Mice expressing a "hyper-sensitive" form of the CB1 cannabinoid receptor (CB1) show modestly enhanced alcohol preference and consumption

Abstract

We recently characterized S426A/S430A mutant mice expressing a desensitization-resistant form of the CB1 receptor. These mice display an enhanced response to endocannabinoids and ∆9-THC. In this study, S426A/S430A mutants were used as a novel model to test whether ethanol consumption, morphine dependence, and reward for these drugs are potentiated in mice with a "hyper-sensitive" form of CB1. Using an unlimited-access, two-bottle choice, voluntary drinking paradigm, S426A/S430A mutants exhibit modestly increased intake and preference for low (6%) but not higher concentrations of ethanol. S426A/S430A mutants and wild-type mice show similar taste preference for sucrose and quinine, exhibit normal sensitivity to the hypothermic and ataxic effects of ethanol, and have normal blood ethanol concentrations following administration of ethanol. S426A/S430A mutants develop robust conditioned place preference for ethanol (2 g/kg), morphine (10 mg/kg), and cocaine (10 mg/kg), demonstrating that drug reward is not changed in S426A/S430A mutants. Precipitated morphine withdrawal is also unchanged in opioid-dependent S426A/S430A mutant mice. Although ethanol consumption is modestly changed by enhanced CB1 signaling, reward, tolerance, and acute sensitivity to ethanol and morphine are normal in this model.

Fig 1. S426A/S430A mutants prefer and consume more ethanol.

A. Consumption (g/kg) of a low (6%) concentration of ethanol was increased for S426A/S430A mutants (red circles and line) relative to wild-type littermates (black lines and squares). Mice were given 24 hour unlimited access to 3%, 6%, 9%, 12%, 15%, and 18% ethanol (v/v) using a two-bottle choice assay. B. S426A/S430A mutants also prefer 6% ethanol more than wild-type littermates. C. Total liquid intake (water plus ethanol) was identical for S426A/S430A mutants and wild-type mice. D. Consumption of 1.75% and 4.25% sucrose was equivalent for S426A/S430A mutants (red bars) and wild-type littermates (black bars). E. S426A/S430A mutants (red bars) and wild-type littermates (black bars) also consume similar amounts of 0.03 mM and 0.1 mM quinine solutions. Averages across the 8 days of intake are shown for each concentration; for both A and B, intakes (collected every 48 hours) are shown in the inset for 6% ethanol. Error bars represent the SEM and data analyses were performed using two-way repeated-measure ANOVA with Bonferroni post-hoc tests (*p<0.05). Sample sizes for each group are in parentheses.

Fig 2. S426A/S430A mutants show normal conditioned place preference for ethanol.

CPP for 1 and 2 g/kg of ethanol was examined in S426A/S430A mutants and wild-type littermates. Pre-conditioning (black bars) and post-conditioning (red bars) place preference was measured before and after eight days of place preference conditioning for (A) 1 and (B) 2 g/kg of ethanol. Error bars represent the SEM and data analyses were performed using a two-way repeated measures ANOVAs with Bonferroni post-hoc tests (*p<0.05). Sample sizes for each group are in parentheses.

Fig 3. Sensitivity to ethanol is not altered for S426A/S430A mutants.

A. The ataxic effects of 0 (saline only), 1, 1.5, 2, 2.5, 3, and 3.5 g/kg of ethanol were measured in S426A/S430A mutants (red circles and line) and wild-type littermates (black lines and squares) using an accelerating rotarod. S426A/S430A mutant mice show the same sensitivity to the ataxic effects of ethanol as wild-type mice. B. S426A/S430A mutants also display the same sensitivity to the hypothermic effect of ethanol as wild-type mice. Error bars represent the SEM and data analyses were performed using two-way repeated measure ANOVAs with Bonferroni post-hoc tests. Sample sizes for each group are in parentheses.

Fig 4. Time to recover the righting reflex is not altered in S426A/S430A mutants.

The amount of time to regain the righting reflex was measured following administration of an ataxic dose (4 g/kg) of ethanol in both S426A/S430A (red bar) and wild-type (black bar) littermates via the loss of righting reflex assay. Error bars represent the SEM and data analysis was done using a t-test. Sample sizes for each group are in parentheses.

Fig 5. Ethanol metabolism is not altered in S426A/S430A mutants.

Blood ethanol concentrations were measured in S426A/S430A mutants (red bars) and wild-type littermates (black bars) 15, 30, 60 and 120 minutes after a bolus injection (IP) of 2 g/kg of ethanol. Error bars represent the SEM and data analyses were performed using two-way ANOVA with Bonferroni post-hoc tests. Sample sizes for each group are in parentheses.

Fig 6. Conditioned place preference for morphine and cocaine is not changed in S426A/S430A mutants.

CPP for either 10 g/kg of morphine (A) or 10 mg/kg cocaine (B) was examined in S426A/S430A mutants and wild-type littermates. Pre-conditioning (black bars) and post-conditioning (red bars) place preference was measured before and after eight days of place preference conditioning. Error bars represent the SEM and data analyses were performed using two-way repeated measure ANOVAs with Bonferroni post-hoc tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Sample sizes for each group are in parentheses.

Fig 7. Tolerance to the antinociceptive and hypothermic effects of morphine is normal in S426A/S430A mutant mice.

Tolerance to the antinociceptive and hypothermic effects of daily treatment with morphine were examined in S426A/S430A mutant mice (red line and circles) and wild-type littermates (black line and squares). Tail-flick antinociception (A), hotplate antinociception (B), and body temperature (C) were measured 60 minutes after SC injection of 10 mg/kg morphine. Data are expressed as mean ± S.E.M and sample sizes for each group are in parentheses. Data analyses were performed using two-way repeated measure ANOVAs with Bonferroni post-hoc tests.