Win55 212 2

IQD analogues PNR-4-20 and PNR-4-02 were synthesized as previously described (30 (link)) and were dissolved in 100% DMSO to 10 mM stock concentrations for in vitro assays. WIN-55,212-2, JWH-018, CP-55,940, and DAMGO were obtained from Tocris Bioscience (Bristol, United Kingdom). GTPγS was purchased from EMD Chemical (Gibbstown, NJ). [³H]CP-55,940 (52.6 Ci/mmol) was acquired from PerkinElmer (Waltham, MA) and [³⁵S]GTPγS (1250 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). PathHunter™ enzyme fragment complementation (EFC) experimental reagent was purchased from DiscoverRx Corporation (Fremont, CA). All other reagents used were purchased from Fisher Scientific (Pittsburgh, PA).

Reagents for in vitro experiments included SR141716A (Rimonabant hydrochloride, 5 μM, Sigma), WIN55,212–2 (Tocris), GAT211 (5 μM, Sigma) were dissolved in 0.5% DMSO.

The CB1R-null mouse line was validated by demonstrating the absence of CB1R mRNA expression in brain sections using in situ hybridization histochemistry (ISHH), as previously described (Vianna, et al. 2012 (link)). The [³⁵S]UTP antisense cRNA riboprobe used in ISHH was transcribed from a template generated by PCR amplification of genomic DNA with the following CB1R-specific primers: forward, 5′-CTGCAAGAAGCTGCAATCTG-3′ and reverse, 5′-TGGCGATCTTAACAGTGCTC-3′. ISHH was performed in 3 CB1R-null mice and 3 wild-type controls. The line was functionally validated by demonstrating the absence of hyperphagia in response to administration of a potent CB1R-selective agonist WIN-55,212–2 (Tocris Bioscience, Minneapolis, MN). WIN 55,212–2 was prepared in saline solution containing 5% DMSO and Tween 80 (1drop/5mL). Adult CB1R-null mice and wild-type littermates were administered vehicle and then with WIN 55,212–2 (0.6 mg/kg BW i.p.) 24h later. The experiments were performed during the first hour after the lights are switched on. One-hour and 2h food intake was measured after both vehicle and WIN 55,212–2 administration.