Figure 4.

In vitro NMJs are functionally active

(A) Schematic overview of transient fluorescent Ca²⁺ imaging in XC150 devices on day 28. MN compartment (green) is stimulated with 50 mM KCl, which initiates an intracellular response through MN axons evoking an increase in Ca²⁺ influx in the Fluo-4-loaded myotubes (red compartment). Cell illustrations are modified from Smart Servier Medical Art licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

(B) Fluorescent micrograph examples of before, during, and after stimulation depicting a wave of increase in intracellular Ca²⁺ in myotubes upon MN stimulation. Inset shows a magnification of an innervated active myotube. Scale bars, 100 μm and 200 μm (insets).

(C) Representative Ca²⁺ influx curves in myotubes after KCl activation (arrow) of MNs before (myotube 1–3) and after 10 min treatment with NMJ blocker tubocurarine (myotube A-C DTC).

(D) Ratio of MN-stimulated active myotubes to directly KCl-activated myotubes.

(E) Effect of DTC on the intensity fluorescent increase due to Ca²⁺ influx induced by KCl increase in the MN compartment. Mean ± SEM of three to four biological replicates. Mann-Whitney test. ^∗∗∗∗p < 0.0001.

See also Figure S4.