Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve–muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5±0.6 pS) and mean open time (8.1±0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3±5.9 pS) and short mean open time (2.6±0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve–muscle co-culture system as a reliable model for the study of murine muscular differentiation and function.