Nicotine (nicotine hydrogen tartrate salt; Sigma-Aldrich, St. Louis, MO) or vehicle (saline) was given subcutaneously (2 mg/kg per 12 h) in a total volume of 100 μL (25). Rats received a nicotine or vehicle injection every 12 h in both subchronic (48 h) and chronic (17 days) experiments. To validate the EE data, all rats were matched for body weight before treatment began. To control nicotine-induced respiratory depression (RD), rats were treated with nicotine and symptoms of RD were visually controlled from 2 min before to 12 h after treatment. For the antagonism of α3β4 nicotinic acetylcholine receptors, rats were treated previously (30 min) intraperitoneally with mecamylamine (mecamylamine hydrochloride, 1 mg/kg; Sigma-Aldrich) as described recently (3) and subcutaneously with nicotine during 48 h.
