Characterising developmental and activity-dependent myelination in neocortical brain slice cultures

Bestall, Natasha (2023). Characterising developmental and activity-dependent myelination in neocortical brain slice cultures. University of Birmingham. M.Sc.

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Abstract

Studying the process of myelination during early brain development is an essential part of understanding several key topics, such as memory, learning, demyelinating diseases, traumatic brain injury, and brain plasticity. In this study, a novel method of culturing murine brain slices is presented and characterised which provides a stable, three-dimensional, in vitro model for studying oligodendrocyte maturation in the neocortex. The slices were from transgenic PLP-dsRED mice and were stained using a fluorescent anti-MBP (Myelin basic protein) marker to visualise oligodendrocyte morphology and track the progress of myelination.
Oligodendrocyte differentiation and myelination were examined qualitatively where images were ranked on a scale of 1-4 based on differentiation and myelination levels. It was found that the optimal time window for making viable slice cultures was at postnatal days 3-4, as this gave better oligodendrocyte maturation than slices taken later, at postnatal day 7.
To appraise this method’s success as a technique for testing the effects of drug treatments on oligodendrocytes, slices were treated with factors expected to modulate myelination. Both TTX (Tetrodotoxin), which has been shown to block oligodendrocyte proliferation and myelination, and BDNF (Brain-derived neurotrophic factor), which enhances the maturation and myelination of oligodendrocytes, were used in this study. Using the fluorescent imaging technique and ranking system, it was concluded that TTX had a negative impact on the level of differentiation and myelination in the neocortex, whereas BDNF had no effect.
These findings were followed by analysing the expression of key oligodendrocyte genes. qPCR was used to determine how TTX and BDNF treatments influenced the expression of oligodendrocyte lineage maturation markers PDGFRα (Platelet-derived growth factor receptor A), Enpp6 (Ectonucleotide pyrophosphatase/phosphodiesterase 6), or MOG (Myelin oligodendrocyte glycoprotein). In agreement with the imaging data, BDNF showed no significant effect on oligodendrocyte genes. In contrast, TTX treatment reduced the expression of MOG in the brain slices, indicating that this neurotoxin reduced the number of oligodendrocyte lineage cells that reached the final myelinating stage of maturity. These results are consistent with findings that oligodendrocyte differentiation is stimulated by axon activity, and that blocking that with TTX reduces myelination. Moreover, they confirm that the neocortical slice culture system is a suitable platform for ex vivo studies of oligodendrocyte and myelin plasticity and should prove useful for further research to increase the depth of knowledge of myelin development, and for testing potential drug treatments for oligodendrocyte-related disorders.

Type of Work:

Thesis (Masters by Research > M.Sc.)

Award Type:

Masters by Research > M.Sc.

Supervisor(s):