Toward Building the Neuromuscular Junction: In Vitro Models To Study Synaptogenesis and Neurodegeneration

Abstract

The neuromuscular junction (NMJ) is a unique, specialized chemical synapse that plays a crucial role in transmitting and amplifying information from spinal motor neurons to skeletal muscles. NMJ complexity ensures closely intertwined interactions between numerous synaptic vesicles, signaling molecules, ion channels, motor neurons, glia, and muscle fibers, making it difficult to dissect the underlying mechanisms and factors affecting neurodegeneration and muscle loss. Muscle fiber or motor neuron cell death followed by rapid axonal degeneration due to injury or disease has a debilitating effect on movement and behavior, which adversely affects the quality of life. It thus becomes imperative to study the synapse and intercellular signaling processes that regulate plasticity at the NMJ and elucidate mechanisms and pathways at the cellular level. Studies using in vitro 2D cell cultures have allowed us to gain a fundamental understanding of how the NMJ functions. However, they do not provide information on the intricate signaling networks that exist between NMJs and the biological environment. The advent of 3D cell cultures and microfluidic lab-on-a-chip technologies has opened whole new avenues to explore the NMJ. In this perspective, we look at the challenges involved in building a functional NMJ and the progress made in generating models for studying the NMJ, highlighting the current and future applications of these models.

Figure 1

The NMJ: influx of calcium (Ca²⁺) into the motor neuron leads to release of the neurotransmitter, ACh into the synaptic cleft. ACh binds to the receptors on skeletal muscle membranes opening up ion channels, leading to muscle depolarization and contraction.

Figure 2

The complexity involved in a functional NMJ: the complexity of the NMJ is highlighted here, indicating how species, muscle type, synapse location, type of neurotransmitters, glial cells, and signaling pathways all play a role in defining function and development.

Figure 3

2D cocultures of dissociated motor neuron and muscle cells. (a) 2D culture: top view of muscle myocytes and motor neurons plated on a coverslip. The motor neurons are usually cultured on top of the muscle cells, which are seeded on a coverslip coated with ECM proteins. (b) 2D culture: side view of muscle cells and motor neurons seeded on a coverslip.

Figure 4

3D culture: side view of muscle cells and motor neurons cocultured in matrigel. The muscle cells are usually allowed to differentiate into muscle fibers in the matrigel before the motor neurons are added on top of them and differentiated to form neuron-muscle contacts.

Figure 5

Combining 3D hydrogel cultures with microfluidic PDMS chambers and anchoring pillars to separate both cell types as well as isolate and study the NMJ.