Varenicline

Poly-D-Lysine (P7886), Nicotine (N3876), Varenicline (PZ0004), Lobeline (141879), Epibatidine (E1145), Mecamylamine (M9020), Ammonium Chloride (A0171), Chloroquine (C6628) and Bafilomycin A1 (B1793) were purchased from Sigma, MO. Dihydro beta-erythroidine (DHβE) (2349) was purchased from Tocris, MN. Neurobasal medium, B27, HBSS and DMEM were purchased from Life technologies (Thermofisher scientific, MA).

Male mice (8 weeks old) were administered vehicle (saline) or 2 mg/kg (s.c.) varenicline (Sigma, Sydney, NSW, Australia) 15 min prior to receiving a vehicle or 2 mg/kg (i.p.) yohimbine (Tocris, Noble Park, VIC, Australia) injection. Approximately 100 μL of tail blood was collected into Ethylenediaminetetraacetic acid (EDTA)-treated tubes, under isoflurane anaesthesia (2–3%), 1 h following the last injection. Twenty hours later, a lethal injection of Pentobarbital was administered and once anaesthetized, a second tail blood sample was collected. Immediately following blood collection, transcardial perfusion was performed using phosphate buffered saline (PBS), the brain was harvested and the nucleus accumbens was dissected on ice.

Substrate specificity of NicA2 was analyzed by the Amplex Red assay described above, using 10 μM of test compound and 160 nM NicA2 enzyme. Compounds tested were: (2’S)-nicotine-1’-N-oxide (Toronto Research Chemicals; TRC), (±)-nornicotine, nicotinamide, β-nicotinamide adenine dinucleotide (NAD), acetylcholine, choline, (−)-cotinine, varenicline, bupropion, (−)-cytisine, mecamylamine (TRC), dopamine, serotonin, (±)-norepinephrine, L-glutamate, γ-amino-N-butyric acid (GABA), (R,S)-anatabine (TRC), (R,S)-anabasine (TRC), and myosmine (compounds obtained from Sigma-Aldrich unless otherwise stated). Activities were expressed relative to the activity found for S-(−)-nicotine run in parallel.