Mgcl2

Isolated MP networks were carefully resuspended in 5 μl collagen N gel (Amedrix, Esslingen, Germany). The MP network/collagen suspension was plated out as a thin layer on a 35 mm petri dish with a glass bottom (Greiner Bio-One) in order to fix the MP networks in one plane. The MP networks were carefully dispersed with forceps if necessary. The samples were incubated 30 min at 37°C for polymerization of the collagen N gel. Then the networks were washed three times with recording buffer. The recording buffer was composed of 150mM NaCl (Applichem), 5mM KCl (Roth), 2mM CaCl₂ (Applichem), 1mM MgCl₂ (Applichem), 10mM Hepes (Sigma Aldrich) and 1,5 g/LGlucose (Applichem). The staining solution consisting of recording buffer, 200 μg/ml Pluronic (Sigma Aldrich) dissolved in DMSO (Applichem), 2 μg/ml Fluo-8 (AAT Bioquest) dissolved in DMSO and 0,5% FCS (PAN) was added for 30 min at RT protected from light. The MP networks were again rinsed three times in recording buffer and used for the measurements. Potassium chloride was used for depolarization. The measurement was carried out with the CellObserver Z1 using the Physiology module of the Axiovision software (Zeiss).

The assay buffer consisted of MilliQ water, 11 mM Na-HEPES (pH = 7.4) (Sigma-Aldrich), 135 mM NaCl (AppliChem), 1 mM CaCl₂ (AppliChem), 5 mM KCl (AppliChem), 1 mM MgCl₂ (AppliChem), protease inhibitor cocktail (according to the manufacturer’s description, Roche) and 0.1% Pluronic^® F-127 (Sigma-Aldrich). Dithiothreitol (DTT) (AppliChem) was added to the assay buffer for experiments with Dopamine and apomorphine with an end concentration of 1 mM.
D₃ receptor ligands 7-hydroxy-DPAT hydrobromide were purchased from TOCRIS and Spiperone (Sigma S7395), Haloperidol (Sigma H1512), Dopamine, apomorphine, and Butaclamol were from Sigma-Aldrich. The fluorescent ligand CELT-419, its pharmacophore P-165 and pharmacophore with linker PL-384 were kindly provided by Celtarys Research. Stock solutions of these ligands were prepared in DMSO (Applichem) or Milli-Q water in the case of Dopamine.

Assay buffer consisted of MilliQ water, 135 mM NaCl (AppliChem, Darmstadt, Germany), 1 mM CaCl₂ (AppliChem), 5 mM KCl (AppliChem), 1 mM MgCl₂ (AppliChem), 11 mM Na-HEPES (pH = 7.4) (Sigma-Aldrich, Taufkirchen, Germany), protease inhibitor cocktail (according to the manufacturer's description, Roche, Basel, Switzerland) and 0.1% Pluronic F-127 (Sigma-Aldrich).
Muscarinic acetylcholine receptor ligands acetylcholine, arecoline, pirenzepine, pilocarpine, atropine and scopolamine were purchased from Sigma-Aldrich and carbachol from Tocris Bioscience (Abingdon, UK). The syntheses of the fluorescent ligands UR-MK342 and UR-CG072 [16 (link)] and the dualsteric M₂R ligands UR-SK59 [50 (link)], UR-SK75 [50 (link)] and UNSW-MK259 [51 (link)], showing also high M₄R affinity, were described previously. All ligand stock solutions were prepared using cell culture grade DMSO (AppliChem) and stored at −20°C.