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Development
2020 Nov 16;14722:. doi: 10.1242/dev.193920.
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Building neuromuscular junctions in vitro.
Barbeau S
,
Tahraoui-Bories J
,
Legay C
,
Martinat C
.
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The neuromuscular junction (NMJ) has been the model of choice to understand the principles of communication at chemical synapses. Following groundbreaking experiments carried out over 60 years ago, many studies have focused on the molecular mechanisms underlying the development and physiology of these synapses. This Review summarizes the progress made to date towards obtaining faithful models of NMJs in vitro We provide a historical approach discussing initial experiments investigating NMJ development and function from Xenopus to mice, the creation of chimeric co-cultures, in vivo approaches and co-culture methods from ex vivo and in vitro derived cells, as well as the most recent developments to generate human NMJs. We discuss the benefits of these techniques and the challenges to be addressed in the future for promoting our understanding of development and human disease.
The stages of NMJ formation in mice. (A) During development, prior to and independent of innervation, AChRs are clustered in the prospective region of muscle. Thus, the muscle cell plays an instructive role from a very early stage of development, in positioning and restricting synapses to a central band in the muscle, and building a post-synaptic density prior to axonal contact. (B) Axons do not contact muscle fibers at random locations, but rather in a narrow central region of the muscle fiber characterized by the presence of aneural ‘pre-patterned’ AChR clusters (Lin et al., 2001; Yang et al., 2001). Axons then become apposed to AChR clusters. (C) Additionally three or four nuclei accumulate under the post-synaptic membrane in the muscle cells (which are defined as multinucleated syncytia formed during development by fusion of mononucleated precursor cells) and become specialized for transcription of synaptic proteins. Concomitantly, folds form in the muscle membrane increasing the area of contact between the post-synaptic membrane and the extracellular matrix (Guarino et al., 2019). (D) Muscle fibers are poly-innervated until the end of the second week after birth in rodents. This is when the multiple innervations of individual post-synaptic densities are reduced to a single innervating motor axon through a process called synapse elimination (Sanes and Lichtman, 2001). AChR, acetylcholine receptor; adult AChR, α2βεδ; E, embryonic day; embryonic AChR, α2βγδ; MuSK, muscle-specific kinase; P, post-natal day; tSC, terminal Schwann cell.
Muscle, neuronal and synaptic hallmarks of the mature NMJ. The main markers that can be used to characterize the mature NMJ are shown. ACh, acetylcholine; AChE, acetylcholinesterase; AChR, acetylcholine receptor; AZ, active zone; BL, basal lamina; ColQ, collagen Q; Dok7, downstream of tyrosine kinase 7; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific kinase; tSC, terminal Schwann cell; VDCC, voltage-dependent Ca2+ channel; VGSC, voltage-gated Na+ channel.
Different technical approaches to studying the NMJ in vitro. (A) Organ explant culture. (B) Dissociated explant culture. (C) Mixed cell and explant culture. (D) Dissociated cell culture. (C,D) Instead of explants or dissociated cells, immortalized cell lines or motoneurons (MNs)/muscle cells generated from induced pluripotent stem cells can be used. *Chimeric model possible (different species of origin for muscle cells and MNs).
