(A–B) Neuronal stimulation by nicotine exposure (A) or increased synaptic input with bicuculline treatment (B) increased levels of wheat germ agglutinin (WGA) surface staining. Scale bars, 10 µm. (A) Cortical cultures were transfected with HA-tagged α4β2R subunits, treated with or without 1 μM nicotine for 17 hr and cultures were live-labeled with WGA-Alexa 647 for 20 min. Images displaying surface WGA labeling were obtained after cultures were fixed. Pseudocolor intensity profiles are shown in the right panels. (B) Following overnight bicuculline treatment, untransfected cultures were processed, surface labeled, and displayed as in A. (C) Quantification of surface WGA intensities from A and B. Data are shown as normalized mean ± SEM, left; nicotine experiment, control neurons, 1.0 ± 0.02; nicotine-treated neurons, 1.4 ± 0.07 (n = 7–11 neurons per group, *p < 0.0007), right; bicuculline experiment, control neurons, 1.0 ± 0.1; bicuculline-treated neurons, 1.4 ± 0.09 (n = 14–16 neurons per group, *p < 0.006). Quantitative analysis was conducted on two independent culture preparations. (D) Differences between Con-A and WGA surface labeling when endocytosis of lectins occurs. Cortical cultures transfected with St3-GFP were surface-labeled with WGA-Alexa 568 and Con A-Alexa 647 for 20 min, washed, then incubated at 37°C for 2 hr. WGA, but not Con A, was rapidly internalized and overlapped with St3-containing Golgi satellites (yellow arrows). Insets are higher magnification of hatched boxes. Scale bar, 10 µm. Inset scale bar, 2.5 µm. (E) Neuronal excitation leads to increased association of dispersed Golgi in dendrites with early endosomal membranes. Cultures were transfected with St3-Halo, treated with or without bicuculline for 17 hr, and fixed, permeabilized, and immunostained with anti-EEA1 antibody (green). Insets are higher magnification of hatched boxes. Scale bar, 10 µm. (F) Quantification of the percentage of St3 puncta that overlapped with EEA1. Data are shown as mean ± SEM, control neurons, 26.4 ± 4.8; bicuculline-treated neurons, 53.5 ± 2.3 (n = 10 fields per group, *p < 0.0007). (G) WGA is endocytosed with α4β2Rs and traffics into Golgi satellites. Cortical neurons were transfected with St3-Halo, HA-tagged α4β2R subunits and treated with 1 μM nicotine for 17 hr. α4β2R endocytosis was imaged by antibody feeding as in Figure 7A and B (anti-HA, green), and ST3-Halo labeled with Halo JF 594 (red), and WGA with WGA-Alexa 647 (blue). After labeling at time 0, neurons were incubated at 37°C for the indicated times to follow their endocytosis. As in D, WGA showed rapid internalization into St3 vesicles during the 30 min incubation period (0 hr), and over the time course, endocytosed β2HA showed increased co-localization with vesicles containing both WGA and St3-Halo. Scale bar, 10 µm. (H) Cultures were transfected with St3-Halo alone, or along with α4 and β2HA subunits (nicotine treatment), for 24 hr, then treated with and without bicuculline or nicotine for 17 hr, and surface labeled for 30 min with Halo JF 594 and WGA-Alexa 647. Cells were washed and incubated further for 2 hr to allow for WGA internalization. Scale bar, 10 µm. (I) Quantification of WGA puncta densities from F. Data are shown as mean ± SEM, left; nicotine experiment, control neurons, 4.1 ± 0.4; nicotine-treated neurons, 6.3 ± 0.5 (n = 6 fields per group, *p < 0.00003), right; bicuculline experiment, control neurons, 4.0 ± 0.5; bicuculline-treated neurons, 5.6 ± 0.4 (n = 6–8 fields per group, *p < 0.02). Quantitative analysis was conducted on two independent culture preparations.