Streamlined correlative live-confocal and SBF-SEM imaging reveals early inflammatory dendritic changes induced by...

As cells that constantly survey their environment, microglia rely on contact with neurons, non-neuronal cells and extracellular substances. The diversity of microglial receptors allows them to elicit either localized or widespread responses to developmental cues, functional states, tissue damage, or pathogens. These responses primarily affect microglia immediate vicinity. In neuroinflammation, aberrantly activated microglia are considered to damage adjacent neurons and impair their function. Understanding the dynamic relationship between microglia and neurons is essential for elucidating the full spectrum of functional and transcriptional microglial states.

This study presents a straightforward yet effective workflow correlative live-cell confocal microscopy and serial block-face scanning electron microscopy (SBF-SEM) to visualize microglia-neuron contacts in cell culture. This approach enables the identification and three-dimensional high-resolution imaging of sites of intense cellular interaction.

We visualized initial membrane contact zones and early organelle alterations at of microglia-neuron interfaces. Our data showed that stimulation with Gram-positive bacterial lysates (from Streptococcus pneumoniae) or Gram-negative lipopolysaccharide (from Escherichia coli) induced distinct patterns of microglial activation. These were characterized by increased adherence to neurites and reduced motility. Notably, local mitochondrial swelling and dendrite shrinkage, were observed shortly after microglial attachment.

Our workflow substantially improves the resolution to study microglia-neuron interactions at and enables detection of changes unvisualizable by light microscopy.

The proposed method provides a powerful platform for investigating functional states of microglia and their interactions with surrounding cells, and it is readily adaptable to research context beyond neuroinflammation.