FIGURE 2.

Validation of pH-dependence of SEP-m2R. A living hippocampal neuron transfected with SEP-m2R was observed by spinning disk confocal microscopy for 30 min. A stack of 20 images (0.5 μM in depth) were collected at 30 s intervals. A projection of the stack images was performed and an equatorial image was extracted (insert) and illustrated on this panel (A–D). The effect of pH was observed on the fluorescence level with or without NH4Cl. (A) At pH7.4, the SEP-m2R is detected at the membrane of cell body and proximal dendrites. (B) At acidic pH, SEP-m2R labeling strongly decreases. (C) The SEP-m2R labeling is seen again the plasma membrane when the medium is back to pH7.4. When NH4Cl (50 mM) that is known to reveal receptors associated with acidic intraneuronal organelles is added in the medium, punctiform m2R labeling was also seen in the cytoplasm. (D) At acidic pH with NH4Cl, the SEP-m2R labeling is very weak again. (E) Quantification of the fluorescence level. Fluorescence was measured at the level of the whole neuron (corresponding mainly to plasma membranes) and in soma using the Fiji software. Data are expressed as normalized values compared to the fluorescence level at pH7.4 at 0 min. SEP-m2R labeling strongly decreases at acidic pH at plasma membranes and in the cytoplasm. NH4Cl at pH7.4 induces an increase of the staining close to the control values at the plasma membranes and much higher in soma. The acidic pH with NH4Cl strongly decreases fluorescence at membranes and in soma. The recovery of SEP-m2R fluorescence is shown in both compartments when the medium is back to pH7.4.