Neuromuscular Junction Model Optimized for Electrical Platforms

Abstract

Neuromuscular junctions (NMJs), specialized synapses between motor neurons and muscle fibers, are essential for muscle activity. A simple and reproducible cell-based in vitro NMJ platform is needed to test the impact of chemicals on the neuron-muscle communication. Our platform utilizes genetically modified neurons and muscle cells, optimized culture conditions, and commercially available multielectrode array system for recording action potentials. Neuronal cells (NSC34) were optogenetically modified with channelrhodopsin chimera to allow for simultaneous, light-mediated, millisecond-precise activation of neuronal population. This signal is propagated through functional synapses to the muscle fibers. Muscle cells (C2C12) were modified by incorporating gap junction protein (Connexin-43) to improve intracellular communication without affecting muscle differentiation. This communication between muscle fibers resulted in better signal propagation and signal strength. Optimized culture medium facilitated the growth and differentiation of both cell types together. Our system was validated using vecuronium, a muscle relaxant, which abolished the muscle response. This in vitro model provides a unique tool for establishing a NMJ platform that is easy to record and analyze. Potential applications include nondestructive long-term screening of drugs affecting the NMJ.

Impact statement

A neuromuscular junction (NMJ) in vitro system is a useful tool to measure the effect of chemicals on the motor neuron—skeletal muscle connection. Here, we describe the use of genetically modified cells and optimized culture medium to establish a reliable and simple analytical system. Nondestructive neuronal stimulation and easy recording of electrical response has a broad range of applications including faster long-term screening of drugs affecting the NMJ.