In this study, we employed DAT-Cnr2 and Cx3Cr1-Cnr2 cKO mice which are created in our lab [28]. The mice were generated through a breeding approach involving Cnr2-floxed mice and DAT-Cre and Cx3-Cre mice. We confirmed the specific deletion of CB2Rs in dopamine cells and microglia in homozygous cKO mice through genotyping and RNAscope in situ hybridization, while no deletion occurred in the WT mice. The experiments were conducted on adult male mice weighing between 20 g and 30 g, all bred in the mouse laboratory at William Paterson University of New Jersey. These mice were kept under controlled conditions, including room temperature (25°C ± 2°C), a 12:12 h light-dark cycle, and ad libitum access to food and water. Our study adhered to the guidelines in the Guide for the Care and Use of Laboratory Animals and received approval from the William Paterson University Animal Care and Use Committee (IACUC).

Absolute ethanol was purchased from Pharmaco-AAper in Bristol, PA. 8% of the absolute alcohol was mixed with distilled water and administered as 0.8 g/kg dose into the peritoneum (i.p.) at a volume of 10 mL/kg body weight. The non-selective cannabinoid receptor agonist, WIN55,212-2 (WIN), was purchased from Cayman Chemical Co. located in Ann Arbor, MI. After dissolving WIN in a mixture of DMSO, tween 80, and saline in a ratio of 1:2:7, a dosage of 3 mg/kg was administered. The doses of alcohol and WIN were determined based on previous research [21, 28–30]. Both alcohol and WIN were injected i.p. in a volume of 10 mL/kg body weight.

To evaluate total distance travelled in the activity box, the locomotor activity monitoring apparatus (ENV-510: Med Associates Inc., St. Albans, VT, USA) was utilized. Thirty minutes after acute alcohol injection, the animals were placed gently into separate test boxes (measuring 43.2 × 43.2 × 30.5 cm) that were connected to a computer. Total distance traveled by mice was recorded and analyzed over a 10-min period [21]. Prior to the test, the mice were given three consecutive days to freely explore the open field chambers for 10 min each day in order to acclimate to the environment.

The wheel running activity of the mice was observed using a spontaneous wheel-running monitor (Wahmann, Geo. H., Manufacturing Company, Baltimore, MD, USA) after 40 min of acute alcohol administration. Each mouse was placed in the monitor, and their wheel running behavior was tracked using auto-counters, which recorded the total number of revolutions made by each animal during the 10-min testing session [21].

Mice were placed on a stationary rota rod (AccuRotor Rotarod, AccuScan Instruments Inc.) by gently gripping their tails, positioning them away from the direction of rotation. To maintain balance, the mice had to walk forward on the rod. The rota rod was set at a height of 30 cm above the ground and featured a rotating rod with a 3 cm diameter. The duration each mouse managed to stay on the rod for 180 s was recorded, excluding falls occurring within the initial 5 s due to improper placement by the experimenter [21]. A soft padded surface was positioned at the base of the apparatus to cushion any falls.

For preference testing, individually housed mice (N = 10 mice per group) were used. Throughout a 24 h period, the mice had access to two conical tubes with a drinking spout attached filled with water. In order to institute a baseline, both tubes were initially filled with 40 mL of water and placed above the cages for three consecutive days. During the preference measurement phase, one of the tubes was replaced with a solution containing 8% alcohol. The amount of alcohol consumed by each animal was recorded over five consecutive days between 10 and 11 AM. To ensure unbiased positioning, the placement of the tubes within the various cages was randomized with regard to the side of the cages they were placed on. In all experiments, the ratio of alcohol to water consumed, and the total fluid consumption, were calculated to obtain a preference ratio. Additionally, half of the animals in each group (N = 5) were injected with WIN daily for five consecutive days. The alcohol preference ratio was determined by dividing the amount of alcohol consumed by the total fluid (alcohol + water) consumption [21].

Mice involved in the acute behavioral experiments were continuously administered either the vehicle or alcohol for seven consecutive days. On the eighth day, the mice were decapitated, and their brains were removed from the skull. To aid dissection, the brains were promptly frozen in liquid nitrogen. Specific brain regions containing the hippocampus were dissected and placed in cell lysis buffer. Using an ultrasonic homogenizer, the tissue was homogenized. The resulting homogenates were then centrifuged at 10,000 RPM for 5 min to separate the tissue debris. Samples of the resulting supernatants were collected and, after determining the protein concentration, frozen and stored at −80°C until needed for cytokine analysis. To profile the expression of IL-1α (interleukin-1α), IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α), a Mouse Inflammation ELISA Strip kit (Signosis, Sunnyvale CA, USA) was employed. In brief, 100 μL of the diluted cell lysate sample was added to wells coated with a specific primary antibody against each cytokine. After incubation for 1 h at room temperature, the wells were aspirated and washed three times with 200 μL of assay wash buffer. Subsequently, 100 μL of a biotin-labeled antibody mixture was added to each well and incubated for 1 h at room temperature. The wells were again aspirated and washed three times with 200 μL of assay wash buffer. Then, 100 μL of streptavidin-HRP conjugate was added to each well and incubated for 45 min at room temperature. Following aspiration and another round of washes, 100 μL of substrate was added and incubated for 10 min, followed by the addition of 50 μL of stop solution to each well. The optical density of each well was measured using a microplate reader at 450 nm [21].

Data are presented as mean ± SEM. Sigma Plot 12.0 statistical program was used. Prior to performing the tests, we conducted a normality test (Shapiro-Wilk) to verify the distribution of the data. The statistical analysis was performed by the two-way analysis of variance (ANOVA). Post hoc comparisons of means were carried out with Tukey’s test for multiple comparisons when appropriate. We used two-way ANOVA for the analysis of behavioral and cytokine assay data. Data from the alcohol preference study were analyzed by using repeated measures two-way ANOVA. The confidence limit of p < 0.05 was considered statistically significant. One of the factors of the ANOVA was the genotype (DAT-Cnr2, Cx3Cr1-Cnr2 or WT mice) and the other factor was treatment groups (vehicle or alcohol).

We evaluated acute motor activity in C57, DAT-Cnr2, and Cx3Cr1-Cnr2 mice following the administration of 8% alcohol using an activity monitor apparatus. The results showed significant main effects for both treatment and genotype (F _{1, 30} = 70.30, p < 0.001 and F _{2, 30} = 81.53, p < 0.001, respectively), as well as a significant interaction between treatment and genotype (F _{2, 30} = 16.22, p < 0.001). Post-hoc analysis using Tukey’s test for multiple comparisons revealed that alcohol administration significantly increased the total distance traveled in the activity box compared to the control group treated with vehicle. Interestingly, the results also indicated that specific deletion of CB2R in dopamine neurons (DAT-Cnr2 cKO) enhanced alcohol-induced locomotor activity, with a statistically significant (p < 0.01) increase in the total distance traveled compared to WT mice. In contrast, the cell-type specific deletion of CB2R in microglia (Cx3Cr1-Cnr2 cKO) reduced alcohol-induced locomotor activity, showing a statistically significant (p < 0.05) decrease in the total distance traveled compared to WT mice (Figure 1A).

In this study, we investigated acute wheel running behavior in C57, DAT-Cnr2, and Cx3Cr1-Cnr2 mice following the administration of 8% alcohol using a mechanical wheel running apparatus. The number of revolutions exhibited a significant association with the treatment groups (F _{1, 30} = 112.2, p < 0.001) and genotype (F _{2, 30} = 56.12, p < 0.001). Post-hoc analysis using Tukey’s test revealed that both the vehicle and alcohol treatment of DAT-Cnr2 mice resulted in a significant (p < 0.01) increase in the absolute number of revolutions compared to the control group of WT mice (Figure 1B).

We examined the ability of mice to maintain their position on a rotating cylinder following the administration of 8% alcohol. We employed a constant speed rotarod apparatus for this assessment in C57, DAT-Cnr2, and Cx3Cr1-Cnr2 mice. The results showed that cell type specific deletion of CB2R in dopamine neurons enhanced alcohol induced reduction in fall latency in the rotarod test of DAT-Cnr2 mice, whereas this effect was not observed in the deletion of CB2R in microglia of Cx3Cr1-Cnr2 mice. There was a significant main effect for both treatment and genotype (F _{1, 30} = 28.43, p < 0.001 and F _{1, 30} = 62.52, p < 0.001, respectively). Post-hoc analysis using Tukey’s test indicated a statistically significant (p < 0.05) increase in fall latency in DAT-Cnr2 mice compared to the WT controls. However, the cell-type specific deletion of CB2R in microglia did not affect the alcohol-induced changes in fall latency when compared to the WT controls (Figure 1C).

We further investigated the potential association between subacute treatment with WIN and alcohol preference. In WT mice, the results demonstrated a significant effect of both treatment and time on the alcohol preference ratio (F _{1, 20} = 79.229, p < 0.001 and F _{4, 20} = 3.172, p < 0.05, respectively), as well as a significant interaction between treatment and time (F _{4, 20} = 6.421, p < 0.05). Post hoc analysis revealed a significant (p < 0.01) reduction in alcohol preference in mice treated with WIN compared to the vehicle-treated controls (Figure 2A). Similarly, in DAT-Cnr2 mice, there was a significant effect of both treatment and time on the alcohol preference ratio (F _{1, 20} = 233.855, p < 0.001 and F _{4, 20} = 4.956, p < 0.05, respectively), along with a significant interaction between treatment and time (F _{4, 20} = 9.042, p < 0.001). Post hoc analysis indicated a significant (p < 0.01) reduction in alcohol preference in mice treated with WIN compared to the vehicle-treated controls (Figure 2B). In Cx3Cr1-Cnr2 mice, statistical analysis also revealed a significant effect of both treatment and time on the alcohol preference ratio (F _{1, 20} = 68.225, p < 0.001 and F _{4, 20} = 5.716, p < 0.05, respectively), as well as a significant interaction between treatment and time (F _{4, 20} = 2.812, p < 0.05). Post hoc analysis demonstrated a significant (p < 0.05) reduction in alcohol preference in mice treated with WIN compared to the vehicle-treated controls (Figure 2C).

The result form the cytokine study showed both treatment and genotype significantly affected the expression of TNF-α [treatment effect: F _{1, 30} = 29.33, p < 0.001; genotype effect: F _{2, 30} = 20.51, p < 0.001; treatment X genotype interaction: F _{2, 30} = 5.43, p < 0.05] and IL-1β [treatment effect: F _{1, 30} = 12.27, p < 0.001; genotype effect: F _{2, 30} = 16.43, p < 0.001; treatment X genotype interaction: F _{2, 30} = 9.62, p < 0.01]. Compared to the WT controls, Tukey’s test revealed that there was statistically significant increase in the levels of TNF-α and IL-β, as evidenced by enhanced absorbance values, in DAT-Cnr2 and Cx3Cr1-Cnr2 mice treated with alcohol (Figures 3A–D).