Brain Extracellular Space: The Final Frontier of Neuroscience

Abstract

Brain extracellular space is the narrow microenvironment that surrounds every cell of the central nervous system. It contains a solution that closely resembles cerebrospinal fluid with the addition of extracellular matrix molecules. The space provides a reservoir for ions essential to the electrical activity of neurons and forms an intercellular chemical communication channel. Attempts to reveal the size and structure of the extracellular space using electron microscopy have had limited success; however, a biophysical approach based on diffusion of selected probe molecules has proved useful. A point-source paradigm, realized in the real-time iontophoresis method using tetramethylammonium, as well as earlier radiotracer methods, have shown that the extracellular space occupies ∼20% of brain tissue and small molecules have an effective diffusion coefficient that is two-fifths that in a free solution. Monte Carlo modeling indicates that geometrical constraints, including dead-space microdomains, contribute to the hindrance to diffusion. Imaging the spread of macromolecules shows them increasingly hindered as a function of size and suggests that the gaps between cells are predominantly ∼40 nm with wider local expansions that may represent dead-spaces. Diffusion measurements also characterize interactions of ions and proteins with the chondroitin and heparan sulfate components of the extracellular matrix; however, the many roles of the matrix are only starting to become apparent. The existence and magnitude of bulk flow and the so-called glymphatic system are topics of current interest and controversy. The extracellular space is an exciting area for research that will be propelled by emerging technologies.

Figure 1

Structure of ECS. (A) Factors affecting the diffusion of molecules. These are: (a) geometry of ECS, which enforces a longer path-length on a diffusing molecule compared to a free medium; (b) dead-space microdomain, where molecules lose time exploring a dead-end (which may be an invagination, as shown, or glial wrapping, or a local expansion of the ECS); (c) obstruction by extracellular matrix molecules such as hyaluronan; (d) binding sites for the diffusing molecule either on cell membranes or extracellular matrix; and (e) fixed negative charges on the extracellular matrix that may affect the diffusion of charged molecules. Graphic image taken from Syková and Nicholson (1). (B) Shown here is the EM of cryofixed neuropil. (C) Shown here is the EM of conventional chemically fixed neuropil. Comparison of (B) and (C) shows reduction in ECS (pseudo-colored in blue) after chemical fixation. (B and C) These images were taken from adult mouse cerebral cortex; the common calibration bar represents 1 μm. Photographs are from Korogod et al. (13). To see this figure in color, go online.

Figure 2

Real-time iontophoresis and Monte Carlo modeling reveal ECS structure. (A) Given here is the recent implementation of the RTI-TMA method. TMA⁺ was delivered from left-hand source micropipette by iontophoresis in awake or asleep mouse cortex and concentration of TMA⁺ recorded with an ISM 150 μm from the source. The microelectrodes contained fluorescent dyes so the tips could be visualized with two-photon light microscopy (2 PLM). Graphic image taken from Xie et al. (28); reprinted with permission from AAAS. (B) Shown here are the theoretical TMA⁺ concentration curves ([TMA]), for a distance of 100 μm from the source in three examples. (a) Given here is the postnatal (P3–P4) rat anesthetized (i.e., sleep) cortex with α = 0.36 (36%) (26). (b) Given here is the adult anesthetized (i.e., sleep) cortex, with average data for rat or mouse α = 0.21 (21%) (1). (c) Given here is the awake adult mouse cortex α = 0.14 (14%) (28). (C) The amplitudes of the curves are related to α, and the shapes similar because λ ∼ 1.6 in all three cases. (D) Shown here is the Monte Carlo simulation in ensemble of 3D cubic cells packing with small, uniform, separation to form an ECS. A set of molecules (red; size exaggerated) has been released at the center of the ensemble and spread outwards to explore the local microenvironment; image taken from Syková and Nicholson (1). (E) The plot of values of λ versus α extracted from Monte Carlo simulations using three different ensembles is shown. These were: cubes (squares), truncated octahedra (hexagonal symbol), and mixture of three differently shaped convex cells (triangular symbol). In each geometry, α was varied from 0.05 to 0.90, D^∗ was estimated from the molecule distribution, and λ was calculated. All three media had an identical λ-α relation (Eq. 3); plot taken from Tao and Nicholson (32) with permission from Elsevier. (F) Given here are factors that may increase λ from the maximum seen in (E) to the value seen in experiments. These are: dead-space microdomains in the form of local expansion of ECS, i.e., voids (V), invaginations (I) of cellular elements, or glial wrapping (red) around cells; graphic image taken from Nicholson et al. (35) with permission of Springer. To see this figure in color, go online.

Figure 3

Imaging methods to study ECS. (A) Given here is a plot of λ measured with IOI in cortex (rat or mouse) versus hydrodynamic diameter (d_H) for macromolecules from several studies (see Table 3 in (1) and (39)). Open circles represent dextrans, solid circles represent proteins, and the open cube is Alexa Fluor 488 dye. The dashed line shows a best fit to these data of RDT for planar or sheetlike pores. (Inset) Pseudo-color diffusion images were obtained with IOI for 3 kDa dextran labeled with Texas Red in agarose (Agar) and in vivo rat cortex (Ctx) at different times after initial injection of molecule from a micropipette. Scale bars indicate 200 μm. The corresponding Gaussian intensity profiles are below. (Inset) Image taken from Thorne and Nicholson (40); copyright 2006 National Academy of Sciences. (B) Shown here is a color-coded trajectory of a single-walled carbon nanotube diffusing in ECS in living cortical slice from a neonatal rat brain (20,000 data points). Image is from Godin et al. (42) and is reprinted by permission from Macmillan Publishers, Ltd: Nature Nanotechnology, copyright 2017. (C) Given here is the diffusivity in interstitial space in slices of rat hippocampus measured with the time-resolved fluorescence anisotropy imaging technique. Values of D_vis/D averaged 0.7, where D_vis is the effective diffusion coefficient attributable to interstitial viscosity, and D is the free diffusion coefficient in ACSF (artificial cerebrospinal fluid), giving an average λ_vis = 1.2 measured across stratum radiatum (sr), stratum pyramidale (sp), and stratum oriens (so). Image was reproduced from Zheng et al. (43). To see this figure in color, go online.

Figure 4

Interaction of diffusing molecules with the extracellular matrix. (A) Shown here is the structure of the perineuronal net. Hyaluronan, secreted by membrane-bound HA synthase (HAS), binds to members of the lectican family (aggrecan, versican, neurocan, and brevican) and is cross-linked by link proteins and tenascin-R to form supramolecular aggregates on the surface of neurons (chondroitin sulfate-glycosaminoglycans, CS-GAGs). Graphic image is taken from Tsien (62) and derived from Kwok et al. (48) with permission of John Wiley & Sons. (B) Shown here is the diffusion of Ca²⁺ before (Control) (left curve, blue) and after CS glycan had been cleaved with chondroitinase ABC (chABC) (right curve, red) in rat cortical slice. Records were obtained with the RTP method, with time of injection marked by black dot in graphs. Plots are taken from Hrabětová et al. (54) with permission from John Wiley & Sons. (C) Shown here are diffusion measurements with the IOI method in vivo to measure interaction with HSPGs. Lactoferrin (Lf) or Lactoferrin + heparin (Lf + H) solutions were pressure-ejected from a micropipette at a depth of 200 μm in rat neocortex. The larger complex (Lf + H) diffused away more rapidly than the smaller Lf, indicating that the latter was binding to matrix components. Scale bar indicates 200 μm. Graphic panels are taken from Thorne et al. (55), copyright 2008 National Academy of Sciences. (D) Given here are the theoretical diffusion curves at 100 μm from source (Eq. 2, k′ = 0), showing effect of chABC on Ca²⁺ diffusion and comparison of diffusion of transferrin (no binding) and lactoferrin (binds to matrix). Note also the difference in diffusions times for the small Ca²⁺ ion compared to the larger proteins (∼80 kDa). Timescale is logarithmic. To see this figure in color, go online.