FIGURE 8.

Absence of internalization of m2R in caveole in fixed neuron after stimulation by CCh. Hippocampal neurons were co-transfected with a plasmid encoding the wild-type receptor (WT-m2R: A,B) and CAV1-mCherry (A’,B’) fixed, and processed for visualization by confocal microscopy. In control and treated neurons, CAV1-mCherry is detected in the cytoplasm as a punctiform labeling (A’,A”, B’,B”). Some m2R and CAV1-mCherry clusters colocalize (arrows) in both treated and untreated neurons. (C) The quantitative analysis of the colocalization of m2R and CAV1-mCherry in neurons was performed using the Jacop Plugin of ImageJ and statistical data are reported from the Costes’s randomization-based colocalization module (see Materials and Methods). Data are expressed as a Pearson’s coefficient (pc) and pc were compared using the Kruskal–Wallis test followed by the Dunn’s Multiple Comparison Test. Our analysis shows that pc values do not significantly differ in control neurons and neurons treated with CCh for 6, 12, and 15 min (NS, not significant).