Characterization of a Mouse Model lacking Acetylcholine Receptor Activity during Embryonic Development

Abstract

During embryonic development, acetylcholine (ACh) and the acetylcholine receptor (AChR) play a pivotal role in the establishment, maturation and maintenance of the neuromuscular junction (NMJ). In humans, genetic mutations affecting AChR-mediated signal transduction give rise to a variety of phenotypes mainly defined by muscle weakness, and known as congenital myasthenic syndromes (CMS), demonstrating the importance of AChR for the correct development of nerve/muscle contacts. However, thus far it has been impossible to determine the specific role played by AChR-mediated postsynaptic activity in NMJs during embryonic development. In this work, the effects caused by lack of postsynaptic activity in the NMJ were studied on a reporter mouse line generated by homologous recombination and expressing a structurally intact but functionally silent GFP-tagged AChR. In these animals, the  subunit of the AChR, normally expressed during embryonic development, carries a point mutation (P121L) which causes fast-channel CMS in human patients. Homozygous -fc animals die at birth, and a wide variety of severe physiological abnormalities appears during embryonic development, caused by the silencing of AChR-mediated postsynaptic potentials. The size, shape and density of the NMJ were profoundly altered by lack of postsynaptic activity, although the overall number of receptors did not seem to change. A vastly increased outgrowth of motor axons could also be detected, and this alteration is associated with the absence of motoneuron death at late embryonic stages. Further alterations could be found in the disorganization of muscle fiber architecture, and the presence of multiple innervation sites on single muscle fibers. These results clarify the role of AChR-mediated postsynaptic activity in the proper development and maturation of nerve/muscle contacts, and support the possibility that presynaptic development is influenced by putative reciprocal signaling between nerve and muscle. The reporter mice provide a new tool to distinguish in an as-yet unknown resolution between activity-dependent and putative structurally-dependent pathways during NMJ maturation, leading to important implications in the study of synapse formation and maintenance, as well as in the field of receptor studies.