“These new results open an experimental window into understanding the brain’s ECS,” explains co-team leader Laurent Cognet of the LP2N at the University of Bordeaux. “They reveal that in live brains at the nanoscale, the extracellular maze is composed of interconnected polymorphic compartments with different characteristics.”

The results also show that the viscosity of the ECS is different in different parts of the brain, a finding that will be important for understanding how cells communicate with each other – for example, how they control neurotransmitter diffusion and clear toxic metabolites in specific areas. The findings could even help in the design of new and improved drug-delivery strategies for treating brain disease, says Cognet.

A dynamic structure

The ECS makes up almost a quarter of the volume of the brain and it is here that cells communicate with each other via signalling molecules. Neurotransmitters and nutrients also transit through the ECS and toxic metabolites are cleared here. It is a dynamic structure that varies during sleep, development and ageing, and its structure is likely affected by neuropsychiatric and neurodegenerative diseases too.

Although techniques such as electron microscopy can be used to image the ECS, they cannot reach sub-micron scales in live brains. By tracking how luminescent carbon nanotubes diffuse through the ECS for time periods of tens of minutes, Cognet and colleagues have now succeeded in doing just this.

Wide-field fluorescent near-infrared microscopy

The researchers performed their study by injecting single-walled luminescent carbon nanotubes coated with phospholipid-polyethylene glycol (PL-PEG) into the lateral cerebroventrides of young rat brains. They killed the rats half an hour after the injection and prepared acute brain slices that they looked at under a wide-field fluorescent near-infrared microscope. These slices are to all intents and purposes like “frozen” samples of the live brain.

The team detected the CNTs in different areas of the slices (for example, in the neocortex, hippocampus and striatum). They imaged the brain at different depths – from a few tens of microns to hundreds of microns – and thanks to the interactions between the nanotubes and biological tissue, were able to extract important information about the size of channels in, and the viscosity of, different ECS regions.

“Unsuspected powerful application of CNTs”

“These results indeed demonstrate an unsuspected powerful application of CNTs in the field of nano-bioimaging that other approaches cannot achieve – namely near-infrared single-molecule imaging in deep live tissue,” co-team leader Laurent Groc tells nanotechweb.org. “What is more, by chemically altering the extracellular matrix of the brain in live animals, we found that only the local rheological properties of the ECS were affected and that these changes did not spread beyond our small nano-imaging scale.

“The technique we describe here will allow for new conceptual and experimental exploration of the brain’s ECS during development and learning, and in neurodegenerative and neuropsychiatric diseases, in which subtle non-uniform and localized modifications are likely to occur in brain tissue.”

The research is detailed in Nature Nanotechnology doi:10.1038/nnano.2016.248.