FIGURE 5.

Phosphorylation of Ser²²⁸ on the M1 DREADD receptor could be detected in the hippocampus following receptor activation with a selective agonist. A, illustration of the two point mutations (Y106C and A195G) used to generation the HA epitope-tagged M1 DREADD receptor mutant in which activation by acetylcholine (ACh) is abolished but instead the receptor could be activated by CNO. B, CHO FlpIn cells expressing the HA-tagged M1 DREADD receptor were stimulated with increasing concentrations of acetylcholine or CNO. Western blots (WB) were probed with either anti-HA (as a loading control) or phospho-specific serine 228 antibodies. NT, non-transfected cells. C and D, M1 DREADD knock-in mice or M₁ mAChR-knock-out mice (M1-KO) were injected (intraperitoneally) with CNO (0.3 mg/kg). After 30 min tissue was fixed by transcardial perfusion and sections stained with anti-HA antibodies (C) or phospho-specific serine 228 antibodies (D). E, fixed sections from M₁ mAChR knock-out mice (M1-KO) or M1 DREADD knock-in mice treated with vehicle or CNO (0.3 mg/kg) were co-stained with anti-HA (green) and anti-phospho-specific serine 228 (red) antibodies. Two neurons where the staining for the receptor and the phosphorylated receptor occur in the same neuron are indicated by the arrows. The areas marked by the white box are magnified in the lower panels.