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Abstract
Aims: Previous studies have shown that CB1 cannabinoid receptors are involved in the behavioural effects induced by chronic ethanol administration in Wistar rats by using SR 141716, a CB1 cannabinoid receptor antagonist. These studies have now been extended to investigate the effect of acute and chronic alcoholization on blood ethanol concentration (BEC) and ethanol preference in CB1 knockout (−/−) mice. Methods: BEC was monitored for a period of 8 h in both male mice and CB1 male wild-type (+/+) mice, which had received an acute i.p. injection of ethanol in 1, 3 or 5 g/kg doses. Ethanol preference was assayed in both groups of male mice in non-forced ethanol administration and forced chronic pulmonary alcohol administration for 14 and 39 days, respectively. Results: After an acute intraperitoneal ethanol injection of 5 g/kg, mice showed a significant higher BEC during the ethanol elimination stage than the mice. However, those in the 1 and 3 g/kg groups showed no significant difference. A 2—3 fold increase in BEC was observed in mice on days 10 and 11 after commencement of forced chronic pulmonary alcoholization in comparison with mice, although comparable BEC values were assayed in both groups on day 12. In addition, these mice showed a significantly lower preference for ethanol than mice. Conclusions: The studies on and mice have clearly confirmed the involvement of CB1 receptor on ethanol induced behavioural effects and also revealed that CB1 receptors may be implicated in ethanol absorption/distribution, particularly after administration of high ethanol doses.
INTRODUCTION
The endocannabinoid system consists not only of G-protein-coupled cannabinoid receptors but also other components such as enzymes [i.e. fatty acid amidohydrolase (FAAH)] and endogenous ligands. There are two types of cannabinoid receptors, CB1 and CB2. The CB1 cannabinoid (CB1) receptor is predominantly expressed in brain at relatively high levels in hippocampus, cerebellum and spinal cord and thus, often referred to as the brain cannabinoid receptor. CB1 receptors are also expressed at low levels in peripheral tissues including spleen, testis and leucocytes (Herkenham et al., 1991; Bouaboula et al., 1993; Lévénès et al., 1998; Navarro et al., 1998). The CB2 receptor is referred to as the peripheral cannabinoid receptor because it mainly shows peripheral expression in immune cells (Munro et al., 1993; Facci et al., 1995). To date, there are two endogenous ligands of CB1 receptors, namely anandamide and 2-arachydonylglycerol, which mimic the pharmacological action of Δ9-tetrahydrocannabinol, the active compound of marijuana and other synthetic agonists (Devane et al., 1992; Mechoulam and Fride, 1995; Mechoulam et al., 1995; Stella et al., 1997). There is growing evidence for the implication of endogenous cannabinoids in biological functions, including the control of appetite and food intake (Di Marzo et al., 2001), the modulation of some of the pharmacological effects of ethanol (Basavarajappa et al., 1998, 2000; Basavarajappa and Hungund, 1999a,b, 2002; Hungund and Basavarajappa, 2000a,b; Hungund et al., 2002) and drinking behaviour (Arnone et al., 1997; Colombo et al., 1998; Gallate et al., 1999; Gallate and McGregor, 1999; RodrÃguez de Fonseca et al., 1999; Freedland et al., 2001; Colombo et al., 2002; Hungund et al., 2002).
Chronic ethanol administration has a dual effect on the cannabinoid receptor, increasing the level of both endogenous cannabinoid agonists, anandamide and 2-arachidonylglycerol, while downregulating the CB1 receptor number and function, thereby suggesting a role for the endocannabinoid system in the neurobiological effects of ethanol (Basavarajappa et al., 1998b, 2000; Basavarajappa and Hungund, 1999a,b). Nonetheless, ethanol did not produce any effects on CB1 receptor binding and mRNA levels in rats (Gonzalez et al., 2002). However, a recent study by Ortiz et al. (2004) showed that forced consumption of high quantity of ethanol for a long period significantly decreased the gene expression of the CB1 receptors in the caudate-putamen, the ventromedial nucleus of hypothalamus and both CA1 and CA2 fields of the hippocampus. The last finding is in accordance with Basavarajappa et al. (1998) and Basavarajappa and Hungund (1999a). The result obtained by Gonzalez et al. (2002) could be due to differences in the quantity of ethanol and the duration of ethanol administration.
CP-55,940, a CB1 receptor agonist, promoted alcohol craving in rats (Gallate et al., 1999), as well as voluntary ethanol intake in Sardinian alcohol-preferring (sP) rats (Colombo et al., 2002). WIN-55,212—2, another CB1 receptor agonist, also promoted voluntary ethanol intake in sP rats (Colombo et al., 2002).
Numerous studies have shown that the CB1 receptor antagonist SR 141716 reduces ethanol intake (Arnone et al., 1997; Colombo et al., 1998; RodrÃguez de Fonseca et al., 1999; Freedland et al., 2001) and ethanol craving (Gallate and McGregor, 1999) in different rat strains. In addition, SR 141716 suppressed the ethanol deprivation effects (i.e. the temporary increase in ethanol intake after a period of ethanol withdrawal) in sP rats (Serra et al., 2002). All these results suggest that the blocking of CB1 receptor decreases the consumption of ethanol. Nonetheless, it is also important to mention that in our previous study in Wistar rats, we showed that the cannabinoid receptor antagonist SR 141716 profoundly altered ethanol preference in chronically pulmonary alcoholised rats depending on the dose and time of administration. Doses of 3 or 10 mg/kg/day, administered during chronic pulmonary alcoholization enhanced ethanol preference whereas its administration during the ethanol withdrawal stage after alcoholization induced a decrease in ethanol preference (Lallemand and De Witte, 2001). We have also shown that the action of SR 141716 was dependent on a number of factors, including the duration of ethanol intoxication as well as the number of ethanol re-exposures and ethanol withdrawals (Lallemand et al., 2004).
All these previous studies used antagonists and agonists of CB1 receptors. In certain circumstances, some antagonists have side-effects, which could alter/modify their actions. For example, SR-141716, a CB1 receptor antagonist, can show agonist property (Shire et al., 1999).
An alternative to avoid these possible pharmacological side-effects is the use of null mutant mice. The development of transgenic CB1 knockout mice has provided the opportunity to study the role of the CB1 receptor system in the regulation of ethanol consumption (Ledent et al., 1999; Zimmer et al., 1999).
mice with CD1 background showed decreased ethanol intake and preference. These effects were associated with a dramatic sensitivity to the hypothermic and hypolocomotor effects in response to low doses of ethanol (Naassila et al., 2004). These mice also showed an increased intensity of ethanol withdrawal-induced convulsions. Female mice consumed more ethanol than male mice; in addition, this gender difference was observed in both genotypes – female mice showed a decreased ethanol consumption compared with that of female mice, but did consume the same quantity of ethanol as did male mice. Hungund et al. (2003) observed similar results, although the gender difference in ethanol consumption observed between female and male mice was abolished in mice. These results were also observed in the study of Poncelet et al. (2003) using mice with C57BL/6 × 129/Ola F2 background.
with C57BL/6J background had a higher preference for ethanol but only for a few days (Racz et al., 2003). After the cessation of chronic ethanol administration, these mice did not exhibit withdrawal symptoms. After mild intermittent foot-shock stress, alcoholized mice did not consume an increased amount of ethanol as did the mice for the next 24 h. The activation of CB1 receptors in wild-type mice will also contribute to the high ethanol preference exhibited by C57BL/6J mice (Wang et al., 2003) as SR 141716 is able to reduce ethanol drinking when administered to these mice and not in mice. Young and old mice with this genetic background displayed low ethanol preference. On the contrary, mice presented an age-dependent decline in ethanol preference, suggesting that the decline in ethanol preference is related to a loss of cannabinoid signalling in the limbic forebrain.
It could be hypothesized that there was an interaction of gender and expression of phenotype associated with the CB1 gene mutation. The total fluid intake was similar between the different genotypes, although differences were evident between males and females within the same genotype. male mice did not show the acute ethanol-induced increase in dopamine levels in nucleus accumbens compared with mice, which would indicate that activation of the limbic system was required for the reinforcing effects of ethanol (Hungund et al., 2003).
The purpose of our study was to investigate the effect of a low to high acute intraperitoneal ethanol injection on blood ethanol concentration (BEC), as well as the effects of non-forced ethanol administration and forced chronic pulmonary ethanol intoxication on ethanol preference by comparing and mice to ascertain the precise involvement of the cannabinoid system on ethanol-related behavioural effects.
MATERIALS AND METHODS
Homozygous male mice were compared with homozygous male wild-type mice. and mice were from a C57BL/6 J × 129/Ola (Harlan) F2 genetic background and generated as described previously (Robbe et al., 2002; Ravinet-Trillou et al., 2003). No backcrosses were performed. These mice were provided by Sanofi-Recherche Synthélabo (Montpellier, France). The mice were housed in clear plastic cages with steel wire fitted tops and wood chip bedding under standard conditions (normal 12 h light—dark cycles, light on at 08:00 h, constant room temperature of 25 ± 1°C) with commercial lab chow diet and tap water available ad libitum during the entire experiments.
Acute ethanol experiments
BEC was assayed in and , 30—32 g, 12-weeks-old, male mice, housed 5/cage, after an intraperitoneal injection of ethanol. The experiment was carried out in their home cages. Blood samples were collected from the retro-orbital sinus under slight ether anaesthesia where necessary, into haematocrit tubes at 20, 40 min and 1, 2, 4, 9 and 12 h, after either 1 or 3 g/kg ethanol doses (15% v/v), while an additional two samples at 14 and 16 h were also collected after the 5 g/kg dose. This procedure followed the schedule of blood drawing used by Bruguerolle and Dubus (1993), Bruguerolle et al. (1994) and Hettiarachchi et al. (2001). Blood from each haematocrit tube was transferred into microcentrifuge tubes containing sodium fluoride as an anticoagulant. The concentration of blood ethanol was assayed by an alcohol-dehydrogenase-based method (Aufrère et al., 1997).
Chronic ethanol experiments
Non-forced ethanol administration experiments. and , 30—32 g, 12-weeks-old, male mice, were housed 2/cage. Fluid intake (water and 10% v/v ethanol when present) was recorded every 1 or 2 days, and body weight every week.
Free-choice period. Two drinking bottles were placed in each cage, one containing tap water and the other, 10% v/v ethanol solution. The mice had continuous access to the drinking tips of both tubes. The position of the tubes was changed every day, in order to avoid possible bias due to place preference. The ratio of the 24 h intake from the ethanol bottle versus total fluid intake was used to define preference and the absolute amount (g/kg body weight/day) of ethanol consumed was also calculated.
Forced chronic pulmonary ethanol administration procedure. The motility of and , 30—32 g, 12 weeks old, male mice, was recorded, after 3 weeks of acclimatization, for 18 h by the MacLab system, the recordings being combined for each hourly interval. The apparatus has been described in detail previously (Lallemand and De Witte, 2001).
Forced chronic alcoholization was induced in these mice, housed in pairs of two, within a plastic chamber (120 × 60 × 60 cm) by pulmonary inhalation of a mixture of ethanol and air. The mixture was pulsed into the chamber via a mixing system that allowed the quantity of ethanol to be increased every day, so that the average BEC continued to rise (Le Bourhis, 1975; Aufrère et al., 1997) during the experimental procedure. The animals remained for 12 days in the alcohol chamber. The chamber temperature ranged between 28—30°C (Terdal and Crabbe, 1994; Finn and Crabbe, 1999). BECs were determined regularly during the chronic alcoholization. Blood from each haematocrit tube was transferred into microcentrifuge tubes containing sodium fluoride as an anticoagulant. The concentration of blood ethanol was assayed by an alcohol-dehydrogenase-based method.
Withdrawal motility and free-choice period. At the end of the forced chronic pulmonary alcoholisation period the motility and ethanol preference was studied in these two groups of mice. For the measurement of ethanol preference, the mice from each strain underwent three successive steps (Le Bourhis, 1977) on cessation of the chronic ethanol intoxication. First, full beverage deprivations, i.e. the drinking bottles were removed during the last 6 h of the chronic alcoholization procedure and the following 18 h of the withdrawal period. The motility of each mouse was recorded during these 18 h using the same apparatus described above. Secondly, a 10% (v/v) ethanol solution was given as the sole drinking fluid during the following 24 h. Thirdly, a free-choice beverage situation [water vs 10% (v/v) ethanol solution] was presented for a period of 39 days. During this free-choice period, the fluid consumptions were recorded daily and ethanol consumption expressed as a percentage of total fluid intakes and as ethanol intake in g/kg of body weight. The positions of the drinking bottles were changed every day to avoid position preference. BECs were assayed at different time points during the free-choice period by the method described above. The weight of animals was recorded every 3 or 4 days.
In all experiments, the results are presented as mean ± standard error (SE) except where stated otherwise. In all experiments, groups were compared by two-way analysis of variance (ANOVA) (genotype; time) with repeated measures on time. Where appropriate, post hoc pair wise comparisons were analysed by the least-significant difference test of multiple comparisons (Fisher LSD protected t-test) (GB-STAT 5.3 for Windows, Dynamic Microsystems, Silver Spring, MD, USA). Criterion for significance was set at P < 0.05 for all tests.
The Belgian Governmental Agency under the authorized number LA 1220028 as well as the European Communities Council Directive concerning the Use of Laboratory Animals approved these experiments.
Products
Absolute ethanol, used in the free-choice paradigm and acute experiment, was obtained from Labotec (La Gleize, Belgium). Ethanol at 15% (w/v) was prepared for i.p. injection in 0.9% saline. Ethanol at 97% was obtained from Belgalco SA (Belgium). Sodium fluoride was from Sigma Aldrich, (Steinheim, Germany).
RESULTS
Acute ethanol experiments
The BECs were similar in both groups of mice after either 1 or 3 g ethanol/kg doses [F(1,56) = 0.0005, P = 0.982 and F(1,56) = 0.1161, P = 0.7421 respectively]. However, after an acute injection of 5 g/kg of ethanol, mice showed significant differences compared to the mice [F(1,8) = 19.254, P = 0.0022], with a significantly higher BEC than the mice [F(9,72) = 2.981, P = 0.0045] (Fig. 1).
Chronic ethanol experiments
Non-forced ethanol administration experiments. After 1 week of measurements, mice showed a significantly higher water consumption in comparison to mice [F(1,54) = 6.8364, P = 0.0176] (data not shown). The mean water consumptions over the time of the experiment were 10.8 ± 1.24 and 13.8 ± 0.87 ml, respectively for and mice.
At the conclusion of the study, the mice showed a significantly lower mean weight in comparison to controls [F(1,38) = 7.3466, P = 0.01]. The mean weights were 32.18 ± 0.62 and 30.16 ± 0.36 g, respectively, for and mice.
Free choice. mice showed a significantly reduced ethanol preference (expressed as a percentage of total fluid intake) in comparison to control mice [F(1,12) = 8.6787; P = 0.0122] (Fig. 2A). There was also a significant interaction between genotype and time [F(8,96) = 2.1965, P = 0.0342]. Nonetheless, when ethanol preference is expressed as ethanol intake in g/kg of body weight, the genotype significance disappeared totally and only the interaction remained [F(38,418) = 3.9539; P < 0.0001] (Fig. 2B). The mean ethanol intake over the time of the experiment was 12.28 ± 0.48 and 13.12 ± 0.59 g/kg/day, respectively, for and mice.
When comparing liquid type consumptions, i.e. water and ethanol, ethanol volume consumed in mice of either genotype was not significantly different [F(1,12) = 0.2861, P = 0.6025]. On the contrary, mice consumed significantly more water than mice [F(1,12) = 14.1872, P = 0.0027].
During the free-choice period the total consumption (water + 10% v/v ethanol) of mice was not significantly different in comparison to control mice [F(1,26) = 3.3544, P = 0.0785] (data not shown), but there was a significant interaction between genotype and time [F(8,208) = 4.3224, P < 0.0001]. Nonetheless, the total consumption of mice was always above that of control mice. The mean consumptions over the time of the experiment were 9.67 ± 0.70 and 7.23 ± 0.33 ml/24 h, respectively, for and male mice.
Forced chronic ethanol pulmonary administration experiments. The motility of mice, prior to forced chronic ethanol pulmonary administration, was not significantly different in comparison to mice [F(1,510) = 0.7872, P = 0.382].
In the mice the mean BEC assayed at different time points during the forced chronic alcoholization regime were significantly different than the mean levels in the mice [F(1,26) = 25.887, P < 0.0001] characterized by a significant higher BEC level at both 10 and 11 days after the commencement of forced chronic pulmonary alcoholization. [F(6,156) = 7.931, P < 0.0001] (Fig. 3). At 10 days, the mean BEC was 3 fold higher in the mice than in the mice, whereas at 11 days, it showed a 2 fold increase. However, on Day 13 no significant difference in mean BEC was assayed.
During the forced chronic pulmonary alcoholization period, water consumption of mice was not significantly different [F(1,18) = 1.7514, P = 0.2023] (data not shown). The mean water consumptions over the time of the experiment were 9.82 ± 0.5 and 8.76 ± 0.48 ml/24 h, respectively, for and mice.
mice had a significantly lower body weight than mice [F(1,38) = 7.3466, P = 0.01]. The mean weights over the time of the experiment were 30 ± 0.46 and 32 ± 0.62 g, respectively, for and mice.
Following forced chronic pulmonary alcoholization, similar motilities were assayed for both and control mice [F(1,340) = 0.8442, P = 0.3704] as they were also similar prior to the chronic pulmonary alcoholization.
Free choice. During the first 24 h period after forced chronic pulmonary alcoholisation, there were no significant differences in ethanol consumption between and mice [F(1,11) = 0.4936, P = 0.4969]. The mean ethanol consumptions in the alcoholized group and in the alcoholized group were, 18.69 ± 1.69 ml and 16.75 ± 1.29 ml, respectively. During the free-choice period, ethanol preference, expressed as percentage of total fluid consumption, of mice showed no significance at the genotype level when compared with mice [F(1,11) = 2.1819, P = 0.1677] (Fig. 4A). There was also absence of significance when ethanol preference was expressed as ethanol intake/kg body weight [F(1,11) = 1.6614, P = 0.2239] (Fig. 4B). Nonetheless, in both representations of ethanol preference, there were always significant interactions between genotype and time [F(38,418) = 2.345, P < 0.0001 and F(38,418) = 3.9539, P < 0.0001, respectively, for percentage of total fluid consumption (Fig. 4A) and ethanol intake expressed in g/kg body weight (Fig. 4B)]. The mean ethanol intakes in all experiments were 17.96 ± 0.52 and 22.05 ± 0.69 g/kg, respectively, for and mice.
When the ethanol and water consumptions in each genotype were compared, no significant differences between the two liquids in both [F(1,12) = 0.0356, P = 0.8534] and mice [F(1,10) = 3.5586, P = 0.0886] were apparent. Nonetheless, there were always significant interactions between liquid type and time in both genotypes [F(38,456) = 8.1675, P < 0.0001 and F(38,380) = 6.2574, P < 0.0001, respectively, for and ]. In mice, the consumptions of water and ethanol were very similar (8.42 ± 0.25 and 8.93 ± 0.23 ml, respectively, for ethanol and water). In contrast, in mice, the ethanol consumption was always lower than the intake of water (8.81 ± 0.27 and 11.21 ± 0.21 ml, respectively, for ethanol and water). In addition, water intake of mice was higher than in the mice.
The total liquid consumption was not significantly different in and mice [F(1,11) = 1.3981, P = 0.262], although there was a significant interaction between genotype and time [F(38,418) = 3.5209, P < 0.0001]. The total consumption values for mice were always greater or at the same level for those of the mice.
During the free-choice paradigm, there was no significant difference between the BEC values at the genotype level [F(1,4) = 3.092, P = 0.1535]; the values assayed being less than 0.02 g/l in both groups of mice (data not shown). However, there was a significant interaction between genotype and time [F(5,20) = 2.849, P = 0.0422], as well as for time [F(5,20) = 20.192, P < 0.0001].
During the free-choice period, mice showed a significantly lower body weight than the mice [F(1,18) = 9.5004, P = 0.0064] (data not shown). There was also a significant interaction between genotype and time [F(10,180) = 6.851, P < 0.0001]. The body weight of mice at the beginning of the study was 31.2 ± 0.6 and 31.14 ± 0.67 g at its conclusion. The body weight of mice was 32.36 ± 0.75 g at the beginning of the study and was 37.64 ± 1.39 g at the end.
DISCUSSION
Recently, mouse specific gene deletions have been used to investigate the role of the endocannabinoid system in alcohol research. In this study we assessed the effect of CB1 receptor null mutation on ethanol preference in both non-alcoholized and chronically alcoholized mice as well as ethanol clearance after an acute ethanol i.p. injection. The acute ethanol injection in mice lacking the CB1 receptor showed an unexpected result in that the ethanol peak concentration for the high ethanol dose, 5 g/kg, induced a significantly higher ethanol peak concentration in mice. However, the ethanol elimination rates for the lower doses, 1 and 3 g/kg, were similar in both and mice. This has not been described previously in the literature for mice. Nonetheless, the influence of the cannabinoid system on the metabolism of ethanol was reported in one study where the administration of cannabinoid receptor inhibitor SR 141716 induced no changes in ethanol metabolism in rats (Colombo et al., 1998). It is difficult to interpret these present results. As the 1 and 3 g/kg ethanol doses showed no significant change between and , we hypothesized that, with respect to the high dose of ethanol used, the lack of CB1 cannabinoid receptors in the enteric nervous system, particularly at the level of the gastrointestinal tract of mice, might interfere with the absorption/distribution of ethanol (Batkai et al., 2001; Pertwee, 2001). However, this lack would intervene only with a high acute dose of ethanol.
In mice with non-forced ethanol administration, ethanol preference ratio was significantly reduced in mice, but when ethanol preference was expressed as g/kg body weight per day, no significances appeared. These results are in agreement with those obtained by Wang et al. (2003) for ethanol preference ratio. Nonetheless, other studies by Hungund et al. (2003) and Poncelet et al. (2003) observed that ethanol intake expressed in g/kg body weight/day was significantly reduced in mice as well. This discrepancy on the preference in ethanol intake was unclear. In our study, the absence of significance in preference as expressed in g/kg body weight/day is mainly the result of a higher, but not significant, total liquid intake of the mice.
In chronically forced alcoholized mice, the BEC in mice peaked faster than in mice, although the maximum values obtained were not significantly different. This result has not been observed in previous studies, although Colombo et al. (1998b) showed that the antagonism at CB1 cannabinoid receptors did not modify ethanol metabolism. In our study, the difference between and mice was noted only during the increase of BEC but not at the end of the chronic alcoholization period. Unlike other chronic alcoholization procedures, our protocol of chronic alcoholization is a forced one, i.e. animals were unable to adjust the amount of ethanol ingested by themselves. Our procedure of chronic alcoholization induced other mechanisms involved in ethanol metabolism microsomal ethanol oxidizing system MeOS/cytochrome P450IIE (Lieber, 1999) and alcohol dehydrogenase (Kishimoto et al., 1995), which have not been studied to date in these knockout animals.
After forced chronic pulmonary alcoholization, the ethanol consumption in mice was similar to that of when access to 10% (v/v) ethanol solution was given. In contrast, when mice had access to both drinking bottles, i.e. free choice, their ethanol preference was significantly lower than mice when expressed as percentage of total consumption. This result is in agreement with our previous study in Wistar rats of the action of the CB1 cannabinoid receptor inhibitor SR 141716 (Lallemand and De Witte, 2001) and data reported recently by Hungund et al. (2003), Poncelet et al. (2003), Racz et al. (2003), Wang et al. (2003) and Naassila et al. (2004), which show that a CB1 receptor antagonist decreases ethanol consumption in rats and mice. Nevertheless, when ethanol preference is expressed as g/kg body weight/day, mice presented significant ethanol intake time point higher than mice.
In both non-forced alcoholized and chronically forced alcoholized experiment, the mice showed a significantly lower weight than the mice. This result was in contradiction to the results from a previous study (Wang et al., 2003) where no difference was observed when the animals had free access to the food. A weight difference between and mice has been described between gender (Hungund et al., 2003) when there is restricted food access. In another study, mice gained less weight than mice when fed with high fat diet (Ravinet-Trillou et al., 2003). Conversely, these data could be interpreted as a higher weight gain by mice, which is in accordance with the results of Wang et al. (2003), although in our experiments the mice had full access to the food. This effect could be due to the length of the experiment and the presence of ethanol, which modulates endocannabinoid levels in neuronal cells (Gonzalez et al., 2002).
Both CB1 genotypes showed no significant differences in their motility irrespective of whether they were chronically forced alcoholized or not. There was also no difference in motilities before and after chronic alcoholization. These results are in agreement with those observed in the study of Racz et al. (2003) where mice showed no withdrawal symptoms when compared with mice. In contrast, Naassila et al. (2004) reported an increased ethanol withdrawal severity in mice. This discrepancy in the results obtained in those studies may be caused by the use of different measures for alcohol withdrawal symptoms.
In conclusion, these data showed: (1) a higher BEC in mice after a high acute ethanol dose of 5 g/kg; (2) during forced chronic pulmonary alcoholization, higher BEC levels are reached at an earlier time point in mice, and (3) mice show a lower ethanol preference. These results strongly support an important role for the endocannabinoid—CB1 receptor system in ethanol drinking behaviour as well as other actions of ethanol. Further studies of enzymes involved in the pharmacokinetics of ethanol are needed to explain the apparent differences in ethanol absorption/distribution observed in mice after high doses of ethanol.
Source, Graphs and Figures: ETHANOL INDUCES HIGHER BEC IN CB1 CANNABINOID RECEPTOR KNOCKOUT MICE WHILE DECREASING ETHANOL PREFERENCE
Aims: Previous studies have shown that CB1 cannabinoid receptors are involved in the behavioural effects induced by chronic ethanol administration in Wistar rats by using SR 141716, a CB1 cannabinoid receptor antagonist. These studies have now been extended to investigate the effect of acute and chronic alcoholization on blood ethanol concentration (BEC) and ethanol preference in CB1 knockout (−/−) mice. Methods: BEC was monitored for a period of 8 h in both male mice and CB1 male wild-type (+/+) mice, which had received an acute i.p. injection of ethanol in 1, 3 or 5 g/kg doses. Ethanol preference was assayed in both groups of male mice in non-forced ethanol administration and forced chronic pulmonary alcohol administration for 14 and 39 days, respectively. Results: After an acute intraperitoneal ethanol injection of 5 g/kg, mice showed a significant higher BEC during the ethanol elimination stage than the mice. However, those in the 1 and 3 g/kg groups showed no significant difference. A 2—3 fold increase in BEC was observed in mice on days 10 and 11 after commencement of forced chronic pulmonary alcoholization in comparison with mice, although comparable BEC values were assayed in both groups on day 12. In addition, these mice showed a significantly lower preference for ethanol than mice. Conclusions: The studies on and mice have clearly confirmed the involvement of CB1 receptor on ethanol induced behavioural effects and also revealed that CB1 receptors may be implicated in ethanol absorption/distribution, particularly after administration of high ethanol doses.
INTRODUCTION
The endocannabinoid system consists not only of G-protein-coupled cannabinoid receptors but also other components such as enzymes [i.e. fatty acid amidohydrolase (FAAH)] and endogenous ligands. There are two types of cannabinoid receptors, CB1 and CB2. The CB1 cannabinoid (CB1) receptor is predominantly expressed in brain at relatively high levels in hippocampus, cerebellum and spinal cord and thus, often referred to as the brain cannabinoid receptor. CB1 receptors are also expressed at low levels in peripheral tissues including spleen, testis and leucocytes (Herkenham et al., 1991; Bouaboula et al., 1993; Lévénès et al., 1998; Navarro et al., 1998). The CB2 receptor is referred to as the peripheral cannabinoid receptor because it mainly shows peripheral expression in immune cells (Munro et al., 1993; Facci et al., 1995). To date, there are two endogenous ligands of CB1 receptors, namely anandamide and 2-arachydonylglycerol, which mimic the pharmacological action of Δ9-tetrahydrocannabinol, the active compound of marijuana and other synthetic agonists (Devane et al., 1992; Mechoulam and Fride, 1995; Mechoulam et al., 1995; Stella et al., 1997). There is growing evidence for the implication of endogenous cannabinoids in biological functions, including the control of appetite and food intake (Di Marzo et al., 2001), the modulation of some of the pharmacological effects of ethanol (Basavarajappa et al., 1998, 2000; Basavarajappa and Hungund, 1999a,b, 2002; Hungund and Basavarajappa, 2000a,b; Hungund et al., 2002) and drinking behaviour (Arnone et al., 1997; Colombo et al., 1998; Gallate et al., 1999; Gallate and McGregor, 1999; RodrÃguez de Fonseca et al., 1999; Freedland et al., 2001; Colombo et al., 2002; Hungund et al., 2002).
Chronic ethanol administration has a dual effect on the cannabinoid receptor, increasing the level of both endogenous cannabinoid agonists, anandamide and 2-arachidonylglycerol, while downregulating the CB1 receptor number and function, thereby suggesting a role for the endocannabinoid system in the neurobiological effects of ethanol (Basavarajappa et al., 1998b, 2000; Basavarajappa and Hungund, 1999a,b). Nonetheless, ethanol did not produce any effects on CB1 receptor binding and mRNA levels in rats (Gonzalez et al., 2002). However, a recent study by Ortiz et al. (2004) showed that forced consumption of high quantity of ethanol for a long period significantly decreased the gene expression of the CB1 receptors in the caudate-putamen, the ventromedial nucleus of hypothalamus and both CA1 and CA2 fields of the hippocampus. The last finding is in accordance with Basavarajappa et al. (1998) and Basavarajappa and Hungund (1999a). The result obtained by Gonzalez et al. (2002) could be due to differences in the quantity of ethanol and the duration of ethanol administration.
CP-55,940, a CB1 receptor agonist, promoted alcohol craving in rats (Gallate et al., 1999), as well as voluntary ethanol intake in Sardinian alcohol-preferring (sP) rats (Colombo et al., 2002). WIN-55,212—2, another CB1 receptor agonist, also promoted voluntary ethanol intake in sP rats (Colombo et al., 2002).
Numerous studies have shown that the CB1 receptor antagonist SR 141716 reduces ethanol intake (Arnone et al., 1997; Colombo et al., 1998; RodrÃguez de Fonseca et al., 1999; Freedland et al., 2001) and ethanol craving (Gallate and McGregor, 1999) in different rat strains. In addition, SR 141716 suppressed the ethanol deprivation effects (i.e. the temporary increase in ethanol intake after a period of ethanol withdrawal) in sP rats (Serra et al., 2002). All these results suggest that the blocking of CB1 receptor decreases the consumption of ethanol. Nonetheless, it is also important to mention that in our previous study in Wistar rats, we showed that the cannabinoid receptor antagonist SR 141716 profoundly altered ethanol preference in chronically pulmonary alcoholised rats depending on the dose and time of administration. Doses of 3 or 10 mg/kg/day, administered during chronic pulmonary alcoholization enhanced ethanol preference whereas its administration during the ethanol withdrawal stage after alcoholization induced a decrease in ethanol preference (Lallemand and De Witte, 2001). We have also shown that the action of SR 141716 was dependent on a number of factors, including the duration of ethanol intoxication as well as the number of ethanol re-exposures and ethanol withdrawals (Lallemand et al., 2004).
All these previous studies used antagonists and agonists of CB1 receptors. In certain circumstances, some antagonists have side-effects, which could alter/modify their actions. For example, SR-141716, a CB1 receptor antagonist, can show agonist property (Shire et al., 1999).
An alternative to avoid these possible pharmacological side-effects is the use of null mutant mice. The development of transgenic CB1 knockout mice has provided the opportunity to study the role of the CB1 receptor system in the regulation of ethanol consumption (Ledent et al., 1999; Zimmer et al., 1999).
mice with CD1 background showed decreased ethanol intake and preference. These effects were associated with a dramatic sensitivity to the hypothermic and hypolocomotor effects in response to low doses of ethanol (Naassila et al., 2004). These mice also showed an increased intensity of ethanol withdrawal-induced convulsions. Female mice consumed more ethanol than male mice; in addition, this gender difference was observed in both genotypes – female mice showed a decreased ethanol consumption compared with that of female mice, but did consume the same quantity of ethanol as did male mice. Hungund et al. (2003) observed similar results, although the gender difference in ethanol consumption observed between female and male mice was abolished in mice. These results were also observed in the study of Poncelet et al. (2003) using mice with C57BL/6 × 129/Ola F2 background.
with C57BL/6J background had a higher preference for ethanol but only for a few days (Racz et al., 2003). After the cessation of chronic ethanol administration, these mice did not exhibit withdrawal symptoms. After mild intermittent foot-shock stress, alcoholized mice did not consume an increased amount of ethanol as did the mice for the next 24 h. The activation of CB1 receptors in wild-type mice will also contribute to the high ethanol preference exhibited by C57BL/6J mice (Wang et al., 2003) as SR 141716 is able to reduce ethanol drinking when administered to these mice and not in mice. Young and old mice with this genetic background displayed low ethanol preference. On the contrary, mice presented an age-dependent decline in ethanol preference, suggesting that the decline in ethanol preference is related to a loss of cannabinoid signalling in the limbic forebrain.
It could be hypothesized that there was an interaction of gender and expression of phenotype associated with the CB1 gene mutation. The total fluid intake was similar between the different genotypes, although differences were evident between males and females within the same genotype. male mice did not show the acute ethanol-induced increase in dopamine levels in nucleus accumbens compared with mice, which would indicate that activation of the limbic system was required for the reinforcing effects of ethanol (Hungund et al., 2003).
The purpose of our study was to investigate the effect of a low to high acute intraperitoneal ethanol injection on blood ethanol concentration (BEC), as well as the effects of non-forced ethanol administration and forced chronic pulmonary ethanol intoxication on ethanol preference by comparing and mice to ascertain the precise involvement of the cannabinoid system on ethanol-related behavioural effects.
MATERIALS AND METHODS
Homozygous male mice were compared with homozygous male wild-type mice. and mice were from a C57BL/6 J × 129/Ola (Harlan) F2 genetic background and generated as described previously (Robbe et al., 2002; Ravinet-Trillou et al., 2003). No backcrosses were performed. These mice were provided by Sanofi-Recherche Synthélabo (Montpellier, France). The mice were housed in clear plastic cages with steel wire fitted tops and wood chip bedding under standard conditions (normal 12 h light—dark cycles, light on at 08:00 h, constant room temperature of 25 ± 1°C) with commercial lab chow diet and tap water available ad libitum during the entire experiments.
Acute ethanol experiments
BEC was assayed in and , 30—32 g, 12-weeks-old, male mice, housed 5/cage, after an intraperitoneal injection of ethanol. The experiment was carried out in their home cages. Blood samples were collected from the retro-orbital sinus under slight ether anaesthesia where necessary, into haematocrit tubes at 20, 40 min and 1, 2, 4, 9 and 12 h, after either 1 or 3 g/kg ethanol doses (15% v/v), while an additional two samples at 14 and 16 h were also collected after the 5 g/kg dose. This procedure followed the schedule of blood drawing used by Bruguerolle and Dubus (1993), Bruguerolle et al. (1994) and Hettiarachchi et al. (2001). Blood from each haematocrit tube was transferred into microcentrifuge tubes containing sodium fluoride as an anticoagulant. The concentration of blood ethanol was assayed by an alcohol-dehydrogenase-based method (Aufrère et al., 1997).
Chronic ethanol experiments
Non-forced ethanol administration experiments. and , 30—32 g, 12-weeks-old, male mice, were housed 2/cage. Fluid intake (water and 10% v/v ethanol when present) was recorded every 1 or 2 days, and body weight every week.
Free-choice period. Two drinking bottles were placed in each cage, one containing tap water and the other, 10% v/v ethanol solution. The mice had continuous access to the drinking tips of both tubes. The position of the tubes was changed every day, in order to avoid possible bias due to place preference. The ratio of the 24 h intake from the ethanol bottle versus total fluid intake was used to define preference and the absolute amount (g/kg body weight/day) of ethanol consumed was also calculated.
Forced chronic pulmonary ethanol administration procedure. The motility of and , 30—32 g, 12 weeks old, male mice, was recorded, after 3 weeks of acclimatization, for 18 h by the MacLab system, the recordings being combined for each hourly interval. The apparatus has been described in detail previously (Lallemand and De Witte, 2001).
Forced chronic alcoholization was induced in these mice, housed in pairs of two, within a plastic chamber (120 × 60 × 60 cm) by pulmonary inhalation of a mixture of ethanol and air. The mixture was pulsed into the chamber via a mixing system that allowed the quantity of ethanol to be increased every day, so that the average BEC continued to rise (Le Bourhis, 1975; Aufrère et al., 1997) during the experimental procedure. The animals remained for 12 days in the alcohol chamber. The chamber temperature ranged between 28—30°C (Terdal and Crabbe, 1994; Finn and Crabbe, 1999). BECs were determined regularly during the chronic alcoholization. Blood from each haematocrit tube was transferred into microcentrifuge tubes containing sodium fluoride as an anticoagulant. The concentration of blood ethanol was assayed by an alcohol-dehydrogenase-based method.
Withdrawal motility and free-choice period. At the end of the forced chronic pulmonary alcoholisation period the motility and ethanol preference was studied in these two groups of mice. For the measurement of ethanol preference, the mice from each strain underwent three successive steps (Le Bourhis, 1977) on cessation of the chronic ethanol intoxication. First, full beverage deprivations, i.e. the drinking bottles were removed during the last 6 h of the chronic alcoholization procedure and the following 18 h of the withdrawal period. The motility of each mouse was recorded during these 18 h using the same apparatus described above. Secondly, a 10% (v/v) ethanol solution was given as the sole drinking fluid during the following 24 h. Thirdly, a free-choice beverage situation [water vs 10% (v/v) ethanol solution] was presented for a period of 39 days. During this free-choice period, the fluid consumptions were recorded daily and ethanol consumption expressed as a percentage of total fluid intakes and as ethanol intake in g/kg of body weight. The positions of the drinking bottles were changed every day to avoid position preference. BECs were assayed at different time points during the free-choice period by the method described above. The weight of animals was recorded every 3 or 4 days.
In all experiments, the results are presented as mean ± standard error (SE) except where stated otherwise. In all experiments, groups were compared by two-way analysis of variance (ANOVA) (genotype; time) with repeated measures on time. Where appropriate, post hoc pair wise comparisons were analysed by the least-significant difference test of multiple comparisons (Fisher LSD protected t-test) (GB-STAT 5.3 for Windows, Dynamic Microsystems, Silver Spring, MD, USA). Criterion for significance was set at P < 0.05 for all tests.
The Belgian Governmental Agency under the authorized number LA 1220028 as well as the European Communities Council Directive concerning the Use of Laboratory Animals approved these experiments.
Products
Absolute ethanol, used in the free-choice paradigm and acute experiment, was obtained from Labotec (La Gleize, Belgium). Ethanol at 15% (w/v) was prepared for i.p. injection in 0.9% saline. Ethanol at 97% was obtained from Belgalco SA (Belgium). Sodium fluoride was from Sigma Aldrich, (Steinheim, Germany).
RESULTS
Acute ethanol experiments
The BECs were similar in both groups of mice after either 1 or 3 g ethanol/kg doses [F(1,56) = 0.0005, P = 0.982 and F(1,56) = 0.1161, P = 0.7421 respectively]. However, after an acute injection of 5 g/kg of ethanol, mice showed significant differences compared to the mice [F(1,8) = 19.254, P = 0.0022], with a significantly higher BEC than the mice [F(9,72) = 2.981, P = 0.0045] (Fig. 1).
Chronic ethanol experiments
Non-forced ethanol administration experiments. After 1 week of measurements, mice showed a significantly higher water consumption in comparison to mice [F(1,54) = 6.8364, P = 0.0176] (data not shown). The mean water consumptions over the time of the experiment were 10.8 ± 1.24 and 13.8 ± 0.87 ml, respectively for and mice.
At the conclusion of the study, the mice showed a significantly lower mean weight in comparison to controls [F(1,38) = 7.3466, P = 0.01]. The mean weights were 32.18 ± 0.62 and 30.16 ± 0.36 g, respectively, for and mice.
Free choice. mice showed a significantly reduced ethanol preference (expressed as a percentage of total fluid intake) in comparison to control mice [F(1,12) = 8.6787; P = 0.0122] (Fig. 2A). There was also a significant interaction between genotype and time [F(8,96) = 2.1965, P = 0.0342]. Nonetheless, when ethanol preference is expressed as ethanol intake in g/kg of body weight, the genotype significance disappeared totally and only the interaction remained [F(38,418) = 3.9539; P < 0.0001] (Fig. 2B). The mean ethanol intake over the time of the experiment was 12.28 ± 0.48 and 13.12 ± 0.59 g/kg/day, respectively, for and mice.
When comparing liquid type consumptions, i.e. water and ethanol, ethanol volume consumed in mice of either genotype was not significantly different [F(1,12) = 0.2861, P = 0.6025]. On the contrary, mice consumed significantly more water than mice [F(1,12) = 14.1872, P = 0.0027].
During the free-choice period the total consumption (water + 10% v/v ethanol) of mice was not significantly different in comparison to control mice [F(1,26) = 3.3544, P = 0.0785] (data not shown), but there was a significant interaction between genotype and time [F(8,208) = 4.3224, P < 0.0001]. Nonetheless, the total consumption of mice was always above that of control mice. The mean consumptions over the time of the experiment were 9.67 ± 0.70 and 7.23 ± 0.33 ml/24 h, respectively, for and male mice.
Forced chronic ethanol pulmonary administration experiments. The motility of mice, prior to forced chronic ethanol pulmonary administration, was not significantly different in comparison to mice [F(1,510) = 0.7872, P = 0.382].
In the mice the mean BEC assayed at different time points during the forced chronic alcoholization regime were significantly different than the mean levels in the mice [F(1,26) = 25.887, P < 0.0001] characterized by a significant higher BEC level at both 10 and 11 days after the commencement of forced chronic pulmonary alcoholization. [F(6,156) = 7.931, P < 0.0001] (Fig. 3). At 10 days, the mean BEC was 3 fold higher in the mice than in the mice, whereas at 11 days, it showed a 2 fold increase. However, on Day 13 no significant difference in mean BEC was assayed.
During the forced chronic pulmonary alcoholization period, water consumption of mice was not significantly different [F(1,18) = 1.7514, P = 0.2023] (data not shown). The mean water consumptions over the time of the experiment were 9.82 ± 0.5 and 8.76 ± 0.48 ml/24 h, respectively, for and mice.
mice had a significantly lower body weight than mice [F(1,38) = 7.3466, P = 0.01]. The mean weights over the time of the experiment were 30 ± 0.46 and 32 ± 0.62 g, respectively, for and mice.
Following forced chronic pulmonary alcoholization, similar motilities were assayed for both and control mice [F(1,340) = 0.8442, P = 0.3704] as they were also similar prior to the chronic pulmonary alcoholization.
Free choice. During the first 24 h period after forced chronic pulmonary alcoholisation, there were no significant differences in ethanol consumption between and mice [F(1,11) = 0.4936, P = 0.4969]. The mean ethanol consumptions in the alcoholized group and in the alcoholized group were, 18.69 ± 1.69 ml and 16.75 ± 1.29 ml, respectively. During the free-choice period, ethanol preference, expressed as percentage of total fluid consumption, of mice showed no significance at the genotype level when compared with mice [F(1,11) = 2.1819, P = 0.1677] (Fig. 4A). There was also absence of significance when ethanol preference was expressed as ethanol intake/kg body weight [F(1,11) = 1.6614, P = 0.2239] (Fig. 4B). Nonetheless, in both representations of ethanol preference, there were always significant interactions between genotype and time [F(38,418) = 2.345, P < 0.0001 and F(38,418) = 3.9539, P < 0.0001, respectively, for percentage of total fluid consumption (Fig. 4A) and ethanol intake expressed in g/kg body weight (Fig. 4B)]. The mean ethanol intakes in all experiments were 17.96 ± 0.52 and 22.05 ± 0.69 g/kg, respectively, for and mice.
When the ethanol and water consumptions in each genotype were compared, no significant differences between the two liquids in both [F(1,12) = 0.0356, P = 0.8534] and mice [F(1,10) = 3.5586, P = 0.0886] were apparent. Nonetheless, there were always significant interactions between liquid type and time in both genotypes [F(38,456) = 8.1675, P < 0.0001 and F(38,380) = 6.2574, P < 0.0001, respectively, for and ]. In mice, the consumptions of water and ethanol were very similar (8.42 ± 0.25 and 8.93 ± 0.23 ml, respectively, for ethanol and water). In contrast, in mice, the ethanol consumption was always lower than the intake of water (8.81 ± 0.27 and 11.21 ± 0.21 ml, respectively, for ethanol and water). In addition, water intake of mice was higher than in the mice.
The total liquid consumption was not significantly different in and mice [F(1,11) = 1.3981, P = 0.262], although there was a significant interaction between genotype and time [F(38,418) = 3.5209, P < 0.0001]. The total consumption values for mice were always greater or at the same level for those of the mice.
During the free-choice paradigm, there was no significant difference between the BEC values at the genotype level [F(1,4) = 3.092, P = 0.1535]; the values assayed being less than 0.02 g/l in both groups of mice (data not shown). However, there was a significant interaction between genotype and time [F(5,20) = 2.849, P = 0.0422], as well as for time [F(5,20) = 20.192, P < 0.0001].
During the free-choice period, mice showed a significantly lower body weight than the mice [F(1,18) = 9.5004, P = 0.0064] (data not shown). There was also a significant interaction between genotype and time [F(10,180) = 6.851, P < 0.0001]. The body weight of mice at the beginning of the study was 31.2 ± 0.6 and 31.14 ± 0.67 g at its conclusion. The body weight of mice was 32.36 ± 0.75 g at the beginning of the study and was 37.64 ± 1.39 g at the end.
DISCUSSION
Recently, mouse specific gene deletions have been used to investigate the role of the endocannabinoid system in alcohol research. In this study we assessed the effect of CB1 receptor null mutation on ethanol preference in both non-alcoholized and chronically alcoholized mice as well as ethanol clearance after an acute ethanol i.p. injection. The acute ethanol injection in mice lacking the CB1 receptor showed an unexpected result in that the ethanol peak concentration for the high ethanol dose, 5 g/kg, induced a significantly higher ethanol peak concentration in mice. However, the ethanol elimination rates for the lower doses, 1 and 3 g/kg, were similar in both and mice. This has not been described previously in the literature for mice. Nonetheless, the influence of the cannabinoid system on the metabolism of ethanol was reported in one study where the administration of cannabinoid receptor inhibitor SR 141716 induced no changes in ethanol metabolism in rats (Colombo et al., 1998). It is difficult to interpret these present results. As the 1 and 3 g/kg ethanol doses showed no significant change between and , we hypothesized that, with respect to the high dose of ethanol used, the lack of CB1 cannabinoid receptors in the enteric nervous system, particularly at the level of the gastrointestinal tract of mice, might interfere with the absorption/distribution of ethanol (Batkai et al., 2001; Pertwee, 2001). However, this lack would intervene only with a high acute dose of ethanol.
In mice with non-forced ethanol administration, ethanol preference ratio was significantly reduced in mice, but when ethanol preference was expressed as g/kg body weight per day, no significances appeared. These results are in agreement with those obtained by Wang et al. (2003) for ethanol preference ratio. Nonetheless, other studies by Hungund et al. (2003) and Poncelet et al. (2003) observed that ethanol intake expressed in g/kg body weight/day was significantly reduced in mice as well. This discrepancy on the preference in ethanol intake was unclear. In our study, the absence of significance in preference as expressed in g/kg body weight/day is mainly the result of a higher, but not significant, total liquid intake of the mice.
In chronically forced alcoholized mice, the BEC in mice peaked faster than in mice, although the maximum values obtained were not significantly different. This result has not been observed in previous studies, although Colombo et al. (1998b) showed that the antagonism at CB1 cannabinoid receptors did not modify ethanol metabolism. In our study, the difference between and mice was noted only during the increase of BEC but not at the end of the chronic alcoholization period. Unlike other chronic alcoholization procedures, our protocol of chronic alcoholization is a forced one, i.e. animals were unable to adjust the amount of ethanol ingested by themselves. Our procedure of chronic alcoholization induced other mechanisms involved in ethanol metabolism microsomal ethanol oxidizing system MeOS/cytochrome P450IIE (Lieber, 1999) and alcohol dehydrogenase (Kishimoto et al., 1995), which have not been studied to date in these knockout animals.
After forced chronic pulmonary alcoholization, the ethanol consumption in mice was similar to that of when access to 10% (v/v) ethanol solution was given. In contrast, when mice had access to both drinking bottles, i.e. free choice, their ethanol preference was significantly lower than mice when expressed as percentage of total consumption. This result is in agreement with our previous study in Wistar rats of the action of the CB1 cannabinoid receptor inhibitor SR 141716 (Lallemand and De Witte, 2001) and data reported recently by Hungund et al. (2003), Poncelet et al. (2003), Racz et al. (2003), Wang et al. (2003) and Naassila et al. (2004), which show that a CB1 receptor antagonist decreases ethanol consumption in rats and mice. Nevertheless, when ethanol preference is expressed as g/kg body weight/day, mice presented significant ethanol intake time point higher than mice.
In both non-forced alcoholized and chronically forced alcoholized experiment, the mice showed a significantly lower weight than the mice. This result was in contradiction to the results from a previous study (Wang et al., 2003) where no difference was observed when the animals had free access to the food. A weight difference between and mice has been described between gender (Hungund et al., 2003) when there is restricted food access. In another study, mice gained less weight than mice when fed with high fat diet (Ravinet-Trillou et al., 2003). Conversely, these data could be interpreted as a higher weight gain by mice, which is in accordance with the results of Wang et al. (2003), although in our experiments the mice had full access to the food. This effect could be due to the length of the experiment and the presence of ethanol, which modulates endocannabinoid levels in neuronal cells (Gonzalez et al., 2002).
Both CB1 genotypes showed no significant differences in their motility irrespective of whether they were chronically forced alcoholized or not. There was also no difference in motilities before and after chronic alcoholization. These results are in agreement with those observed in the study of Racz et al. (2003) where mice showed no withdrawal symptoms when compared with mice. In contrast, Naassila et al. (2004) reported an increased ethanol withdrawal severity in mice. This discrepancy in the results obtained in those studies may be caused by the use of different measures for alcohol withdrawal symptoms.
In conclusion, these data showed: (1) a higher BEC in mice after a high acute ethanol dose of 5 g/kg; (2) during forced chronic pulmonary alcoholization, higher BEC levels are reached at an earlier time point in mice, and (3) mice show a lower ethanol preference. These results strongly support an important role for the endocannabinoid—CB1 receptor system in ethanol drinking behaviour as well as other actions of ethanol. Further studies of enzymes involved in the pharmacokinetics of ethanol are needed to explain the apparent differences in ethanol absorption/distribution observed in mice after high doses of ethanol.
Source, Graphs and Figures: ETHANOL INDUCES HIGHER BEC IN CB1 CANNABINOID RECEPTOR KNOCKOUT MICE WHILE DECREASING ETHANOL PREFERENCE
