Long-term in vivo three-photon imaging reveals region-specific differences in healthy and regenerative oligodendrogenesis
This paper reports an original study on the dynamics of oligodendrocytes in the mouse cortex and white matter using three-photon microscopy.
Introduction: Oligodendrocytes are the cells that produce myelin in the central nervous system, which is crucial for neural transmission, cognitive function, and injury repair. Previous studies have shown regional differences in the generation and differentiation of oligodendrocytes in different brain regions, but the mechanism of this region-specific regulation remains unclear due to the inability to observe deep structures in vivo over an extended period. This study took advantage of the deep imaging capabilities of three-photon microscopy to achieve long-term in situ imaging of oligodendrocytes in the mouse cortical columns and white matter.
Source:
Authors: Michael A. Thornton et al.
Institutions: Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Center, etc.
Journal: Nature Neuroscience
Publication Date: May 2024
Research Procedure: (a) Transgenic mobp-EGFP mice expressing enhanced green fluorescent protein (EGFP) were implanted with a transparent cranial window, allowing mature oligodendrocytes and their myelin sheaths to emit fluorescence in vivo.
(b) A three-photon microscope system was established, using a 1300 nm near-infrared laser to excite the three-photon process and obtain high-resolution three-dimensional images up to a depth of 1000 micrometers.
© Three-photon imaging parameters were optimized to avoid laser-induced tissue damage, including adjusting laser energy, using an adaptive optics correction system to suppress aberrations, and controlling the scanning rate.
(d) Long-term in situ imaging was performed to track individual oligodendrocytes and study their dynamic changes under healthy and demyelinating (cuprizone-induced) conditions.
(e) Three-photon imaging data, including EGFP-labeled cell bodies and third-harmonic generation signals labeling myelin structures, were collected.
(f) Image data were analyzed to calculate the number of newly generated, surviving, and lost oligodendrocytes, and to simulate the dynamics of the cell population.
(g) RNA in situ hybridization and other methods were used to investigate the expression of specific genes in different oligodendrocyte subpopulations across brain regions.
Key Findings: 1. Under healthy conditions, more newly generated oligodendrocytes were produced in the white matter, but the oligodendrocyte population expanded more rapidly in the gray matter.
Cuprizone-induced oligodendrocyte loss was comparable in the gray and white matter regions. However, the regenerative capacity of oligodendrocytes in the white matter was stronger after injury.
Oligodendrocytes in the deep cortical layers (5⁄6) exhibited reduced regenerative capacity after injury, and the recovery of the MOL5/6 molecular subtype was also impaired.
In the healthy state, the distribution of oligodendrocyte molecular subtypes (MOL1/2/3/5/6) varied across brain regions, and this heterogeneity was partially restored after injury.
Research Significance: 1. Established a new method for long-term in situ three-photon imaging of oligodendrocytes, overcoming the depth limitations of traditional two-photon imaging. 2. Discovered region-specific regulation of oligodendrocyte generation, differentiation, and regeneration, suggesting the involvement of different microenvironmental mechanisms. 3. The reduced regeneration of deep cortical oligodendrocytes may be related to cognitive impairment in the later stages of white matter diseases, highlighting the importance of promoting repair in this region. 4. Laid the foundation for exploring the link between oligodendrocyte heterogeneity and function, aiding in the elucidation of molecular mechanisms underlying normal myelination and repair.
In conclusion, this study systematically elucidated the region-specific regulation of oligodendrocyte dynamics, demonstrating the important application value of three-photon imaging in neuroscience research.