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. 2018 Nov 30;12:450. doi: 10.3389/fncel.2018.00450

FIGURE 3.

FIGURE 3

Time lapse imaging and quantification of SEP-m2R membrane labeling in a living neuron after stimulation by CCh, a muscarinic receptor agonist. A living hippocampal neuron transfected with SEP-m2R was observed by spinning disk confocal microscopy for 75 min. A stack of 20 images were collected at 30 s intervals. An equatorial image (0.5 μM in depth) was selected and illustrated on this panel (A–C). (A) In control condition, the SEP-m2R staining is detected at the membrane of the cell body and proximal dendrites. A faint signal is also shown in the cytoplasm. (B) CCh (100 μM) induces a decrease of SEP-m2R labeling at cell body and dendrites levels. (C) Application of NH4Cl (50 mM) that reveals receptors associated with acidic intraneuronal organelles induces an abundant and intense punctiform staining in the cytoplasm. (D) Quantification of the effect of CCh on the fluorescence level +/- SEM using the Fiji software. Fluorescence was measured on three different neurons on a projection of the stack images at the level of the whole neuron using the Fiji software. Data are expressed as normalized values compared to the fluorescence level at 9 min before CCh application. The quantification shows a significant difference of m2R fluorescence with time. The statistical analysis (Repeated measures ANOVA test followed by the Dunnett post hoc test), performed on raw data, shows that CCh induces a significative decrease of fluorescence 39, 54, and 69 min after the beginning of the treatment. Post hoc anlayses were performed on two segments of the slop to analyze (1) the effect of CCh (from T = 0 min until 84 min) and (2) the effect of NH4Cl (from T = 84 min until 120 min) on fluorescence levels. The values are compared to the values at T = 9 min, the initiation point of CCh application for the CCh effect and at T = 84 min, the initiation of NH4Cl application, for NH4Cl effect. Results show a significant decrease of the fluorescent level from 39 to 54 min after CCh stimulation. From 54 min, fluorescence slowly returns to normal values. In contrast, NH4Cl, which reveals m2R attached to acidic vesicles, induces a significant increase of SEP-m2R fluorescence levels. NS, not significant, ∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001.