Figure 5.

Nicotinic stimulation in cell culture in the absence of functional β2*-nAChRs increases the number of functional glutamatergic synapses that form on dendritic shafts, as opposed to dendritic spines. A, Neurites of rat hippocampal neurons transfected to express GFP and incubated with (Nic) or without (Con) 1 μm nicotine for 20 d and imaged. B, Immunostaining cultures for PSD-95 (red) overlaid with GFP (green) in a nicotine-treated culture as in A. C, Spine counts on control cells or cells treated with nicotine, 100 nm αBgt, and 10 μm DHβE. D, Quantification of spines in cultures grown 10 d with the indicated compounds present for the last 7 d. Drebrin immunostaining was used to visualize spines. Drug concentrations are as in C; 50 nm MLA. E, Confocal images of FM4-64-labeled neurons (red) expressing lenti-GFP (green). Arrows and asterisks indicate examples of FM4-64 puncta on spines and dendritic shafts, respectively (puncta lying centrally over shafts were not scored). F, Quantification of FM4-64 puncta on spines (Spine) and shafts (Shaft) for the conditions indicated, scored as in E (3–5 cultures; 5 cells per culture). Scale bars: A, E, 5 μm; B, 10 μm. Con, Control; Nic, nicotine.