, Nine minutes after the initiation 1042 of CCh treatment, some m2R clusters colocalize with native CCP (arrows). C : The analysis 1043 of the colocalization of clathrin and m2R-ICC was performed on fixed neurons using the 1044, neuron displays no m2R and clathrin colocalization

, The quantification of m2R and clathrin colocalization 1046 and the statistical analysis demonstrates a significant increase of the Pearson's coefficient in 1047 treated neurons (n=20) compared to cells treated with CCh (n=20) (Mann Whitney U test; 1048 * : p<0.0001). ). Control neurons : n=20, Jacop Plugin of ImageJ are reported from the Costes's randomization-based colocalization 1045 module (see Materials and methods), p.21

, Figure 6 : 1051 Internalization of m2R in clathrin-coated pits in a living neuron after stimulation by 1052

. Cch, A living hippocampal neuron transfected with GFP-m2R and Ds-Red clathrin was 1053 observed by spinning disk confocal microscopy before and during a 30-min-long carbachol 1054 (30µM) treatment. A stack of 10 images was collected and selected images at the indicated 1055 times show a representative dendrite and a selected area in a cell body

, Six mimutes after the begining of CCh treatment, m2R clusters appear at loci of clathrin 1057 coated-pit (CCP) spots (arrows) in the dendrite and the cell body (A''-E''). Note that the 1058 m2R clusters form in preexisting CCP

, Al594-Tf uptake and m2R clathrin-dependent endocytosis in hippocampal neurons. A1062 D' : Hippocampal neurons were transfected with m2R-WT, pre-incubated with Tf-Al594, 10 1063 min before CCh treatment. Cells were fixed after 15min treatment, p.1064

, CCh treatment 1065 induces a strong decrease of membrane m2R labeling and the appearence of m2R 1066 punctiform staining (B',B''). Al594-Tf and m2R-ICC signal often colocalize, Al594-Tf is detected in the cytoplasm as a punctiform labelling (A'-A'')

, The quantitative analysis of the colocalization of m2R and Al594-Tf in neurons was 1068 performed using the Jacop Plugin of ImageJ and statistical data are, p.1069

, Costes's randomization-based colocalization module (see methods). Data are expressed as a 1070

, Our analysis shows that the colocalization observed 1072 between the m2R immunofluorescent signal and Al594-Tf is higher after treatment with 1073 CCh compared to untreated neurons (***: p<0.0001). Atropine prevents the increase of m2R 1074 and Al594-Tf colocalization (Atropine treatment vs Control : NS : not significant), p.9

, The day after 1082 transfection, neurons were treated with CCh (30µM) for 15min and fixed. The m2R was 1083 detected by ICC. When the mutants are expressed (labeling in A', B'), m2R labeling is seen 1084 at the plasma membrane of neurons (A,B). The expression of the control plasmid (labeling 1085 in C') does not block m2R internalization (C). D : Intracellular immunofluorescent clusters, 1086 representing m2R in endosomes, were segmented and counted using the FIJI/ImageJ 1087 software. Results are expressed as intracellular immunofluorescent clusters per ?m 2 1088 cytoplasmic surface in Eps15 dominant negative-treated and control neurons.The statistical 1089 analysis shows that the expression of the EH29 and DIII mutants blocks m2R clusterization 1090 (Mann-Whitney U test: NS : Not significant, Blockade of m2R clathrin-dependent endocytosis in hippocampal neurons with 1080 negative dominant of Eps15. Hippocampal living neurons were co-transfected with WT1081 m2R plasmid and GFP-tagged EH29 and DIII mutants or their control (D3?2), vol.29

, Absence of internalization of m2R in caveole in fixed neuron after stimulation by CCh

, Some m2R and CAV1-mCherry 1101 clusters colocalize (arrows) in both treated and untreated neurons. The quantitative analysis 1102 of the colocalization of m2R and CAV1-mCherry in neurons was performed using the Jacop 1103 Plugin of ImageJ and statistical data are reported from the Costes's randomization-based 1104 colocalization module (see methods). Data are expressed as a Pearson's coefficient (pc) and 1105, Hippocampal neurons were co-transfected with a plasmid encoding the wild-type receptor 1098 (WT-SS-m2R : A,B) and CAV1-mCherry

, Our analysis shows that pc values do not significantly differ in control 1107 neurons and neurons treated with CCh for 6, 12 and 15 min (NS : not significant)

, Immunohistochemical localization of m2R in neuronal compartments involved in 1111 endocytosis, synthesis, maturation and degradation in fixed hippocampal neurons

, Neurons were stimulated with CCh 1113 at 30µM for 6, 20 min and 1hr fixed, and processed for visualization of m2R together with 1114 markers of intraneuronal compartments and observed by confocal microscopy. A-C'': 6min 1115 after CCh stimulation (30µM), some m2R immunopositive punta colocalize with CHC in 1116 clathrin-coated pits, Hippocampal neurons were transfected with m2R-WT

D. , we failed to detect no colocalization of m2R 1118 with PDI, a marker of endoplasmic reticulum and GM130 and TGN38, markers of Golgi 1119 apparatus. G-G'': 1hr after CCh stimulation (30µM), some m2R immunopositive puncta 1120 colocalize with CathD, a marker of lysosomes (arrow heads). The quantitative analysis of 1121 the colocalization of m2R and markers of subcellular compartment in neurons was 1122 performed using the Jacop Plugin of

, Costes's randomization-based colocalization module (see methods). Data are expressed as a 1124

, Our 1125 analysis shows that the colocalization of the immunofluorescent signals for m2R with CHC, 1126 EEA1, Rab9 and CathD is higher after treatment with CCh compared to untreated neurons 1127 (CHC, Rab9 and CathD : ***: p<0.0001; EEA1 : ** :p<0.01). In contrast, the colocalization 1128 of the immunofluorescent signals for m2R with PDI and GM130 do not significantly differ 1129 in CCh-treated neurons compared to untreated Control neurons : CHC n=20, EEA1 n=16, 1130 Rab9 n=15, PDI n=21, GM130 n=17, CathD n=17, Pearson's coefficient (pc) and pc were compared using the Mann-Whitney U test