Am281

The phytocannabinoids THC and CBD were obtained from THCPharm (Frankfurt, Germany), AM281 and AM630 were from Cayman Chemicals (Ann Arbor, MI, USA). Stock solutions of all drugs were prepared in dimethyl sulfoxide. Drug injection solutions were made up immediately prior to administration. Drugs were administered as an intrathecal injection using a Hamilton syringe and 25 G needle (volume = 10 µL in a vehicle consisting of 25% DMSO, 15% ethanol and 2% randomly methylated beta-cyclodextrin (RAMEB) in saline). Intrathecal injections were made under brief anesthesia (2% Isoflurane in saturated oxygen) and recovered immediately following drug administration. For intrathecal injection, mice were placed in dorsal recumbency and the fur over the lumbar-sacral spine was clipped, and the skin wiped with ethanol. The needle was inserted in the intervertebral gap between L4 and L5 until a ‘popping’ sensation and/or twitch of the tail was observed, and the drug solution slowly injected. The needle was then slowly removed, and the animal placed in its home cage for recovery.

KML29 (MAGL inhibitor; 1-piperidinecarboxylic acid, 4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-, 2,2,2-trifluoro-1-(trifluoromethyl)ethyl ester) was obtained from Med Chem Express Ltd. (Monmouth Junction, NJ, USA). Celecoxib (CXB; COX-2 inhibitor; 4-[5-(4-methylphenyl0-3-(trifluoromthyl)-1H-pyrazol-1-yl]-benzenesulfonamide), AM281 (CB1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide), and AM630 (CB2 receptor antagonist; 6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) were obtained from Cayman Chemicals (Ann Arbor, MI, USA). Rhodamine 6G, cremophor, dimethyl sulphoxide (DMSO), urethane, and sodium monoiodoacetate (MIA) were obtained from Sigma Aldrich (St. Louis, MO, USA). Solutions of KML29, AM281, AM630, and Celecoxib were prepared in vehicle (1:1:18; DMSO:cremophor:saline) on the day of use. Rhodamine 6G (0.05%) and MIA were dissolved in saline. Physiological buffer (135 mM NaCl, 20 mM NaHCO₃, 5 mM KCl, 1 mM MgSO₄·7H₂O, pH = 7.4) was prepared in the lab.

Myrcene (7-methyl-3-methylene-1,6-octadiene) in soy oil was purchased from Toronto Research Chemicals (Toronto, ON, Canada). CBD (2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) was obtained from Tocris Bioscience (Bio-Techne, Abingdon, UK). AM281 (CB1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and AM630 (CB2 receptor antagonist; 6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) were obtained from Cayman Chemicals (Ann Arbor, MI, USA). The selectivity of AM281 is 12 nM: 4200 nM for CB₁:CB₂, and for AM630 5µM: 31 nM for CB₁:CB₂. Rhodamine 6 G, cremophor, dimethyl sulphoxide (DMSO), urethane, and Freund’s complete adjuvant were obtained from Sigma Aldrich (St. Louis, MO, USA). Solutions of CBD, AM281, and AM630 were prepared in vehicle (1:1:18; DMSO:cremophor:saline) on the day of use. Rhodamine 6 G (0.05%) was dissolved in 0.9% saline.

Indomethacin morpholinamide, AM281, a CB1 receptor antagonist and AM630, a CB2 receptor antagonist and the deuterated AEA (AEA-d4), 2-AG (2-AG-d5), and AA (AA-d8) were purchased from Cayman Chemicals (Ann Arbor, MI, United States). All other chemicals and reagents were purchased from Sigma (St. Louis, MO, United States), unless stated otherwise.

The ABHD6 inhibitor WWL70, the CB1R antagonist AM281, and the CB2R antagonist AM630 were purchased from Cayman Chemicals (Ann Arbor, MI). All other chemicals and reagents were purchased from Sigma (St. Louis, MO), unless stated otherwise.

Normal goat serum and lysophosphatidic acid (LPA) were obtained from Abcam (Toronto, ON, Canada), PF05089771 from Tocris Bioscience (Toronto, ON, Canada), AM281 from Cayman Chemical Company (Ann Arbor, MI, USA), PEP-1 from Abbiotec Inc. (San Diego, CA, USA), Fluoro-gold from Fluorochrome LLC (Denver, CO, USA), mouse IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA), and guinea pig anti-Na_v1.7 (for IHC) from Alomone Labs (Jerusalem, Israel).
The mouse anti-Nav1.7 clone N68/6 monoclonal antibody used for behavioural experiments was developed by and obtained from the UC Davis/NIH NeuroMab Facility (Davis, CA, USA). The rabbit anti-guinea pig secondary biotin, streptavidin protein Dylight 488, and fluoromount G were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cremophor, dimethyl sulphoxide (DMSO), naloxone HCl and sodium monoiodoacetate (MIA) were obtained from Sigma-Aldrich (Oakville, ON, Canada). Isoflurane and euthansol were purchased from CDMV (Dartmouth, NS, Canada).