Tolerance to cannabinoid-induced behaviors in mice treated chronically with ethanol

Abstract

Rationale: Chronic ethanol (EtOH) treatment decreases the motor-impairing effects of cannabinoids and downregulates the cannabinoid type 1 (CB1) receptor. However, these studies have been limited to measures of ataxia and analysis of CB1 expression from whole-brain or hippocampal preparations.

Objective: To more fully assess the interactions between ethanol and cannabinoids, a tetrad of four well-characterized cannabinoid-induced behaviors (hypolocomotion, antinociception, hypothermia, and catalepsy) was measured in mice following EtOH treatment. Additionally, immunoblotting assessed CB1 protein in tissue from nine brain regions associated with these behaviors and the addiction neurocircuitry.

Materials and methods: Male C57Bl/6J mice were administered EtOH (0, 2, or 4 g/kg; intraperitoneally (i.p.)) twice daily for 10 days. Tetrad behaviors induced by the CB1 agonist WIN 55212-2 (3 mg/kg, i.p.) were measured in subjects 1 or 10 days following the last EtOH injection. In a separate group of animals, tissue was collected at the same time points for immunoblot analysis.

Results: EtOH-treated mice were less sensitive to the hypothermic, hypolocomotive, and antinociceptive effects of WIN, and this effect reversed to control levels over a 10-day abstinence period. EtOH treatment did not affect WIN-induced catalepsy. CB1 protein expression was significantly altered in several brain areas including the hypothalamus, periaqueductal gray, ventral tegmental area, and cerebellum.

Conclusions: These results show that chronic EtOH treatment significantly affects the behavioral sensitivity to cannabinoid drugs and alters CB1 expression in several brain regions. Furthermore, these effects are selective as some behaviors and brain regions display an altered response while others do not.

Fig. 1

Summary of EtOH treatment and testing regimen. A: Mice were administered saline or EtOH (2 or 4 g/kg) i.p. twice per day for 10 consecutive days. Following the last injection, subjects were returned to their home cage for 24 hrs (bottom panel) or 10 days prior to behavioral testing or tissue collection for CB1 protein quantification (top panel). B: Timeline of behavioral measures taken as part of the mouse tetrad assay.

Fig. 2

Effects of WIN on the mouse tetrad in ethanol-naïve mice. Ordinates represent the mean (± SEM, N=15–24) value for each measurement and are expressed as number of beam breaks per session (locomotor activity), percent maximum possible effect (antinociception), change in body temperature (hypothermia), and percent immobility on the ring stand (catalepsy). Abscissae represents the dose (mg/kg) of WIN administered to a group of subjects. Symbol: (***), values significantly different from the vehicle control (p<0.0001, One-way ANOVA)

Fig. 3

Effects of repeated EtOH treatments on WIN-induced effects in the mouse tetrad. Bars represent the mean (± SEM; N=11–13) value for each measure. The scales on the ordinates are expressed as number of beam breaks per session (locomotor activity), percent maximum possible effect (antinociception), change in body temperature (hypothermia), and percent immobility on the ring stand (catalepsy). Values on the abscissae represent the dose of EtOH administered for pre-treatment. Symbols: value significantly different from saline control (*p<0.05, **p<0.01, ***p<0.001, One-way ANOVA with Bonferroni post-tests)

Fig. 4

Effects of repeated 4.0 g/kg EtOH treatments on WIN-Induced behaviors are reversible. Bars represent the mean (± SEM; N=8–13) value for each measure. The scales on the ordinates are expressed as number of beam breaks per session (locomotor activity), percent maximum possible effect (antinociception), change in body temperature (hypothermia), and percent immobility on the ring stand (catalepsy). Values on the abscissae represent the day behavioral testing was performed. Symbol: value significantly different from corresponding saline control (***p<0.001, Two-way ANOVA with Bonferroni post-test)

Fig. 5

Effects of repeated EtOH treatments on expression of CB1 receptor protein. A) Representative gel showing immunoreactive band corresponding to CB1 receptor (MW≈52 kDa) in wild-type (WT) that is lacking in knockout (KO) mice. B) Quantification of CB1 receptor expression in saline and EtOH treated animals. Bars show mean (± SEM, N=4–5) fraction of control for optical density of CB1R immunoreactive band for each region shown. Values on the abscissae represent the day tissue was havested. Representative blots for each region are shown below the corresponding graph. Symbols: significant interaction (§) and main effect of treatment (†), p<0.05, two-way ANOVA; value significantly different from corresponding saline control (*p<0.05, **p<0.01, Two-way ANOVA with Bonferroni post-test)