Current Techniques for Investigating the Brain Extracellular Space

Abstract

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.

Ultrastructural analysis of neuroimplant-parenchyma interfaces uncover remarkable neuroregeneration along-with barriers that limit the implant electrophysiological functions

“…The seal resistance (R s ) is given by R seal =ρ s •δ /d, where ρ s is the resistivity of the electrolyte solution (ρ s = 0.7 ΩCm), d is the average cleft width between the neuron's plasma membrane and the electrodes' surface, and δ is the overlapping surface coefficient that takes into account the percentage of the electrodes' sensitive area in contact with the microglia (Massobrio et al, 2016). Because of the unavoidable ~20% shrinking artifact of the extracellular spaces due to the chemical fixation of the tissue for TEM imaging (Korogod et al, 2015;Hrabetova et al, 2018;Soria et al, 2020) the actual width (d) of the clefts formed between the microglia and the implanted gMµEs-PPMPs surfaces cannot be extracted with precision from the ultrastructural images. In addition, the fraction of the surface area of the contact between a gMµE or planar electrode and the adhering microglia (δ) cannot be obtained from classical TEM images.…”

Brain Ultrastructure: Putting the Pieces Together

“…Plasma membranes, basement membranes, clefts in gap junctions, actin filaments, intermediate filaments, microtubules, ribosomes, extracellular spaces, glycogen granules, synaptic vesicles, dense-core vesicles, nuclear pores, and lysosomes, are only or best resolved with EM, at the highest resolution (reaching 1 nm) for a biological technique ( Tremblay et al, 2010b ; Savage et al, 2018 ; SynapseWeb, 2021 ). Although super-resolution microscopy, and more recently, expansion microscopy, were developed to resolve small structures, notably in correlation with EM ( Carrier et al, 2020 ; Hoffman et al, 2020 ; Parra-Damas and Saura, 2020 ; Soria et al, 2020 ), the capacity of EM to reveal the ultrastructure of cells and their constituents without selective staining (although staining can be used to provide better visualization of membranes, cytoskeletal elements, and ribosomes, for instance; Dykstra and Reuss, 2003 ; Svitkina, 2009 ) confers an important advantage ( Tremblay et al, 2010b ; Savage et al, 2018 ).…”

Ultrastructural Analysis of Neuroimplant-Parenchyma Interfaces Uncover Remarkable Neuroregeneration Along-With Barriers That Limit the Implant Electrophysiological Functions

“…In retrospect, based on the preservation qualities of the cell membranes and subcellular organelles including the mitochondria, the smooth and rough endoplasmic reticulum, synaptic vesicles, post-synaptic densities and myelin, we concluded that the perfusion of the fixative was not impaired in the surroundings of the implant. It is important to note, however, that as in other ultrastructural studies of the CNS, the extracellular spaces between the various cell types is reduced by approximately 20% ( Korogod et al, 2015 ; Hrabetova et al, 2018 ; Soria et al, 2020 ). Since the volume of the implanted PPMPs is not altered by the fixatives, transcardial fixation led to the generation of mechanical tension around the implant.…”