%0 Journal Article %A Yaron Meirovitch %A Kai Kang %A Ryan W. Draft %A Elisa C. Pavarino %A Maria Fernanda Henao Echeverri %A Fuming Yang %A Stephen G. Turney %A Daniel R. Berger %A Adi Peleg %A Richard L. Schalek %A Ju Lu %A Juan-Carlos Tapia %A Jeff. W. Lichtman %T Neuromuscular connectomes across development reveal synaptic ordering rules %D 2021 %R 10.1101/2021.09.20.460480 %J bioRxiv %P 2021.09.20.460480 %X The connections between motor neurons and muscle fibers are dramatically reorganized in early postnatal life. This work attempts to better understand this synaptic rewiring by using a connectomic approach, i.e., tracing out all the connections between motor neurons and muscle fibers, at successive ages in a small mouse muscle. We reconstructed 31 partial-complete neuromuscular connectomes, using serial section scanning electron microscopy in a neonatal mouse and Brainbow-based and XFP-based fluorescent reconstructions in older animals. Our data included a total of more than 6000 neuromuscular junctions (NMJs), including complete connectomes from one newborn, seven developmental ages (P6-P9), and two adults. Analysis confirmed the massive rewiring that takes place as axons prune their motor units but add more synaptic areas at the NMJs with which they remain in contact. Interestingly, we found synaptic ordering rules that likely underlie this circuit maturation and yield the resulting adult neuromuscular pattern, as manifest in Henneman’s size principle. In particular, by analyzing both the identities of axons sharing NMJs at developing ages and muscle fibers with multiple endplates, we found evidence suggesting an activity-based linear ranking of motor neurons such that neurons co-innervated the same endplates and same muscle fibers (if there were more than one endplate) when the axons were similar in activity and hence rank. In addition, this ranking provided a means for understanding action at a distance in which the activity at one neuromuscular junction can impact the fate of the axons at another junction at a different site on the same muscle fiber. These activity-dependent mechanisms provide insight into the means by which timing of activity among different axons innervating the same population of cells, that start out with nearly all-to-all connectivity, can produce a well-organized system of axons, a system that is necessary for the recruitment order of neurons during a graded behavior like muscle contraction.Competing Interest StatementThe authors have declared no competing interest. %U https://www.biorxiv.org/content/biorxiv/early/2021/09/20/2021.09.20.460480.full.pdf