AM 251

We performed power analyses to determine the number of mice required to provide statistically significant results, based on the levels of differences seen previously [Zhou et al. 2016 (link), 2017 ], and predicted that these studies require 6–8 animals per group. In the experiments with URB597, alcohol intake, water intake, total fluid and preference ratio differences between the different groups were analyzed using two-way ANOVA with repeat measures for treatment (vehicle vs drug) and for time (0–4h, 5–8h, vs 9–24h interval) in each sex (Figure 1). For dose response analysis on URB597, group differences for alcohol intake and preference ratios at the 4-hour recording time were analyzed using one-way ANOVA for treatments with different doses (Figure 2). In the experiments with URB597 with 7.5 and 30% alcohol, alcohol intake and preference ratio differences across the different groups were analyzed using two-way ANOVA for treatment (vehicle vs drug) and for concentration (7.5% vs 30%) (Table 2). In the experiments with URB597 combination with AM251, alcohol intake and preference ratio differences across the different groups were analyzed using one-way ANOVA for different treatments (Table 3). In the experiments with URB597 with 0.2 and 0.4% saccharin, saccharin intake and preference ratio differences across the different groups were analyzed using two-way ANOVA for treatment (vehicle vs drug) and for concentration (0.2% vs 0.4%) (Figure 3). In the ADE experiments, alcohol intake differences across the different groups were analyzed using two-way ANOVA for treatment (vehicle vs drug) and for session (baseline vs ADE) in each sex, with testing our a priori hypothesis that there was an ADE based on the published findings [e.g., Vengeliene et al. 2014 (link); Zhou et al. 2017 ] (Figures 4, 5). For NAE quantification, abundance differences between water and alcohol groups were analyzed using a two-tailed Student’s t-test followed by Bonferroni post-hoc tests (Figure 6) or one-way ANOVA with different time-points (Figure 7). All the ANOVA analysis was followed by Newman-Keuls post-hoc tests. The accepted level of significance for all tests was p < 0.05. All statistical analyses were performed using Statistica (version 5.5, StatSoft Inc, Tulsa, OK).

At 1 week after SNL, an SCS lead was placed epidurally through a small laminectomy at the T13 vertebra, as described in previous studies.^{51 (link),57 (link)} The sterilized lead was placed at the T10-12 spinal levels, which corresponds to T13-L1 spinal cord region. A subcutaneous tunnel was used to position the proximal end of the electrode in the upper thoracic region, where it exited the skin and connected to an external stimulator (model 2100, A-M Systems, Sequim, WA). Animals were allowed to recover from surgery for >1 week. SCS and mechanical pain hypersensitivity were examined at 7–14 days after lead implantation.
We used a crossover design to minimize potential time and order effects of testing. For the 1^st SCS, animals (n=11) were randomly selected to receive intrathecal infusion of CB1 receptor antagonist AM251 (50 μg, 15μl) or vehicle through lumbar puncture injection. One day later, to allow recovery from the previous treatment, the same group of animals was tested to the 2^nd SCS after having the drug assignment switched. The data from the two tests were combined for analysis. A separate group of SNL rats (n=10) received AM251 (50 μg, 15μl) or vehicle treatment followed by sham SCS. We blinded the experimenter to the treatments (e.g., drug, SCS) to reduce selection and observation bias.
On each test day, rats were acclimated for 30 minutes before we measured the baseline PWT. Motor threshold (MoT) was determined first by slowly increasing the amplitude of 4 Hz electrical stimulation from zero until muscle contraction was observed in mid-lower trunk or hind limbs. We set the first and third contacts (rostral to caudal) of the four-contact lead as an anode and the second and fourth as a cathode (“twin-pairs” stimulation) as shown in our previous studies.^{51 (link),57 (link)} Animals then received drug treatment, followed by pre-SCS PWT testing 30 minutes later. SCS (50 Hz, 0.2 millisecond, 80% MoT) or sham stimulation (0 mA) was then applied for 60 minutes. We examined PWTs at 30 and 60 minutes during the SCS (intra-SCS) and at 30 minutes after the completion of SCS to determine the carryover pain inhibitory effect. We used 80% of the MoT because it represents the maximum intensity of SCS that can be applied without causing discomfort in awake animals and it has been used in many previous studies.