Exploring Ca2+ Dynamics in Myelinating Oligodendrocytes through rAAV-Mediated jGCaMP8s Expression in Developing Spinal Cord Organ Cultures

Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and -independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus (rAAV)/Olig001 capsid variant to express the genetically encoded calcium (Ca2+) indicator jGCaMP8s, under the control of the myelin basic protein (MBP) promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live-imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observe a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth is modulated at the level of myelin axon-interface through Ca2+ signaling and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.