Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3 Ϫ/Ϫ mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3, and Has2 CKO , the seizures were most prevalent in Has3 Ϫ/Ϫ mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3Ϫ/Ϫ brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ϳ40% in Has3 Ϫ/Ϫ mice. Finally, osmotic manipulation experiments in brain slices from Has3 Ϫ/Ϫ and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

The concentration of extracellular calcium plays a critical role in synaptic transmission and neuronal excitability as well as other physiological processes. The time course and extent of local fluctuations in the concentration of this ion largely depend on its effective diffusion coefficient (D * ) and it has been speculated that fixed negative charges on chondroitin sulphate proteoglycans (CSPGs) and other components of the extracellular matrix may influence calcium diffusion because it is a divalent cation. In this study we used ion-selective microelectrodes combined with pressure ejection or iontophoresis of ions from a micropipette to quantify diffusion characteristics of neocortex and hippocampus in rat brain slices. We show that D * for calcium is less than the value predicted from the behaviour of the monovalent cation tetramethylammonium (TMA), a commonly used diffusion probe, but D * for calcium increases in both brain regions after the slices are treated with chondroitinase ABC, an enzyme that predominantly cleaves chondroitin sulphate glycans. These results suggest that CSPGs do play a role in determining the local diffusion properties of calcium in brain tissue, most likely through electrostatic interactions mediating rapid equilibrium binding. In contrast, chondroitinase ABC does not affect either the TMA diffusion or the extracellular volume fraction, indicating that the enzyme does not alter the structure of the extracellular space and that the diffusion of small monovalent cations is not affected by CSPGs in the normal brain ionic milieu. Both calcium and CSPGs are known to have many distinct roles in brain physiology, including brain repair, and our study suggests they may be functionally coupled through calcium diffusion properties.

There are a limited number of methods available to quantify the extracellular diffusion of macromolecules in an anisotropic brain region, e.g., an area containing numerous aligned fibers where diffusion is faster along the fibers than across. We applied the integrative optical imaging method to measure diffusion of the fluorophore Alexa Fluor 488 (molecular weight (MW) 547) and fluorophore-labeled flexible random-coil dextran polymers (dex3, MW 3000; dex75, MW 75,000; dex282, MW 282,000; dex525, MW 525,000) in the extracellular space (ECS) of the anisotropic molecular layer of the isolated turtle cerebellum. For all molecules, two-dimensional images acquired an elliptical shape with major and minor axes oriented along and across, respectively, the unmyelinated parallel fibers. The effective diffusion coefficients, D*(major) and D*(minor), decreased with molecular size. The diffusion anisotropy ratio (DAR = D*(major)/D*(minor)) increased for Alexa Fluor 488 through dex75 but then unexpectedly reached a plateau. We argue that dex282 and dex525 approach the ECS width and deform to diffuse. In support of this concept, scaling theory shows the diffusion behavior of dex282 and dex525 to be consistent with transition to a reptation regime, and estimates the average ECS width at approximately 31 nm. These findings have implications for the interstitial transport of molecules and drugs, and for modeling neurotransmitter diffusion during ectopic release and spillover.

Brain extracellular space (ECS) is an interconnected channel that allows diffusion-mediated transport of signaling molecules, metabolites, and drugs. We tested the hypothesis that β-adrenergic receptor (βAR) activation impacts extracellular diffusion-mediated transport of molecules through alterations in the morphology of astrocytes. Two structural parameters of ECS - volume fraction and tortuosity - govern extracellular diffusion. Volume fraction (α) is the volume of ECS relative to the total tissue volume. Tortuosity (λ) is a measure of the hindrance that molecules experience in the ECS, compared to a free medium. The Real-Time Iontophoretic (RTI) method revealed that treatment of acutely prepared visual cortical slices of adult female rats with a βAR agonist, DL-isoproterenol (ISO), decreases α significantly, from 0.22 ± 0.03 (mean ± SD) for controls without agonist to 0.18 ± 0.03 with ISO, without altering λ (control: 1.64 ± 0.04; ISO: 1.63 ± 0.04). Electron microscopy revealed that the ISO treatment significantly increased the cytoplasmic area of astrocytic distal endings per unit area of neuropil by 54%. These findings show that norepinephrine decreases α, in part, through an increase in astrocytic volume following βAR activation. Norepinephrine is recognized to be released within the brain during the awake state and increase neurons’ signal-to-noise ratio through modulation of neurons’ biophysical properties. Our findings uncover a new mechanism for noradrenergic modulation of neuronal signals. Through astrocytic activation leading to a reduction of α, noradrenergic modulation increases extracellular concentration of neurotransmitters and neuromodulators, thereby facilitating neuronal interactions, especially during wakefulness.