Motor neurons fire action potentials along axons to the neuromuscular junction, which transmits the signal to the muscle fibre via calcium controlled neurotransmitter release. Loss of motor neuron function results in motor neuron diseases, which are both degenerative and incurable. Both the neuromuscular junction and muscle have also been found to be involved in motor neuron disease progression. Neurons from the hippocampi of embryonic rats are currently used to analyse the spread of electrical and calcium signals, and can be combined with muscle cells to form a functional neuromuscular junction model.
Why we funded it
This Training Fellowship aims to replace the use of embryonic rats in the study of axon physiology with an in vitro neuron model derived from differentiated human induced pluripotent stem cells (hiPSCs). These neurons will be co-cultured with hiPSC-derived muscle cells on segregated microfluidic chambers, generating a customisable and transferable in vitro model of the human neuromuscular system.
Dr Rigby estimates that his experiments surrounding functional axon physiology require approximately 50 adult rats and 600 embryonic pups per year. The use of hiPSC-derived motor neurons will replace all rats required for these studies. In order for motor neurons to form realistic synapses, the isolated neurons are co-cultured with muscle cells, requiring the use of additional animals. The differentiation of hiPSCs into muscle cells will lead to further replacement of ~200 chicks or ~100 mice per year in Dr Rigby’s experiments.
Dysfunction of mitochondria is known to impact on the pathogenesis of motor neuron diseases, often in the pre-symptomatic stages. Both calcium signalling and neurotransmitter release are affected by mitochondria function but the effect of mitochondria on propagation of an action potential has not yet been explored. Through this fellowship, Dr Rigby aims to use genetically encoded voltage indicators to measure changes in axonal membrane voltage in the in vitro neuromuscular junction model. The generation of the neuromuscular microfluidic device in this proposal can be used further to study motor neuron axon, or general axon, physiology and Dr. Rigby seeks to optimise functional assays to allow for a “plug and play” neuromuscular microfluidic device. hiPSCs can also be isolated from patients with genetic variants linked with motor neuron diseases. The combination of patient-derived hiPSCs with the neuromuscular microfluidic devices, allows for in depth in vitro studies of the pathological mechanisms of neurodegeneration.
Barcellos Machado C, Pluchon P, Harley, P et al. (2019) In Vitro Modeling of Nerve–Muscle Connectivity in a Compartmentalized Tissue Culture Device. Advanced Biosystems 3(7) 1800307-1800335 doi:10.1002/adbi.201800307