Investigating axon-oligodendrocyte interactions during myelinated axon formation in vivo
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Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.