Figure 2
Lithium impairs nAChR turnover in normally innervated mature NMJs. (A) Experimental design used to analyse the effect of lithium on nAChRs turnover. Daily injection of control or lithium-treated groups was performed 3 days before BTX1 labelling. After 7 days, BTX2 was used to stain newly incorporated nAChRs. Confocal imaging of BTX1 or BTX2 within individual mature nAChR structures is represented for control (NaCl) or lithium (LiCl)-treated mice. (B) Pictures show representative confocal images of merged BTX1 and BTX2 staining after lithium or control treatment. Bar = 20 µm. (C) nAChRs turnover was determined by quantifying the BTX2/BTX1 fluorescence intensity ratio. BTX1 (D) and BTX2 (E) fluorescence intensities are shown to determine the contribution of each nAChR pool in all tested conditions. Graphs represent means ± SD along with individual data for control or lithium condition. For statistical analyses, unpaired t-test was used. ***p < 0.001; **p < 0.01. Detailed analyses of lithium effect on nAChRs turnover in segmented endplates (F–H) or segmented pretzel branches (I–K) were performed by quantifying the BTX2/BTX1 fluorescence intensity ratio for each experimental group. Multiple t-tests were carried out for statistical analysis between each segment from control or lithium-treated groups respectively. ****p < 0.0001; **p < 0.01; *p < 0.05. (L) Confocal imaging of BTX1 plus BTX2 fluorescence signals are displayed in gray colour to facilitate comparison on nAChRs density between experimental conditions. The total fluorescence signal of BTX1 plus BTX2 was measured both for endplate (M) (Bar = 20 µm) and postsynaptic branches (N). Graphs in (M) represent mean values ± SD along with individual data for control or lithium condition. t-test was performed for statistical analyses of 30–40 NMJs per animal (N = 3 animals per group). n.s: not significant; p > 0.05.
