Moreover, percentages of open arm time and the numbers of open arm entries did not differ between rats receiving rimonabant in the absence or presence of JZL184 (8 mg/kg) ( 0.05; Fig. and diazepam were purchased from Sigma Aldrich (St. Louis, MO, USA). All drugs were dissolved in a vehicle made up of 20% DMSO and 80% emulphor: ethanol: saline in a 1:1:8 ratio. Drugs, administered either alone or in combination, were usually dissolved in a volume of 1 ml/kg bodyweight to ensure that all studies employed a uniform injection volume and handling manipulation for i.p. drug administration. 2.3 General experimental methods 2.3.1. Experiment 1: Evaluation of anxiolytic effects produced by acute (i.p.) injection of JZL184 in the elevated plus maze under varying conditions of environmental aversiveness Rats were randomly assigned to receive a single intraperitoneal (i.p.) injection of either vehicle, the MGL inhibitor JZL184 (1, 4, or 8, or 16 mg/kg), the benzodiazepine anxiolytic diazepam (1, 2 or 3 3 RPS6KA5 mg/kg), or the cannabinoid CB1 receptor antagonist rimonabant (SR141716; 1mg/kg) in the presence or absence of JZL184 (8 mg/kg). All drugs were administered 30 min before behavioral screening. After injections, rats were placed in a room adjacent to the experimental room until tested. All drugs were prepared new on the day of screening and dispersed in vehicle before use by vortexting. Doses of diazepam (45C47) and JZL184 (42, 48) were selected based on previous studies. Pilot studies in our laboratory in rats indicated that 8 mg/kg was the maximum behaviorally effective dose of JZL184 on elevated plus maze behavior and assessments of nociception (data not shown). Limitations in drug solubility prevented screening of doses exceeding 16 mg/kg i.p. Previous research has shown that MGL blockade using JZL184 produces robust increases in 2-AG, but not AEA, in mouse brain (43). Our laboratory previously validated that, in the rat, JZL184 (local) selectively suppresses MGL but not FAAH activity (49) and produces non-overlapping, modality-specific, and pharmacologically unique antinociceptive effects from that of the FAAH inhibitor URB597 (50). Behavioral screening was conducted during the light phase of the light:dark cycle between 11:30 AM to 6:30 PM. The elevated plus maze (EPM) was a solid black + shaped structure with a black matte painted floor. The apparatus was elevated 50 cm above the floor and included two open (45 9 cm) and two closed (45 9 38 cm) arms extending from a central platform (9 9 cm). Rats were placed in the central platform of the plus-shaped maze, facing an open arm opposite to the experimenter. Test sessions of 5-min duration were digitally-recorded, as previously explained (51). An experimenter blind to treatment conditions quietly remained in the room during screening, hidden behind a room divider, and monitored the session. Measured behaviors were the number of open and closed arm entries and the percentage of time spent in open arms (24). Between assessments, the apparatus was wiped cleaned with a chlorhexidine diacetate answer and was allowed to dry. To induce different levels of environmental aversiveness, fluorescent lighting in the screening room was adjusted to emit low and high levels of illumination in the maze. For the low environmental aversiveness condition, illumination in the open and closed arms of the maze was 15 and 0 lux, respectively. For the high environmental aversiveness condition, illumination in the open and closed arms was 890 and 480 lux, respectively. Screening was conducted in a sound-attenuated (79 dB) environment. 2.3.2. Experiment 2: Effects of chronic and acute administration of the MGL inhibitor JZL184 on anxiety-like behavior in the elevated plus maze Rats received repeated once daily injections of either JZL184 (8 mg/kg i.p.) or Bibf1120 (Nintedanib) vehicle for 6 days. To control for handling Bibf1120 (Nintedanib) effects, a third group of rats (i.e. referred to here as the acute JZL184 Bibf1120 (Nintedanib) condition) received five once daily injections of vehicle followed by a terminal.