Abstract

Myelination of axons can adaptively change in response to nervous system activity, with implications for structure and function of neural circuits. New myelin is made by the differentiation of specified oligodendrocyte precursors cells (OPCs). OPCs constitute an abundant population throughout the central nervous system (CNS) lifelong, but only some of these cells differentiate to myelinating oligodendrocytes at any given time. It is known that OPCs express neurotransmitter receptors through which they can sense neuronal activity, which affects OPC proliferation as well as their differentiation to myelinating oligodendrocytes. However, it remained unclear whether all OPCs communicate with axons in the same way, or whether differences exist between OPCs to integrate and respond to nervous system activity. Therefore, the aim of my PhD work was to investigate mechanisms of axon-OPC interactions to understand whether all OPCs similarly integrate neural activity and if they all contribute to myelination. I used existing transgenic lines and I generated new transgenic reagents to specifically label OPCs and axons with fluorescent reporters and genetically encoded calcium indicators in zebrafish. This approach allowed me to visualise these two cell types in the CNS and to study their calcium signatures to investigate their interaction. Two subpopulations of OPCs with distinct cellular behaviours and fates have been identified in the spinal cord. One OPC subgroup was primed to differentiate, while the other one not. Therefore, I wondered whether OPCs also differentially communicate with axons. I have generated lines that allowed me for the first time to perform in vivo calcium imagining of OPCs at high resolution from subcellular domains to whole tissue population levels using the genetically encoded calcium sensor GCaMP6. My analysis of OPC calcium signatures at the single cell and population level using light-sheet imaging revealed different types of GCaMP signals. Most OPCs exhibited GCaMP transients in process microdomains. However, in some OPCs, calcium transients spread throughout the entire cell. Dual colour imaging using GCaMP in OPCs and the red shifted calcium indicator RGECO in neurons showed that whole cell calcium transients in OPCs frequently occurred in response to neuronal calcium rises. I used pharmacological approaches to manipulate neural activity and found that the frequency of OPC GCaMP signals was increased and decreased when neuronal activity was enhanced and blocked, respectively. I investigated OPC calcium signatures at the population level using volumetric timelapse imaging in animals where all OPCs express GCaMP. These experiments revealed that whole cell GCaMP signals could appear in different patterns, in which only single cells, groups of cells, or the entire OPC population within a field of view light up. In order to investigate these signals over even larger distances, I developed assays for whole animal analysis of the OPC calcium signatures using encoded Calcium Modulated Photoactivatable Ratiometric Integrator (CaMPARI). Using this additional approach, I could detect characteristic boundaries between neighbouring OPCs which displayed similar intracellular calcium level. Together, my single cell and population analysis of OPC calcium signatures showed that the probability and amplitude of somatic calcium transients was significantly higher in non-myelinating OPCs when compared to OPCs that are primed to differentiate. In order to further explore possible functions of these OPCs calcium signatures, I manipulated neural activity using 4-Aminopyridine (4-AP), which specifically increased proliferation of OPCs that do not directly differentiate. Furthermore, by expressing a calcium exporting pump in OPCs, I could demonstrate that OPC divisions triggered by 4-AP required intracellular calcium signaling. In summary, my studies show that OPCs exhibit different types of calcium transients and that OPCs with different properties and fates differentially communicate with axons. This provides new insights into mechanisms of axon-OPC communication and the mechanisms by which oligodendrogenesis is regulated by distinct OPC subpopulations in response to neural activity.