We tested if cannabinoid type 2 receptor (CB2) in the central nervous system plays a role in alcohol abuse/dependence in animal model and then examined an association between the CB2 gene polymorphism and alcoholism in human. Mice experiencing more alcohol preference by drinking showed reduced Cb2 gene expression, whereas mice with little preference showed no changes of it in ventral midbrain. Alcohol preference in conjunction with chronic mild stress were enhanced in mice treated with CB2 agonist JWH015 when subjected to chronic stress, whereas antagonist AM630 prevented development of alcohol preference. There is an association between the Q63R polymorphism of the CB2 gene and alcoholism in a Japanese population (P=0.007; odds ratio 1.25, 95% CI, (1.06–1.47)). CB2 under such environment is associated with the physiologic effects of alcohol and CB2 antagonists may have potential as therapies for alcoholism.
Alcohol is one of the oldest substances abused by human, and significant advances have been made towards understanding the neurobiological effects of alcohol. However, the exact mechanism that underlies alcohol addiction is not completely understood. Although classical genetic studies, such as twin and adoption studies, estimated the genetic contribution to alcoholism as approximately 0.5,1, 2, 3 few family-based linkage studies have yielded consistent linkages at specific loci.4, 5, 6, 7 The genetic polymorphisms in the alcohol metabolizing pathway, such as aldehyde dehydrogenase 2 (ALDH2)8 and alcohol dehydrogenase (ADH)9, 10, 11, 12 appear to be associated with alcoholism vulnerability. Aside from the genes encoding the metabolizing enzyme, there appear to be no single gene that plays a significant role in alcoholism, and small functional gene effects may act in conjunction with environmental factors to promote alcoholism. However, such gene–environment interaction underlie alcoholism have not been revealed yet, although a study reported that forced swim stress led to an increase of ethanol consumption in mice.13
Several lines of experimental evidence support roles for the endocannabinoid system in alcoholism. The endocannabinoid system consists of cannabinoid receptors, endocannabinoids, enzymes for the synthesis and degradation of endocannabinoids, and also perhaps endocannabinoid transporters, which have not been identified. There are two well-characterized cannabinoid receptors (CNRs), CB1/CNR1 and CB2/CNR2, that mediate the effects of endocannabinoid and exocannabinoid from marijuana use. CNR1 is expressed primarily in the central nervous system (CNS) and peripheral tissues, whereas CNR2 is expressed mainly in some peripheral and immune cells and has therefore been traditionally referred to as the peripheral cannabinoid receptor.14, 15 Studies in rodent models have suggested that CNR1 is involved in the neural circuitry regulating alcohol consumption and motivation to consume alcohol. For example, although CNR1 agonists stimulate alcohol intake and the motivational properties of alcohol, the CNR1 antagonist rimonabant suppresses acquisition and maintenance of alcohol drinking behavior, relapse-like drinking, and the motivational properties of alcohol in rats.16 Ethanol self-administration and ethanol-conditioned place preference were reduced in mice lacking Cnr1, and treatment with rimonabant reduced ethanol intake in heterozygotes but had little or no effect in the Cnr1 mutant mice, suggesting that cannabinoid system is an essential component of the molecular pathways that underlie the reinforcing effects of alcohol.17 In CNS, cannabinoids and ethanol activate the same reward pathways, and recent advances in understanding of the neurobiological basis of alcoholism suggest that the CNR1 system may play a key role in the reinforcing effects of ethanol and in modulating ethanol intake.18 CNR1 polymorphisms were found to be associated with polysubstance abuse, including alcoholism.19 Thus, cannabinoids, which are the main component of marijuana, act with exo-cannabinoids on the endocannabinoid system, which plays a significant role in vulnerability to development of addiction and other mental disturbances.20
CNR2 may also be associated with addiction vulnerability as a modulator of the reward system. CNR2 has been observed in the brainstem,21 cerebellum22 and several other regions of the brain.23 We also found that expression of Cnr2 is altered in response to cocaine and heroin (manuscript in preparation). In view of our findings that CNR2 is expressed in the mammalian brain, and that this expression is altered in response to addictive drugs, we hypothesized that genetic variants of CNR2 may have a significant effect on alcohol dependency. Recently, the polymorphism which makes the substitution of glutamine at amino acid position 63 by arginine (Q63R: two base pairs replacement polymorphism, although registered as single base polymorphism, rs2501432 in NCBI SNP database) was reported to be associated with autoimmune disease,24 and human osteoporosis.25 Sipe et al.24 reported its functional change from the polymorphism in the immune system by in vitro study that may suggest a differential function in CNS. Therefore, we examined a possible role of Cnr2 in alcohol preference/abuse/addiction in mice and association between the Q63R polymorphism in the CNR2 gene and alcoholism in a Japanese population.
Expression of Cnr2 is regulated by ethanol exposure in mice
After 15 days of free access to alcohol drink, there were differences in development of reinforcement for ethanol under the same experimental conditions. Although there was little difference in alcohol consumption during the days using 2–8% alcohol concentration, mice consumed alcohol differently when they had access to high concentration alcohol, 16–32% in the last 6 days of the experiment. The total alcohol consumptions during the last 6 days were significantly correlated with Cnr2 expression in ventral midbrain (F=11.2, P=0.005, R2=0.46), but not in other brain regions (Figure 1). These findings suggest that Cnr2 expression in midbrain appears to be related to development of ethanol addiction during reinforcement.
Cnr2 agonist and alcohol consumption in mice subjected to conditions of chronic mild stress (CMS)
One-way analysis of variance (ANOVA) showed a significant difference among four groups of mice treated with chronic mild stress (CMS) and the agonist (F=12.9, P<0.0001). Post hoc comparisons were made using student t-test (three groups compared to the control groups). CMS increased alcohol consumption (P=0.03), and that CMS in conjunction with a Cnr2 agonist dramatically increased alcohol consumption (P=7.5 × 10−7), whereas Cnr2 agonist alone did not appear to have an effect on alcohol consumption in mice (P=0.48) (Figure 2). Stepwise logistic regression analysis showed a strong combined effect from both of CMS and the agonist on alcohol consumption (R2=0.42).
Cnr2 antagonist and alcohol consumption in CMS mice
One-way ANOVA showed no difference between four groups of mice treated with (CMS) and the antagonist (F=1.35, P=0.28). Although CMS increased alcohol consumption (P=0.08), administration of the Cnr2 antagonist AM630 tended to weaken the effect of CMS on alcohol consumption (CMS vs CMS+AM630, P=0.09) (Figure 3). There was no difference in alcohol preference when AM630 alone was used (P=0.95).
Association between the CNR2 Q63R polymorphism and alcoholism
A significant association was found between the CNR2 Q63R polymorphism and Japanese alcoholics (P<0.01, odds ratio 1.25; 95% confidence interval, (1.06–1.47)]. Q recessive model (QQ vs QR+RR) of association between the CNR2 and alcoholism [P=0.005, odds ratio 1.42; 95% confidence interval, (1.11–1.80)] is indicated (Table 1). There was no association between CNR2 genotype and a risk of physiological dependence, such as delirium tremens or epilepsy, or age-onset of their alcoholism.
Because cannabis or pharmaceutical preparations of cannabinoid may have potential as therapeutic agents, it is important to analyze the neurobiological and behavioral consequences of administration of these compounds. We have continued to study the components of the endocannabinoid physiologic control system and the systems' role in brain function, substance abuse disorders and other neuropyschiatric disorders.
The present findings suggest that CNR2 plays a role in alcohol-related behavior. Ethanol altered Cnr2 gene expression in ventral midbrain, areas considered to be involved in brain circuits related to the reward properties of addictive substances, including alcohol. Interestingly, downregulation of Cnr2 expression in midbrain appeared to be related to reinforcement for alcohol (Figure 1). It is interesting that although inbred mice, such as those in the present study, should have the same genetic background, we observed different reinforcement phenotypes, suggesting that slight differences in environmental factors, such as the mild stressors, may affect addictive behavior. Although CMS alone appears to increase ethanol preference, and a Cnr2 agonist, JWH015, enhanced the CMS-increased alcohol preference, a Cnr2 antagonist, AM630, blocked the effect of CMS on ethanol preference.
Our genetic study in Japanese alcoholics indicated that the Q63R polymorphism in the CNR2 gene may be a functional polymorphism that influences alcoholism vulnerability. Although the amino acid 63 site is well-conserved as arginine in rodents, chimpanzee and baboon, there is a common polymorphism in human CNR2 protein which has glutamine.24, 26 Amino acid 63 locates in the first intracellular site. GENETYX (http://www.sdc.co.jp/genetyx) computer analysis of the secondary structure of CNR2 protein predicted that the polymorphism causes a structural change, thus the polymorphism could alter the receptor function. Although there is no population data registered in the public database, by NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or HapMap Project (http://www.hapmap.org/), no ethnic difference in the allelic distribution was observed in our population compared with that in the Caucasian population.24
Our previous study revealed that CMS increased Cnr2 protein expression in the brain.27 In the present study, a Cnr2 agonist enhanced CMS-induced alcohol preference, whereas an antagonist reduced the effect of CMS on alcohol preference. Recently, it was reported that endocannabinoid transmission was increased and Cnr1 expression was decreased as feedback in the prefrontal cortex of alcohol-preferring rats.28 Taken together, these findings suggests that high endocannabinoids activity, which is stimulated by stress, appears to be related to alcohol preference, which induces a compensatory downregulation of Cnr2 in brain. It is possible that endocannabinoids act like endorphin for reward in the brain and that high Cnr2 signal in brain may be a key modulator of susceptibility to alcoholism under condition of stress.
Alcoholism in humans may be caused by both genetics and environmental factors. There is a high incidence (comorbidity) of alcoholism and depression in the human population.29 Because CMS or forced swim stress that increase ethanol preference as shown in this study and in others,13 were also animal model of depression,27, 30 our mice and human data in this study are well suited to a model of addiction to alcoholism. The human CNR2 gene is located on chromosomes 1p36, nearby where only one report has indicated a broad and weak linkage to alcoholism,31 although common linkage area was reported around chromosome 1p31, not very close to CNR2 locus.32 Because our genetics data indicated that CNR2 has a moderate effect on human alcoholism and our mouse studies revealed the contributions of non genetic factors related to development of alcohol preference, it is likely that reinforcement of alcohol preference/addiction and stress may also influence endocannabinoids regulation that alters Cnr2 gene/protein expression in CNS. It is also likely that CNR2 contributes to initiation and development of alcoholism during periods of vulnerability owing to stress. Stress is thought to be an important factor at least in the initial phase in the development of alcoholism.
The Q63R polymorphism in the CNR2 gene was recently reported to be associated with autoimmune disease24 and with osteoporosis.25 Although the biologic and genetic mechanisms common between osteoporosis and alcoholism are not known, heavy alcohol intake and alcoholism disrupt calcium and bone homeostasis, which reduce bone mineral density and increase the incidence of fractures. Alcohol abuse has been suggested as lifestyle factor for secondary osteoporosis.33, 34, 35 Little has been reported about association between autoimmune disorders and alcoholism, however, one of the least-appreciated medical complications of alcohol abuse is altered immune regulation leading to immunodeficiency and autoimmunity.36 It appears that immunocannabinoid activity in the nervous system may play a significant role in controlling stress-responses and alcohol dependence, and CNR2 in the brain may be a novel target to modulate the effects of cannabinoids. Because CNR2 and environmental factors, such as stressors, appear to be involved in susceptibility to alcoholism, CNR2 antagonists may be useful for treatment of addiction.
Materials and methods
Regulation of Cnr2 expression response to alcohol in mice
C57/BJ6 male mice (age, 8–10-week-old; weight, 20–25 g) were housed under 12/12 h light/dark conditions. The group of mice (n=15) was housed separately when those had access to water and ethanol bottles for 15 h each night. During a rest of the day, those mice can access only water in group cages (five mice in each cage). The ethanol concentration was increased from 2–4, 8, 16 and 32% every 3 days. We measured water and ethanol consumption daily. We calculate total ethanol consumptions of each mouse during first 6 days (2-4% ethanol concentration) and those during last 6 days (16–32% ethanol concentration, which is not basically preferred by naïve mice) for further analysis in relation to Cnr2 gene expression. Control mice (n=5) had access only to water during the experimental period. 1 hour after the time of last access to ethanol bottle, mice were killed for RNA extraction from brain.
Brains were dissected into the prefrontal cortex, striatum, hippocampus and ventral midbrain. RNA was extracted with RNeasy kit (Qiagen, KK, Tokyo, Japan), and cDNA was synthesized with Revertra Ace (Toyobo, Tokyo, Japan) and oligo dT primer. Expression of Cnr2 gene was analyzed by TaqMan real-time polymerase chain reaction (PCR) with an ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA), with the TaqMan gene expression assay for Cnr2 (Mm0043826_m1), normalized between the reaction wells by Rodent GAPDH Control Reagents (Applied Biosystems, Foster City, CA, USA). The relative Cnr2 gene expressions in each brain region of alcohol-drinking mice were calculated based on averages of the Cnr2 gene expression in same brain region from control mice.
Alcohol preference under conditions of CMS and Cnr2 agonist administration
A total of 40 BALB/c male mice (age, 24–28 week; weight 25–30 g) were housed under 12/12 h light/dark cycles. CMS was established as described previously.27 Briefly, experimental animals were subjected to the weekly CMS regime of three 10-h periods of 45° cage tilt, three periods of overnight stroboscopic illumination, two 10 hour periods with empty water bottle, two periods of overnight food or water deprivation, two 10 h periods of damp bedding.
The CMS-treated and non-stressed groups comprised of 20 mice each, were then split into two subgroups. The first group (n=10) was subjected to chronic daily injection intraperitoneally (i.p.) of 20 mg/kg Cnr2 agonist JWH015 (Sigma, Tokyo, Japan). The second group (n=10) was subjected to equal volume per body weight saline injections. All non-stressed animals were given food and water ad libitum, as well as comfortable cage surroundings. Animals in the experimental group were housed in a different room. After 4 weeks, all mice were housed in normal cages. Ethanol (4% v/v) consumption of each mouse was then measured 24 h on the second day for comparison between mice with different treatments.
Alcohol preference under conditions of CMS and Cnr2 antagonist administration
A total of 30 BALB/c male mice (age, 10–12 weeks; weight, 25–30 g) were housed under 12/12 h light/dark conditions. An effect of Cnr2 antagonist AM630 (Tocris Cookson, Bristol, UK) was evaluated in twenty mice. We used the same protocol for CMS for 2 weeks, which had been shown to have the same stress level as 4 weeks of CMS in our previous study.27 The groups with/without CMS were subjected to chronic daily injection i.p. of 3 mg/kg Cnr2 antagonist AM630 (n=10 and 5, respectively). Animals with/without CMS were subjected to saline injections (n=5 and 10, respectively). After 2 weeks, all mice were housed in normal cages with access to water on the first day, and then were give access to ethanol (4% v/v) in 12 h at each night. Ethanol consumptions during 5 days, from second to sixth day, were measured and summed for comparison between mice with different treatments.
All animal studies above have been approved by the Review Board of University of Tsukuba.
Human DNA subjects
Alcoholics (n=785, age, 51.2±9 1 year, 667 male and 118 female) and 487 age- and gender-matched controls were research volunteers from mid-north main island area with diagnoses of alcohol dependence based on DSM-IIIR criteria without other psychiatric diagnoses, provided written informed consent to participate in the study. The average age-onset is 38.7±10.7 and 22% of the patients had alcoholic relatives within second degree. The rate of delirium tremens was 37% and that of convulsion was 16% in the population. The human study was approved by the Ethics Committee of University of Tsukuba.
Association study between the Q63R polymorphism of CNR2 and alcoholism
The genotype was determined by restriction fragment length polymorphism (RFLP) method after PCR amplification of the CNR2 region containing the polymorphism, with primers 5′-IndexTermCACCCCATGGAGGAATGCTGGGTGACAG and 5′-IndexTermGAACAGGTATGAGGGCTTTCGGCGG where T was replaced from C to destroy the problematic MspI restriction site for allele discrimination located near the polymorphic MspI restriction site. Amplification conditions were 94°C 10 min for predenaturation, followed by 35 cycles of 94°C 20 s, 67°C 30 s and 72°C 30 s and a single cycle of 72°C for 1 min in a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, USA). Reactions contained 1 ng of genomic DNA, GoldBuffer with 2.5 mM MgCl2, 0.16 mM dNTP and 0.5 U of AmpliTaq Gold Polymerase (Applied Biosystems, Foster City, USA) The PCR amplicons were digested with MspI (New England Biolabs, Beverly, USA) for 4 h, and then separated by 4% agarose gel electrophoresis. The PCR–RFLP genotype method was confirmed in 32 subjects tested by direct sequencing with a Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems, Foster City, USA) and an ABI3100 autosequencer.
Statistics and computer analysis of the gene structure
Deviations of the observed allele and genotype distributions from Hardy–Weinberg equilibrium were calculated with HWE computer program, and differences in allele frequencies between groups were tested for significance with Fisher's exact test on 2 × 2 contingency tables (http://linkage.rockefeller.edu/ott/linkutil.htm). A relationship between the gene expression and alcohol preference was examined by a correlation analysis. The comparisons of alcohol consumption between groups of mice treated with CNR2 agonist/antagonist and stress were made by one-way ANOVA and post hoc test was made using Student's t-test. Stepwise method was used to calculate an effect size on alcohol preference of either or both CMS and CNR2 agonist/antagonist. We considered P<0.05 to indicate significance.
cannabinoid type 2 receptor
chronic mild stress
central nervous system
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This work was supported financially by Ministry of Education, Culture, Sports, Science and Technology of Japan, Japan Science and Technology. Also it was supported by William Paterson, University Center for research and Dean, Dr Sandra DeYoung, who provided student worker support for the maintenance of laboratory animals. We also thank Dr Scott Hall (National Institutes of Health, National Institute on Drug Abuse) for technical advice regarding generation of a mouse model of alcohol preference and brain anatomy.
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Ishiguro, H., Iwasaki, S., Teasenfitz, L. et al. Involvement of cannabinoid CB2 receptor in alcohol preference in mice and alcoholism in humans. Pharmacogenomics J 7, 380–385 (2007). https://doi.org/10.1038/sj.tpj.6500431
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