Nicotine free base

Mecamylamine HCl and (-)-nicotine hydrogen tartrate salt (Sigma-Aldrich, St. Louis, MO) were dissolved in physiological saline (Patterson Veterinary, Devens, MA), and the pH was adjusted to approximately neutral (pH ~ 7), as necessary. (-)-Nicotine free base (Sigma-Aldrich) was mixed with a 50:50 propylene glycol and glycerin solution (Sigma-Aldrich). Doses of nicotine for injection are expressed as mg/kg of the base. Nicotine and mecamylamine were injected subcutaneously (s.c.) at a volume of 10 ml/kg. Concentrations for aerosol administration are expressed as mg/ml in the e-cigarette tank, and may not be representative of the actual amount of nicotine inhaled.
Chemicals and reagents for the analysis of biological samples were purchased commercially and included nicotine (Sigma-Aldrich), cotinine (Toronto Research Chemicals, Toronto, ON), nicotine-d3 (Cambridge Isotope Laboratories, Tewksbury, MA), cotinine-d3 (Santa Cruz Biotechnology, Dallas, TX), ammonium acetate (Sigma-Aldrich), and formic acid and acetonitrile (Fisher Scientific, Fair Lawn, NJ). An internal standard solution was prepared in methanol (Fisher Scientific) containing 48 µg/mL nicotine-d3 and 38 μg/mL cotinine-d3. Working solutions containing both nicotine and cotinine were prepared in methanol at concentrations of 10,000 and 100 ng/mL.

(−)-Nicotine free base (Cat # N3876, Sigma-Aldrich, St. Louis, MO, USA) was mixed with a 50:50 propylene glycol (PG) and vegetable glycerin (VG) blend (Fisher Scientific, Pittsburgh, PA, USA) for aerosol administration (PG:VG ratio was selected based on the fact that e-cigarette liquid formulations used by humans are often composed of 50:50 PG:VG (Peace at al. 2016 (link)). Mecamylamine hydrochloride (Cat # M9020, Sigma-Aldrich), a non-selective and non-competitive antagonist of the nicotinic acetylcholine receptors (nAChRs) (Bacher et al. 2009 (link)) was dissolved in saline and administered via subcutaneous (s.c.) injections.

Nicotine free base was purchased from Sigma (St. Louis, MO, USA) and diluted to the appropriate concentration in tap water for drinking studies. Melatonin was purchased from Tocris (St. Louis, MO, USA) and dissolved in 100% ethanol to a concentration of 400 μg/mL, which was further diluted to 400 ng/mL in tap water (for a final ethanol concentration of 0.1% in drinking solutions). To avoid potential confounds, control groups were also supplemented to 0.1% ethanol in drinking solutions. Quinine and saccharin were obtained from Sigma (St. Louis, MO, USA) and were resuspended in tap water at two different concentrations based on previously published results (Kamens et al., 2010 ; Kamens et al., 2005 (link)). The high concentration for Quinine was 0.03 mM, the low was 0.015 mM. For saccharin, the high concentration was 0.066%, and low was 0.033%.

(−)-Nicotine bitartrate, nicotine freebase, acetylcholine (ACh) iodide, diisopropyl fluorophosphate (DFP), NaCl, KCl, CaCl₂, MgSO₄, glucose, sucrose, HEPES, tetrodotoxin, polyethelenimine, bovine serum albumin (BSA), and cytisine were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO). The radioisotopes [³H]-dopamine (7,8-³H at 20–40 Ci/mmol), carrier-free ⁸⁶RbCl (initial specific activity 13.6–18.5 Ci/μg), and [¹²⁵I]-epibatidine (2200 Ci/mmol) were purchased from Perkin Elmer (Waltham, MA). α-Conotoxin MII (α-CtxMII) was generously provided by J. Michael McIntosh, University of Utah, Salt Lake City, UT.