Nicotine-Evoked Dopamine Release Assay

Previously described methods [27 (link)] were followed using 7,8-[³H]DA (20–40 Ci/mmol) obtained from Perkin Elmer Life Sciences (Boston, MA). Crude synaptosomal preparations from freshly dissected brain regions were allowed to take up tracer [³H]DA prior to superfusion at 0.7 ml/min for 10 min. Release of DA was stimulated by exposure to nicotine for 20s. Parallel aliquots were exposed to 50 nM α-conotoxin MII (α-CtxMII) (generously provided by Dr. J. Michael McIntosh, University of Utah) for 5 min before the nicotine exposure. Fractions (10 s, ~0.1 ml) were collected into 96-well plates using an FC204 fraction collector (Gilson, Inc., Middleton, WI) for 3.8 min starting one min before nicotine exposure. DA release units are calculated as evoked cpm exceeding baseline cpm, summed and normalized to baseline cpm.