Figure 2. Upregulation onset and reversal of cortical neuron nAChRs in real time. *A*, Time course of upregulation. Primary cultures of cortical neurons were treated with 1 μm nicotine for the indicated times. The data are plotted as the fold-increase (the ratio of nicotine-treated to untreated cultures) in ^{125}I-epi binding as a function of time following nicotine exposure ± SEM (*n* = 5). The line represents a least-squares fit to the sum of two exponentials or fold-increase = *m*1 * (1 − exp(−*t*/τ1)) + *m*2 * (1 − exp(−*t*/τ2)) + 1 where *m*1 and *m*2 are the maximal values for the two exponentials (*m*1 = 0.89, *m*2 = 0.74) and τ1 and τ2 are the respective time constants (τ1 = 1.1 h, τ2 = 12.5 h). *B*, Decay of high-affinity nicotine-binding sites following nicotine withdrawal. Primary cultures of cortical neurons were treated with or without 1 μm nicotine for 17 h, and the cultures were washed three times with neurobasal medium and maintained in preconditioned media lacking nicotine for the indicated times. High-affinity binding sites remaining were measured using 1 nm ^{125}I-epi bound for 20 min. Displayed in the main figure are the data for the first 24 h after nicotine removal to show the details of the fast decay. The full time course of the decay over 11 d is displayed in the insert. Data are mean ± SEM of four independent experiments performed in triplicate. The lines through the points represents the least-square fit to the data of the equation: fold increase = *m*1(exp(−*t*/τ1)) + *m*2 (exp(−*t*/τ2)) +1, where *m*1 and *m*2 are the initial values for the two exponentials (*m*1 = 2.4, *m*2 = 1.12) and τ1 and τ2 are the respective time constants (τ1 = 0.45 h, τ2 = 295 h). *C*, Nicotine-induced upregulation after short exposures to nicotine. Primary cultures of cortical neurons were treated with or without 1 μm nicotine for 1, 2, or 4 h and cultures were washed and maintained in nicotine-free media as mentioned above for indicated times and ^{125}I-epi binding performed. Each point is the mean ± SEM (*n* = 3). The data were fit to the equation *m*(exp−*t*/τ) + 1, where *m* is the initial value and τ is the time constant. The τ values are 0.5, 0.9, and 1.1 h, respectively, for 1, 2, and 4 h time points. For comparison, the data from the 24 h treatment from *B* are also displayed. *D*, HEK 293 cells stably expressing α4β2HA receptors were treated with or without 10 μm nicotine for 17 h and washed and maintained in medium without nicotine for the indicated times. High-affinity binding sites remaining were measured using 5 nm ^{125}I-epi bound for 20 min. Data are mean ± SEM (*n* = 5). The lines through the points are the least-squares fit of fold-increase = *m*1(exp(−*t*/τ1)) + *m*2 (exp(−*t*/τ2)) +1, where *m*1 and *m*2 are the initial values for the 2 exponentials (*m*1 = 1.3, *m*2 = 2.5), and τ1 and τ2 are the respective time constants (τ1 = 0.76 h, τ2 = 44 h). *E*, HEK293 cells stably expressing α6Flagβ2HA were treated with 30 μm nicotine for 17 h to achieve maximum upregulation and then grown in the absence of nicotine for the indicated times. High-affinity binding sites remaining were measured using 2.5 nm ^{125}I-epi bound for 20 min. Data are mean ± SEM (*n* = 6). The lines were the fits to the equation in *D* (*m*1 = 1.0, *m*2 = 2.46; τ1 = 0.94 h, τ2 = ∼500 h). Because the HEK cells stably expressing α6β2 receptors did not survive much beyond 48 h after the nicotine removal, the estimate of τ2 is an approximate. Nic, Nicotine.