Demonstration of pharmacological response of MS to cholinergic stimulation.

At day 14, the cholinergic agonist carbachol was added to cultured MS to induce MS depolarization after incubating in calcium indicator (S4 Video). Calcium intensity increased in MS between images taken just before (A) and immediately after (B) adding carbachol. A plot of calcium intensity (C) in the observed muscle strip indicates a pharmacological sensitivity to cholinergic agonists. MS were also incubated with the cholinergic antagonist atropine along with calcium indicator to reduce MS cholinergic sensitivity (S5 Video). MS with atropine were observed before (D) and after addition of carbachol (E). A plot of calcium indicator intensity shows reduced intensity after incubation in atropine compared to MS without atropine, while the addition of carbachol (arrow) did not increase intensity of the calcium indicator. An arrow points to observed MS, an arrowhead indicates addition of cholinergic stimulation on intensity profiles.100x magnification, 200-µm scale bar.