawal after chronic ethanol exposure between the C57BL/6J and DBA/2J mouse strains provides an excellent starting point for dissecting genetic influences involved in physical dependence on ethanol. A quantitative trait locus (QTL) identifies the genomic location of a gene (or genes) affecting a trait of interest. OBJECTIVES: A genome-wide QTL mapping study was carried out to dissect the multifactorial nature of withdrawal after chronic ethanol exposure using 400 B6D2F2 mice. METHODS: To induce physical dependence, we used a standard paradigm in which mice were exposed to ethanol vapor for 72 h. The mice were then tested hourly for handling-induced convulsions (HICs) for 10 h and at hours 24 and 25. Ethanol withdrawal severity was first computed as the area under the 25-h HIC curve. Separate regression residuals were then calculated that corrected for individual differences in blood ethanol concentration at the time of withdrawal and baseline HIC severity (i.e. before ethanol exposure). RESULTS: Statistical mapping yielded significant evidence ( P<0.00005) for QTLs on chromosomes 19 and distal 1 that account for 45% of the genetic variance in ethanol withdrawal severity. The F2 results also provide supporting evidence for a sex-limited QTL on chromosome 13, and QTLs on chromosomes 4 and proximal 1, which may account for an additional 38% of the genetic variance. The distal chromosome 1 QTL is a locus of major effect, accounting for 26% of the genetic variance. Experiments using two congenic strains more precisely mapped this QTL. CONCLUSIONS: The QTLs map near candidate genes involved in neurosteroid biosynthesis and signal transduction. Syntenic homology between human and mouse chromosomes suggests that genes related to physical dependence on ethanol may localize to human chromosome regions 10q23-q26, 1q21-q43, 2q11-q32, 5p15/5q14-q21, and 9p24-p22.     

Genetic analysis of hypothermia induced by morphine in two strains of inbred mice

The degree of hypothermia elicited by morphine was greater in DBA/2 than C57BL/6 strain mice of both sexes. Hypothermia elicited by morphine was antagonized by naloxone in both strains of mice, suggesting the involvement of opioid receptors. To examine the role of genetic factors in the strain difference of morphine-induced hypothermia, the effect of morphine on changes in the rectal temperature was studied in the 6 generations of male mice, including the 2 inbred strains, P1 (DBA/2) and P2 (C57BL/6), their F1 and F2 hybrids, and 2 backcrosses, B1 (F1 X P1) and B2 (F1 X P2). The order of mean temperature decrease determined 40 min after 20 mg/kg morphine injection was P1 greater than B1 greater than F1 = F2 greater than B2 greater than P2. There was no maternal effect on the morphine responses of the F1 generation. Biometrical analysis revealed that DBA/2 (P1) is partially dominant over C57BL/6 (P2) and contribution of polygenes was suggested.     

Naltrexone and alcohol drinking in mice lacking beta-endorphin by site-directed mutagenesis

Alcohol-induced activation of the opioid system may contribute to the reinforcing properties of alcohol. This study investigated whether elimination of beta-endorphin (BE) synthesis via site-directed mutagenesis in embryonic stem cells would alter alcohol intake in mice. Both BE-deficient and wildtype (WT) mice generated from the targeted stem cells were backcrossed for nine generations onto a C57BL/6 background, and were maintained with ad libitum food and water. Mice had access to alcohol (10% v/v) under the following conditions: 24 h, scheduled access for 2 h/day, following acute (1 or 2 days) or chronic (5 weeks) alcohol deprivation, and scheduled access following six doses of naltrexone (0.125-16.0 mg/kg BW, ip) or saline treatment. Alcohol intake was similar in BE-deficient and WT mice given chronic access to alcohol, but greater in BE-deficient compared with WT mice during the first 10 days of scheduled access to alcohol, but not after more extensive experience with scheduled access. BE-deficient, but not WT mice, increased alcohol intake following 2 days, but not 1 day or 5 weeks, of deprivation. Naltrexone reduced alcohol drinking both in BE-deficient and WT mice, suggesting that drinking is mediated, in part, by activation of opioid receptors in both genotypes.     

Dopamine D3 receptor knockout mice and the motivational effects of ethanol

Dopamine D3 receptors have been implicated in the behavioral effects of abused drugs including ethanol. The present experiments characterized the acquisition of ethanol-induced place conditioning and ethanol self-administration in D3 knockout (D3 KO) mice compared with C57BL/6J (C57) mice. For place conditioning, D3 KO and C57 mice received six pairings of a tactile stimulus with ethanol (3 g/kg i.p.). D3 KO mice showed higher basal locomotor activity levels in comparison with the C57 mice during conditioning. Ethanol produced similar magnitudes of conditioned place preference in both genotypes. In a two-bottle drinking procedure, mice of each genotype received 24 h access to water and either 3% or 10% v/v ethanol. No difference was noted between D3 KO and C57 mice in either consumption or preference. In an operant self-administration procedure using 23 h sessions, D3 KO and C57 mice received access to 10% v/v ethanol on an FR4 schedule of reinforcement, food on an FR1 schedule of reinforcement and water from a sipper tube. D3 KO and C57 mice had similar response rates of ethanol and food as well as similar water intakes. Overall, these results indicate that elimination of D3 receptor function has little influence on ethanol reward or intake.     

Pituitary-adrenal axis and oral morphine consumption in rats.

Removal of the pituitary gland in rats leads to suppression of oral morphine and quinine intake behavior. Experiments measuring oral intake of solutions containing graded concentrations of morphine or quinine, revealed that the detection acuity for bitter taste is changed in hypophysectomized (hypox) animals. Treatment of these rats with ACTH 1--24 restored oral morphine intake towards that on intact rats. Morphine consumption in hypox rats was not affected by administration of ACTH 4--10 or ACTH 11--24, but was normalized by treatment with corticosterone. Adrenalectomy also diminished oral morphine intake. It is concluded that hypophysectomized animals refuse a morphine solution because their threshold for bitter taste quality is altered, presumably due to a diminished release of corticosteroids.     

Voluntary consumption of ethanol in 15 inbred mouse strains

To determine genetic differences in ethanol consumption, 15 commonly used inbred strains of mice were given ad libitum two-bottle choice between ethanol, 0.2% saccharin, or ethanol plus saccharin in one bottle versus tap water in the other bottle. Three different concentrations of ethanol were used: 3%, 6% and 10% (v/v). Of the 15 strains, the C57BL/6J, C57BR/cdJ and C57L/J strains showed the most consistent higher intake of ethanol either with or without 0.2% saccharin. In marked contrast, the DBA/1J and DBA/2J strains consistently showed the lowest intake. Consumption of 3% ethanol without saccharin was highly genetically correlated with saccharin consumption (r=0.77), suggesting that low concentrations of ethanol may have a sweet taste that affects voluntary consumption. Most strains showed very different patterns of response to ethanol with or without saccharin. Three patterns of strain responses were identified. Some strains avoided higher concentrations of ethanol whether in water or saccharin; some appeared to be sensitive to the ability of saccharin to mask the odor of ethanol; and some may have reduced consumption only when ethanol concentrations were high enough to produce aversive postingestional effects. Whereas earlier studies generally attempted to explain strain differences in consumption by invoking a single mechanism, our results demonstrate that more than one mechanism is necessary to explain the preferential ethanol intake of all strains studied.     

Morphine preference in individual rats after morphine ingestion

Individual differences in drug intake were investigated. Inbred Sprague-Dawley male rats were choice-tested after various periods of morphine ingestion. Nearly 10% of the rats showed more than 50% preference already after 4 days ingestion on 340 mg morphine/kg/day, while a further 10% had a mean preference less than 30% over 6 days of choice, even after as long as 38 days' treatment on this same dose. High morphine preference was stable for long choice periods. It was also found that a high morphine preference level in an individual rat persisted over several choice tests, even if the animals had been without morphine for several months. The ₂-agonist clonidine diminished high preference to the same extent as it diminished over-all morphine preference. There were no differences in food intake, body weight gain, severity of abstinence reactions, morphine serum levels, taste sensitivity tested with quinine, or learning the choice test behaviour comparing extremely high and low morphine preference rats. Thus, two subgroups of high and low morphine-ingesting rats were identified in the Sprague-Dawley strain.     

Quantitative trait loci for acute behavioral sensitivity to paraoxon

Genetic mechanisms responsible for organophosphate (OP)-induced behavioral changes remain obscure. In the present study, provisional quantitative trait loci (QTL) associated with acute sensitivity or insensitivity to hypolocomotion produced by the OP paraoxon were identified. Naive adult male and female mice of the BXD/Ty series (22 different BXD strains plus C57BL/6J and DBA/2J progenitor strains) received 0 or 0.25 mg/kg paraoxon (IP), immediately before placement in an activity chamber for a 30-min test. As expected, based on dose-response and time course studies with Swiss-Webster, C57BL/6, and DBA/2 mice, paraoxon treatment reduced locomotor activity in most, but not all BXD strains. Heritability (proportion of phenotypic variability attributed to genetic differences) was 0. 58 for the paraoxon treatment effect. Difference scores (strain mean for vehicle activity minus strain mean for paraoxon activity), and percent change in activity of paraoxon-treated mice compared to vehicle-treated mice were calculated for each BXD strain. QTL analyses using activity difference scores and percentage change in activity were conducted using a database with over 1300 unique genetic markers. Several provisional QTL found on different chromosomes were associated with the activity phenotype. Of these, several markers attained p<0.01 or greater. These were as follows: Chr 1: Ly9, p<0.006; Chr 6: D6Ncvs44, p<0.0005; Chr 9: D9Mit15, p<0. 003; Chr 11: D11Ncvs76, p<0.002; Chr 15: Tstap198, p<0.008. In addition, several markers on chromosome 3 approached p<0.01. Identified genes found near these regions include two plasma carboxylesterase alleles on chromosomes 6 and 9, a glutamate receptor subtype on chromosome 11 and a glycine receptor subunit on chromosome 11, raising the possibility that these genes could be the basis for these provisional QTLs.     

Dependence on and preference for morphine. (III) Improvement of spontaneous drug intake after combined treatment with morphine and cocaine]

One group of rats (n equals 6) was allowed free access to food combined with morphine and cocaine for 3 weeks, another group combined with either morphine or cocaine alone for 3 weeks. Intensity of decrease in body weight by withdrawal and ratios of spontaneous morphine and cocaine intake were compared, and the cross-spontaneous drug intake between morphine and cocaine using cocaine and morphine dependent rats was investigated, respectively. The results indicate that (a) decreased rate in body weight by withdrawal after 3 weeks administration with morphine-cocaine mixtures increased slightly, and spontaneous intake ratio for the higher drug-admixed food during administration period was also slightly increased as compared with a morphine added diet alone. (b) However, spontaneous intake ratio of both morphine [morphine (1 mg/g of food) vs. a normal diet (N.D.)] and cocaine [cocaine (2 mg/g of food) vs. N.D] increased 30 and 50 approximately 60% respectively, and these changes were maintained for about two months. (c) Spontaneous cocaine intake ratio after 3 weeks administration of morphine was almost the same level as that in cocaine dependent rats and decreased dependently when the administration period was extended. Spontaneous morphine intake ratio after 3 weeks administration of cocaine was 20% lower in total daily intake ratio as compared to morphine dependent rats, but spontaneous morphine intake ratio as compared to morphine dependent rats was gradually increased as in morphine dependent rats. (d) Furthermore, morphine-quinine combined treatment and cocaine or codeine treatment had no effect on spontaneous intake of quinine. These data suggest that combinations of morphine and cocaine have promoting properties of morphine and cocaine intake, respectively. Cross-spontaneous drug intake between morphine and cocaine was successful only in cocaine dependent rats using this method.