86RbCl (average initial specific activity 15 Ci/mg) as well as Optiphase Supermix scintillation cocktail was purchased from Perkin-Elmer NEN (Boston, MA). The α5 knockout mice were injected with either the Lenti-CHRNA5 or Lenti-Control vector as previously described. Following an incubation period of at least 3 weeks, mice were killed synaptosomes generated from the IPN, habenula, hippocampus, striatum, thalamus and cortex as described previously 7. Samples were loaded with 86Rb+ and acetylcholine-stimulated 86Rb+ efflux was measured as described previously7, with each sample stimulated only once. 86Rb+ efflux was expressed as the increase in signal above basal efflux. A non-linear least squares curve fit to a first order equation (Ct = C0*e−kt), where Ct is the basal efflux counts at time, t, C0 is the estimated efflux counts at t = 0 sec, and k is the first order decay constant) was used to estimate basal efflux for each sample. Counts in fractions preceding and following the peak were used for curve fitting. Acetylcholine-stimulated efflux was calculated by summing the counts in the fractions exceeding basal efflux during ACh exposure and dividing by the corresponding basal efflux counts. This value represents total peak relative to baseline.
