Recent reports have revealed oligodendrocyte precursor cell (OPC) heterogeneity. It remains unclear if such heterogeneity reflects different subtypes of cells with distinct functions, or rather transiently acquired states of cells with the same function. By integrating lineage formation of individual OPC clones, single-cell transcriptomics, calcium imaging and neural activity manipulation, we show that OPCs in the zebrafish spinal cord can be divided into two functionally distinct groups. One subgroup forms elaborate networks of processes and exhibits a high degree of calcium signalling, but infrequently differentiates, despite contact with permissive axons. Instead, these OPCs divide in an activity and calcium dependent manner to produce another subgroup with higher process motility and less calcium signaling, which readily differentiates. Our data show that OPC subgroups are functionally diverse in responding to neurons and reveal that activity regulates proliferation of a subset of OPCs that is distinct from the cells that generate differentiated oligodendrocytes.

Recent reports suggest that rehabilitation measures that increase physical activity of patients can improve functional outcome after incomplete spinal cord injuries (iSCI). To investigate the structural basis of exercise-induced recovery, we examined local and remote consequences of voluntary wheel training in spinal cord injured female mice. In particular, we explored how enhanced voluntary exercise influences the neuronal and glial response at the lesion site as well as the rewiring of supraspinal tracts after iSCI. We chose voluntary exercise initiated by providing mice with free access to running wheels over "forced overuse" paradigms because the latter, at least in some cases, can lead to worsening of functional outcomes after SCI. Our results show that mice extensively use their running wheels not only before but also after injury reaching their pre-lesion exercise levels within five days after injury. Enhanced voluntary exercise improved their overall and skilled motor function after injury. In addition, exercising mice started to recover earlier and reached better sustained performance levels. These improvements in motor performance are accompanied by early changes of axonal and glial response at the lesion site and persistent enhancements of the rewiring of supraspinal connections that resulted in a strengthening of both indirect and direct inputs to lumbar motoneurons.

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glialfibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes axon regrowth and is accompanied by neurogenesis. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ atomic force microscopy-enabled nanoindentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter before returning to baseline values after the completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. This work constitutes the first quantitative mapping of the spatiotemporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.

1 Severe injury to the mammalian spinal cord results in permanent loss of function due 2 to the formation of a glial-fibrotic scar. Both the chemical composition and the 3 mechanical properties of the scar tissue have been implicated to inhibit neuronal 4 regrowth and functional recovery. By contrast, adult zebrafish are able to repair 5 spinal cord tissue and restore motor function after complete spinal cord transection 6 owing to a complex cellular response that includes neurogenesis and axon regrowth.7 The mechanical mechanisms contributing to successful spinal cord repair in adult 8 zebrafish are, however, currently unknown. Here, we employ AFM-enabled nano-9 indentation to determine the spatial distributions of apparent elastic moduli of living 10 spinal cord tissue sections obtained from uninjured zebrafish and at distinct time 11 points after complete spinal cord transection. In uninjured specimens, spinal gray 12 matter regions were stiffer than white matter regions. During regeneration after 13 transection, the spinal cord tissues displayed a significant increase of the respective 14 apparent elastic moduli that transiently obliterated the mechanical difference 15 between the two types of matter, before returning to baseline values after completion 16 of repair. Tissue stiffness correlated variably with cell number density, 17 oligodendrocyte interconnectivity, axonal orientation, and vascularization. The 18 presented work constitutes the first quantitative mapping of the spatio-temporal 19 changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides 20 the tissue mechanical basis for future studies into the role of mechanosensing in 21 spinal cord repair. 22 42 recovery in adult zebrafish within 6-8 weeks post-injury [8]. 43Morphological changes, proliferation, migration and differentiation also constitute 44 responses that mechanosensitive neurons and glia exhibit when exposed to distinct 45 mechanical environments [14][15][16][17]. In vitro studies of neural cells reported an 46 increased branching of neurons on compliant, but directed axonal growth on stiff 47 substrates [14, 18]. Astrocytes and microglia display morphological characteristics of 48 an activated phenotype and upregulate inflammatory genes and proteins when 49 exposed to a mechanical stimulus that deviates from their physiological mechanical 50 4 environment both in vitro and in vivo [17]. Oligodendrocyte precursor cells increase 51 their expression of myelin basic protein and display an elaborated myelin membrane 52 on stiffer substrates as compared to more compliant substrates indicating a preferred 53 mechanical environment for differentiation [15]. 54 In vivo, this mechanical environment is formed by the surrounding nervous tissue 55 whose mechanical properties are determined by factors such as the combined 56 material properties of neighboring cells, cell density, myelin content, collagen 57 content, extra cellular matrix composition and cell interconnectivity [19, 20]. As these 58 may change during develop...

Recent reports revealed heterogeneity of oligodendrocyte precursor cells (OPCs). It remains unclear if heterogeneity reflects different types of cells with distinct functions, or rather transiently acquired states of cells with the same function. By integrating lineage formation of individual OPC clones, single-cell transcriptomics, calcium imaging and manipulation of neural activity, we show that OPCs in the zebrafish spinal cord can be divided into two functionally distinct entities. One subgroup forms elaborate networks of processes and exhibits a high degree of calcium signalling, but infrequently differentiates, despite contact to permissive axons. Instead, these OPCs divide in an activity and calcium dependent manner to produce another subgroup with higher process motility and less calcium signaling, which readily differentiates. Our data show that OPC subgroups are functionally diverse in responding to neurons and reveal that activity regulates proliferation of a subset of OPCs that is distinct from the cells that generate differentiated oligodendrocytes.

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