Blood sampling (tail bleedings) was performed immediately after daily bouts of alcohol vapor exposure in dependent animals. Blood samples were also obtained from nondependent animals (exposed to control air) at the same time to control for handling during blood sampling. Plasma (5 μL) was used for measurement of blood alcohol levels using an Analox AM 1 analyzer (Analox Instruments). The reaction is based on the oxidation of alcohol by alcohol oxidase in the presence of molecular oxygen (alcohol + O2 → acetaldehyde + H2O2). The rate of oxygen consumption is directly proportional to the alcohol concentration. Single-point calibrations were done for each set of samples with reagents provided by Analox Instruments (25–400 mg% or 5.4–87.0 mM). When blood samples were outside the target range (125-250 mg%), vapors levels were adjusted. Blood alcohol levels were always undetectable in nondependent animals (air exposure only).
