Fig. 4

Hyperactivity of ACC pyramidal neurons simultaneously develops in chronic nicotine-treated mice. A (Left) Bilateral distribution of c-Fos⁺ neurons in the ACC in vehicle- and nicotine-treated mice after 28 days. Scale bar, 200 μm (left), 20 μm (right); (Right) number of c-Fos⁺ cells per 0.04mm² area in the ACC. B showing that c-Fos co-labeled neurons within the ACC on day 28 after nicotine-treated were mainly co-localized with glutamatergic immunofluorescence and statistics data. Scale bars, 20 μm. C Schematic for viral injection and whole-cell recordings. D Sample traces (left) and data (middle and right) of action potentials and summarize of rheobase of vehicle- and nicotine-treated mice. E Schematic and timelines of fiber photometry. F Representative imaging of GCaMP6m viral expression in the ACC glutamatergic neurons 3 weeks after viral injection. The boxed region (dashed lines) represents the site of implantable fiber optic cannula. Scale bars, 200 μm (left), 20 μm (right). G The heatmaps (left) and mean (right) show that Ca²⁺ signals rapidly increased in nicotine-treated mice compared with vehicle mice after 28 days. The colored bar on the right indicates ΔF/F (%). H Average ΔF/F of ACC^GCaMP6m signals in vehicle- and nicotine-treated mice. Data were presented as mean ± SEM or Median (IQR). *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant. Details of the statistical analyses are presented in Additional file 1: Table S1