125i epibatidine

Neurobasal media, Minimal essential media (MEM), B27 supplement, GlutamaxTM, inactivated horse serum and TrypLE express (trypsin solution) were purchased from Invitrogen (Carlsbad, CA). [¹²⁵I]epibatidine (2200 Ci/mmol) was purchased from Perkin-Elmer Life Science (Waltham, MA). 2-(2-bromoacetyloxy)-N,N,N-trimethylethanaminium bromide (BrACh), cytisine, cytosine β-D-arabino-furanoside (ARA C), 5,5’-dithio-bis(2-nitrobenzoic acid (DTNB), 1,4-dithio-DL-treithol (DTT), (−)-nicotine hydrogen tartrate, polyethyleneimine (PEI), and poly-l-lysine (> 30,000 kDa) were purchased from Sigma Aldrich Chemical Company (St. Louis, MO). 4-(2-Hydroxyethyl)-piperazineethanesulfonic acid (HEPES) half-sodium salt was from Roche Diagnostics Corporation (Indianapolis, IN).

α4β2* nAChR density was assessed via ¹²⁵I-epibatidine binding to striatal core slices as previously described (Lai et al., 2005 (link); Huang et al., 2009 (link)). Briefly, slides were thawed on a slide warmer (5-10 min) and pre-incubated in binding buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.0 mM MgCl₂, pH 7.5) plus 100 nM αCtxMII {Cartier, 1996 #569} (used to inhibit epibatidine binding to α6β2* nAChR) at room temperature for 30 min. Slides were then incubated for 40 min in binding buffer containing 0.015 nM ¹²⁵I-epibatidine (2200 Ci/mmol, PerkinElmer, Watham, MA) in the presence of 100 nM αCtxMII. In a previous study conducted in the same laboratory, nonspecific binding was determined by slides incubated in binding buffer containing 0.015 nM ¹²⁵I-epibatidine plus 0.1 mM nicotine (Vieira-Brock et al., 2015 ). Slides were rinsed twice in ice-cold buffer for 5 min followed by a 10 s rinse in distilled water and air-dried. Sample slides and standard ¹²⁵I microscale slides (American Radiolabeled Chemicals, St. Louis, MO) were exposed to Kodak MR film (Eastman Kodak Co., Rochester, NY, USA) for 24 h.