Dihydro β erythroidine hydrobromide

TC-2559 difumarate (4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate), sazetidine A dihydrochloride (6-[5-[(2S)-2-azetidinylmethoxy]-3-pyridinyl]-5-hexyn-1-ol dihydrochloride), and dihydro-β-erythroidine hydrobromide ((2S,13bS)-2-methoxy-2,3,5,6,8,9,10,13-octahydro-1H,12H-benzo[i]pyrano[3,4-g]indolizin-12-one hydrobromide; DHβE) were purchased from Tocris Biosciences (Bristol, UK). These agents were dissolved in sterile water at a higher concentration and diluted with sterile PBS for use. Perineural injection was performed according to a method described previously [9 (link), 37 (link)]. In brief, under isoflurane anesthesia, the agents (10 μl) were injected without a skin incision into the region surrounding the SCN, using a microsyringe fitted with a 30-gauge needle connected to a cannula. Injections of TC-2559, sazatidine A, or DHβE were administered consecutively for 4 days during the first week (days 0, 1, 2, and 3; early phase), second week (days 7, 8, 9, and 10; middle/late phase), or fourth week (days 21, 22, 23, and 24; much later phase) after PSL. Bupivacaine hydrochloride (Sigma-Aldrich) was dissolved in sterile saline at 0.5% concentration, which were administered for consecutive 6 days after PSL (days 0–5).

(−)-Nicotine hydrogen tartrate salt was obtained from Sigma Chemical (St. Louis, MO). Varenicline dihydrochloride was obtained from the National Institute on Drug Abuse drug supply program (Rockville, MD). Mecamylamine was obtained from Waterstone Technology (Camel, IN). The epibatidine derivatives 2′-Fluorodeschloroepibatidine (RTI-7527-36), 3′-(3″-dimethylaminophenyl) epibatidine (RTI-7527-76), and 2′-fluoro-3′-(4-nitrophenyl) deschloroepibatidine (RTI-7527-102) were synthesized at the Center for Organic and Medicinal Chemistry, Research Triangle Institute (Research Triangle Park, NC) as previously described [16 (link)–18 (link)]. Dihydro-β-erythroidine hydrobromide was obtained from Tocris (Minneapolis, MN). Drugs were dissolved in 0.9% physiological saline and administered s.c. (except mecamylamine which was administered intraperitoneally) in a volume of 10 mL/kg. The dose of nicotine is expressed as the weight of the free base. Other drug doses are expressed as the weight of the base plus the salt.