Oxotremorine m

Cytosolic and mitochondrial Ca²⁺ levels were measured following IP3R-mediated release from ER stores in HEK293 cells^{20 (link)}. To do so, cells transfected with M3R and either empty vector control plasmid or TDP-43 plasmids were loaded with either 2 μM Fluo4-AM or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM Na-HEPES pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37°C, followed by washing in external solution for 15 min. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) with MetaMorph (Molecular Dynamics) on an Axiovert S100 microscope (Zeiss) equipped with appropriate filtersets (Chroma Technology), a 40x/1.3NA Plan-Neofluar objective (Zeiss) and a Photometrics Cascade-II 512B EMCCD. The cells were kept under constant perfusion with external solution (0.5 ml/min). IP3R-mediated Ca²⁺ release from ER stores was triggered by application of 100 μM Oxotremorine-M (Tocris) for 2 min. Ca²⁺ levels were calculated as relative Fluo4 or Rhod2 fluorescence compared to baseline fluorescence at the start of the measurement.

Clozapine N-oxide (CNO) dihydrochloride (water soluble) and clozapine dihydrochloride (water soluble) were purchased from Hello Bio Inc. (Princeton, NJ, USA). Scopolamine hydrobromide, Oxotremorine M, and aCSF were purchased from Tocris (Minneapolis, MN, USA). Stock solutions of drugs were made and working concentrations of the drugs were prepared freshly on the day of each experiment by appropriate dilutions of the stock solutions in aCSF.