Figure 1
Compartmentalized analyses of nAChRs turnover in mature NMJs. (A) Experimental design used for the time-course analysis of nAChRs turnover. The pre-existing nAChRs pool was labelled with AlexaFluor488-BTX (BTX1) and after different time points, newly incorporated nAChRs were detected by AlexaFluor555-BTX (BTX2) staining. Pictures show representative confocal images of BTX1 and BTX2 staining at different time points ranging from 6h to 14d. Bar = 20 µm. (B) nAChRs turnover analyses in individual endplates were performed by quantifying BTX2 and BTX1 fluorescence intensity at different time points. nAChR turnover was evaluated using the BTX2/BTX1 fluorescence intensity ratio. Graphs represent mean values ± SEM of 25–50 NMJs for each time point. (C) Compartmentalized nAChRs turnover was determined by dividing each endplate in five concentric sections and fluorescence intensity of both BTX1 and BTX2 was measured. The graph represents mean values ± SEM for nAChRs turnover in each segment (1 to 5, from periphery to centre) from 10 endplates per animal. (D) nAChRs turnover within pretzel branches were analysed in three branches per pretzel located in the periphery, middle, and central region of the mature end-plate structure. Each branch was subsequently sectioned in peripheral (1, yellow), middle (2, gray), or centre (3, orange) segments. Fluorescence intensity ratios from BTX2/BTX1 were quantified and plotted. The graph represents the mean values ± SEM for each branch segment from 30 pretzel branches per animal. N = 2 (14d), or 3 (6h–7d). One-way ANOVA with subsequent Tukey’s multiple comparisons tests were performed at each point. ****p < 0.0001; ***p < 0.001; *p < 0.05 when middle (2) or centre (3) branch segments were compared to the peripheral (1) segment.
