FIGURE 4.

Time lapse imaging and quantification of internalization of m2R in a living neuron after stimulation by CCh, a muscarinic receptor agonist. A living hippocampal neuron transfected with GFP-m2R was observed by spinning disk confocal microscopy before and during a 30 min long carbachol (100 μM) treatment. A stack of 20 consecutive confocal images (0.5 μM in depth) were acquired every 30 s. A projection of the stack images was performed and illustrated on this panel (A–F). An equatorial image was selected and an enlargement of a dendritic shaft is shown at the bottom of each image (insert). Here is shown the GFP-m2R labeling in this neuron 3 min before CCh and every 3 min for 30 min. Before CCh addition (A) and at the beginning of agonist treatment (C), m2R was detected mainly at the plasma membrane of soma and dendrites. Agonist induces internalization of membrane-associated m2R and clusterization 6 min after treatment initiation in the cytoplasm of soma and dendrites (arrows in C). (G) Quantification of the effect of CCh on the density of fluorescent clusters +/- SEM in the cytoplasm of 3 neurons. Fluorescence was measured on a projection of the stack images using the Fiji software. The clusters density increases during the first 30 min and stabilized afterward. The statistical analysis (repeated measures ANOVA test followed by the Dunnett post hoc test) shows a significant increase of the number of clusters as early as 10 min after the beginning of CCh stimulation.