A system to study mechanisms of neuromuscular junction development and maintenance

The neuromuscular junction (NMJ), a cellular synapse between a motor neuron and a skeletal muscle fiber, enables the translation of chemical cues into physical activity. The development of this special structure has been subject to numerous investigations, but its complexity renders in vivo studies particularly difficult to perform. In vitro modelling of the neuromuscular junction represents a powerful implement to fully delineate the fine tuning of events that lead to subcellular specialization at the pre-synaptic and post-synaptic sites. Here we describe a novel heterologous co-culture in vitro method using rat spinal cord explants with dorsal root ganglion and murine primary myoblasts to study neuromuscular junctions. This system allows the formation and long-term survival of highly differentiated myofibers, motor neurons, supporting glial cells and functional neuromuscular junctions with postsynaptic specialization. Therefore, fundamental aspects of NMJ formation and maintenance can be studied using the described system which can be adapted to model multiple NMJassociated disorders. D ev el o pm en t • A dv an ce a rt ic le INTRODUCTION The continuous development of new experimental approaches have proved beneficial to model a number of adverse health conditions among which neuromuscular diseases (Chew et al., 2015; Lenzi et al., 2015; Sandoe and Eggan, 2013; Tu et al., 1996). Neuromuscular pathologies encompass a wide-range of sub-groups among which (i) myopathies like Duchenne's and Becker's muscular dystrophies (Flanigan, 2014), (ii) motor-neuron diseases (MNDs) like amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, pseudobulbar palsy and spinal muscular atrophy (SMA) (de Boer et al., 2014; Edens et al., 2015; Karam et al., 2010; Tu et al., 1996) and (iii) auto-immune neuromuscular diseases like myasthenia gravis and Lambert Eaton myastenic syndrome (Ha and Richman, 2015; Lang et al., 2003). Researchers have set up different animal models and cell lines (Chen et al., 2014; Corti et al., 2012; Lenzi et al., 2015) with the hope of recapitulating some features of the latter group and understand the triggers of one of their common hallmarks: the disruption of the neuromuscular junction (NMJ). The NMJ is one of the most studied synapses. It is formed of three key elements: the lower motor neuron (the pre-synaptic compartment), the skeletal muscle (the post-synaptic compartment) and the Schwann cell (Sanes and Lichtman, 1999). The NMJ is formed in a step-wise manner following a series of cues involving these three cellular components and its role is basically to ensure the skeletal muscle functionality. Following an action potential down the motor neuron axon, synaptic vesicles will fuse with the membrane at the half-terminal of the axon releasing neurotransmitters in the synaptic cleft. The post-synaptic membrane of the muscle fiber is specialized to respond efficiently to the neurotransmitter release and will convert the chemical signal to a mechanical signal in the form of muscular contraction (Das et al., 2010). In some cases, the dialogue between these cellular components is compromised and leads to the instability of the NMJ and in worst cases like in ALS and SMA, eventually axon retraction and muscle atrophy. D ev el o pm en t • A dv an ce a rt ic le In order to study NMJ physiology and pathology, different in vivo systems are used such as mouse diaphragm or drosophila abdominal segments(Packard et al., 2002; PerezGarcia and Burden, 2012). However these systems do not allow observation and manipulation over long periods of time in live NMJ. Therefore understanding the NMJ's development often needs transgenic organisms and their manipulation involves generation of transgenic organisms which is time consuming and sometimes impossible. To overcome these problems, different in-vitro co-culture systems have been set up where motor neuron and skeletal muscle are grown together in order to recapitulate the formation and eventual disruption of the NMJ. To date, co-culture methods established from various species have been described: mouse (Morimoto et al., 2013; Zahavi et al., 2015), rat (Das et al., 2010; Southam et al., 2013), Xenopus (Lu et al., 1996; Peng et al., 2003), chick (Frank and Fischbach, 1979) and also heterologous co-cultures built from motor neuron and muscle cells obtained from different species: rat-human (Askanas et al., 1987), mouse-human (Son et al., 2011), mouse-chick (Soundararajan et al., 2007). However, these co-culture methods resulted in the formation of immature myofibers (thin muscle fiber, with centrally localized nuclei and no transversal triads) with immature sarcomeric structures (Das et al., 2007a; Das et al., 2009; Southam et al., 2013). Moreover, previous models did not take advantage of their co-culture system to analyze other post-synaptic structures like the formation of MuSK and rapsyn clusters which are formed as agrin-induced signaling sparks off and which are essential to the formation of AChR clusters. Here, we describe a new functional co-culture system where muscle fibers from primary murine myoblasts are brought to advanced differentiation and form highly matured NMJs with motor neurons derived from rat spinal cord. The muscle fibers show hallmarks of mature skeletal muscle fiber: peripheral nuclei, transversal triads, myofibrils and organization into three-dimensional bundles performing synchronized contraction. Furthermore the NMJ showed pretzel-like morphology reminiscent D ev el o pm en t • A dv an ce a rt ic le of in vivo synapses. We used this co-culture model to investigate the formation of the postsynaptic apparatus beyond the clustering of acetylcholine receptors (AChRs) and we investigated the role of motor neuron firing on muscle development and differentiation. We found that AChRs form clusters at motor-neuron/muscle contacts and that the post and presynapse show hallmarks of maturation and these NMJs are functionally active. D ev el o pm en t • A dv an ce a rt ic le MATERIALS AND METHODS