Current Techniques for Investigating the Brain Extracellular Space

The brain extracellular space (ECS) is a continuous reticular compartment that lies between the cells of the brain. It is vast in extent relative to its resident cells, yet, at the same time the nano- to micrometer dimensions of its channels and reservoirs are commonly finer than the smallest cellular structures. Our conventional view of this compartment as largely static and of secondary importance for brain function is rapidly changing, and its active dynamic roles in signaling and metabolite clearance have come to the fore. It is further emerging that ECS microarchitecture is highly heterogeneous and dynamic and that ECS geometry and diffusional properties directly modulate local diffusional transport, down to the nanoscale around individual synapses. The ECS can therefore be considered an extremely complex and diverse compartment, where numerous physiological events are unfolding in parallel on spatial and temporal scales that span orders of magnitude, from milliseconds to hours, and from nanometers to centimeters. To further understand the physiological roles of the ECS and identify new ones, researchers can choose from a wide array of experimental techniques, which differ greatly in their applicability to a given sample and the type of data they produce. Here, we aim to provide a basic introduction to the available experimental techniques that have been applied to address the brain ECS, highlighting their main characteristics. We include current gold-standard techniques, as well as emerging cutting-edge modalities based on recent super-resolution microscopy. It is clear that each technique comes with unique strengths and limitations and that no single experimental method can unravel the unknown physiological roles of the brain ECS on its own.