Uncovering the biology of myelin with optical imaging of the live brain

Abstract

Myelin has traditionally been considered a static structure that is produced and assembled during early developmental stages. While this characterization is accurate in some contexts, recent studies have revealed that oligodendrocyte generation and patterns of myelination are dynamic and potentially modifiable throughout life. Unique structural and biochemical properties of the myelin sheath provide opportunities for the development and implementation of multimodal label-free and fluorescence optical imaging approaches. When combined with genetically encoded fluorescent tags targeted to distinct cells and subcellular structures, these techniques offer a powerful methodological toolbox for uncovering mechanisms of myelin generation and plasticity in the live brain. Here, we discuss recent advances in these approaches that have allowed the discovery of several forms of myelin plasticity in developing and adult nervous systems. Using these techniques, long-standing questions related to myelin generation, remodeling, and degeneration can now be addressed.

Figure 1:. Approaches for label-free myelin imaging

The myelin sheath is a unique biological structure composed of multiple layers of oligodendrocyte cell membrane wrapped tightly around single axons with very little to no cell cytoplasm between the myelin layers. The differential refractive index of the compact myelin sheath compared to the rest of the brain tissue and the high concentration of phospholipids allows label-free detection of myelin using various optical imaging approaches. These approaches include optical coherence microscopy (OCM), third harmonic generation (THG) microscopy, spectral confocal reflectance (SCoRe) microscopy, coherent anti-Stokes Raman scattering (CARS) microscopy. OCM, THG and CARS require relatively complex instrumentation/light sources while SCoRe can be conducted on conventional confocal laser scanning microscopes (LSMs). Recent advances in each technique using long wavelength light sources (1300–1700nm) have achieved imaging depths above 1mm, potentially allowing intravital imaging of white matter tracts such as the corpus callosum in murine models.

Figure 2:. Intravital imaging of oligodendrocytes and myelin

a) Fluorescence labeling and in vivo imaging of single oligodendrocyte lineage cells in the mouse cortex. Isolated NG2 glia or oligodendrocytes can be visualized after a low dose injection of tamoxifen to induce cre recombination in single cells in Cspg4-creER transgenic mice. Membrane tethered GFP allows visualization of NG2 glia processes or myelin internodes. Arrowheads indicate cell soma b) The small molecule fluorescent dye sulforhodamine 101 (SR101) allows in vivo visualization of astrocyte and oligodendrocyte gap junction coupling. Astrocytes initially take up the dye which then diffuses to coupled myelinating oligodendrocytes. Arrowheads indicate oligodendrocyte cell soma. c) Label-free spectral confocal reflectance (SCoRe) microscopy in the live mouse cortex. The reflection of multiple wavelengths (left image, 448 nm, 552 nm, and 638 nm laser wavelengths used) is combined to visualize myelination (middle image) which maps precisely with membrane localized EGFP in Cnp-mEGFP transgenic mice (right image). Arrowheads indicate oligodendrocyte cell soma. d) Nodes of Ranvier (arrow) can be visualized in vivo using SCoRe and/or mEGFP labeling.

Figure 3:. Forms of myelin plasticity and degeneration in the adult brain

In vivo imaging studies have discovered protracted ongoing oligodendrocyte generation in the adult brain. The continuous oligodendrocyte production results in progressive myelination of previously unmyelinated and partially myelinated axons over days to months. This form of myelin plasticity is robust in the superficial layers of the mouse cortex and extends until late stages of adulthood. A subpopulation of myelin segments exhibit length plasticity (double arrow) however the majority remain stable after initial formation. In aging or after myelin damage the myelin sheath can display several forms of pathology including the formation of myelin spheroids and blebbing. In some cases, the myelin associated with these spheroids will degenerate. In aging or after myelin damage oligodendrocyte death is also prominent and results in myelin loss and accumulation of myelin debris. Questions remain about the dynamics and patterns of remyelination.