Synaptic circuit refinement in the developing neuromuscular system

Neural circuits undergo considerable rearrangement and refinement in early postnatal life. The purpose of these changes may be to shape the nervous system to match the specific demands of the environment it finds itself in. How experience (i.e., neural activity) alters neural circuits in early life is not understood. One approach to this fundamental question would be to ascertain precisely how neural circuits are changed during early postnatal development. Thus, I have developed a method to fully reconstruct immature circuits in the mouse neuromuscular system using multi-color 'Brainbow' reporter mice and high spectral and spatial resolution confocal microscopy. By tagging different motor neuron arbors with unique color labels, I have observed and characterized how an entire set of developing axons interact with each other in several small muscles. In contrast to the tortuous branching and absence of any positional topography in the axonal arborizations, I have discovered a striking higher-level organization in the pattern of synaptic connectivity. I found that individual motor neurons show a highly significant bias to co-innervate neuromuscular junctions with certain synaptic partners over others during early postnatal life. Furthermore, analysis of complete connectomes reveals that this bias is part of a systemic single axis ranking of all the motor axons that project to a muscle. Branches of each motoneuron co-innervate neuromuscular junctions most often with neurons that are "adjacent" in this ranking system, and less often with more remote neurons in proportion to their distance. Results show that the process of synapse elimination has an important role in the creation of this rank order. By comparing the probability of withdrawal to a neuron's position in the rank order, I found that neurons in triply innervated junctions withdraw such that neurons farther apart in the ranking are selectively eliminated, whereas neurons near in the ranking maintain co-innervation on muscle fibers. This shows that the synapse elimination gradually generates singly innervated neuromuscular junctions by an orderly process of winnowing the final competitors to ones that are "close" on the ranking scheme. However, a definitive explanation of what functional properties drive this pattern remains to be resolved.