Correlative light and serial block face electron microscopy for targeting and investigating oligodendrocyte-vasculature interactions

Abstract text (incl. figure legends and references)

Cell-cell specific interactions occur between all major cell types in the brain. Although the investigation of cellular interactions with light microscopy is a standard approach, targeting specific cell-cell interactions with electron microscopy is highly challenging. In this project we investigated cellular interactions of oligodendrocyte cell bodies with the brain vasculature. This interaction has been has been previously identified, but organization and function of oligodendrocytes associated with the brain vasculature remains unknown. In preceding light microscopy experiments we estimated that in the neocortical grey matter about 17% of oligodendrocytes contact in majority capillaries. As the vasculature is highly organized and is formed by different cell types including endothelial cells, pericytes and astrocytes we wanted to further investigate vascular associated oligodendrocytes on the ultrastructural level to resolve the anatomical organization.

To specifically target and analyze oligodendrocyte vascular ensembles and obtain 3D reconstructions with electron microscopy we applied a correlative light and serial sectioning electron microscopy approach. We obtained fluorescent oligodendrocytes by using a transgenic mouse line that expressed membrane targeted EGFP under an oligodendrocytic specific promoter. After transcardial fixation of mice with 4% paraformaldehyde and 2.5% glutaraldehyde, the vasculature was labelled with fluorescent tomato lectin, a specific marker for the vasculature. After fixation, EGFP fluorescence remained and we identified oligodendrocyte-vasculature ensembles in 50 µm thick vibratome sections of the motor cortex using confocal imaging. After identification of ensembles we applied 2-photon branding (910 nm) of ensemble locations and included orientation marks of larger size for improved sample recovery. Subsequently the brain slices were prepared for serial block face electron microscopy (SBF-SEM), embedded in epon and mounted with silver containing conductive resin on sample pins. Using confocal microscopy z-stacks that we acquired during sample preparation, we re-identified the marked positions and proceeded with SBF-EM (Zeiss) to acquire backscattered images of the target regions at nanoscale resolution (pixel size of 10 x 10 x 30 nm). Data analysis included image post-processing, reconstruction of the structures of interest with Microscopy image browser and 3D rendering with Imaris (Fig 1).

By optimizing parameters of the sample preparation process we could detect the laser marked regions and identified oligodendrocyte-vasculature ensembles in electron microscopy. Analysis of the EM images revealed that the oligodendrocyte cell body and the vascular basement membrane are in direct contact (n = 3 cells, n = 2 mice,). Moreover, we detected Golgi apparatus, microtubules, endoplasmic reticulum, mitochondria and lipid inclusions in oligodendrocytes cell bodies (Fig 2). Astrocyte endfeet surrounded the oligodendrocyte cell body at non-contact sites suggesting a close interaction with astrocytes at the neuro-vascular-junction.

Our presented approach shows that a combination of fluorescence labelling, light microscopy and SBF-EM allows the investigation of the nanoscale structure of an oligodendroglial vascular niche. Our data suggest that oligodendrocytes are a component of the neuro-glia-vascular unit raising the possibility of direct signaling pathways and metabolite exchange with vascular cells.