Faculty Bibliography

The extracellular space (ECS) consists of the narrow channels between brain cells together with their geometrical configuration and contents. Despite being only 20-60 nm in width, the ECS typically occupies 20% of the brain volume. Numerous experiments over the last 50 years have established that molecules moving through the ECS obey the laws of diffusion but with an effective diffusion coefficient reduced by a factor of about 2.6 compared to free diffusion. This review considers the origins of the diffusion barrier arising from the ECS and its properties. The paper presents a brief overview of software for implementing two point-source paradigms for measurements of localized diffusion properties: the real-time iontophoresis or pressure method for small ions and the integrative optical imaging method for macromolecules. Selected results are presented. This is followed by a discussion of the application of the MCell Monte Carlo simulation program to determining the importance of geometrical constraints, especially dead-space microdomains, and the possible role of interaction with the extracellular matrix. It is concluded that we can predict the impediment to diffusion of many molecules of practical importance and also use studies of the diffusion of selected molecular probes to reveal the barrier properties of the ECS.

PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events

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

Diffusion in the extracellular space (ECS) of the brain is constrained by the volume fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many molecules in the brain. Deviations from the equation reveal loss of molecules across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromolecules, including dextrans or proteins. Theoretical models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromolecules with ECS channels. Extensive experimental studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the volume fraction of the ECS typically is approximately 20% and the tortuosity is approximately 1.6 (i.e., free diffusion coefficient of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic volume transmission to the development of paradigms for drug delivery to the brain

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 diffusion properties are at present most commonly evaluated by magnetic resonance (MR) diffusion imaging. MR cannot easily distinguish between the extracellular and intracellular signal components, but the older technique of real-time iontophoresis (RTI) detects exclusively extracellular diffusion. Interpretation of the MR results would therefore benefit from auxiliary RTI measurements. This requires a molecular probe detectable by both techniques. Our aim was to specify a minimum set of requirements that such a diffusion probe should fulfill and apply it to two candidate probes: the cation tetramethylammonium (TMA(+)), used routinely in the RTI experiments, and the anion hexafluoroantimonate ( [Formula: see text] ). Desirable characteristics of a molecular diffusion probe include predictable diffusion properties, stability, minimum interaction with cellular physiology, very slow penetration into the cells, and sufficiently strong and selective MR and RTI signals. These properties were evaluated using preparations of rat neocortical slices under normal and ischemic conditions, as well as solutions and agarose gel. While both molecules can be detected by MR and RTI, neither proved an ideal candidate. TMA(+) was very stable but it penetrated into the cells and accumulated there within tens of minutes. [Formula: see text] did not enter the cells as readily but it was not stable, particularly in ischemic tissue and at higher temperatures. Its presence also resulted in a decreased extracellular volume. These probe properties help to interpret previously published MR data on TMA(+) diffusion and might play a role in other diffusion experiments obtained with them

The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced approximately 60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate

Aquaporin-4 (AQP4) is the major water channel expressed at fluid-tissue barriers throughout the brain and plays a crucial role in cerebral water balance. To assess whether these channels influence brain extracellular space (ECS) under resting physiological conditions, we used the established real-time iontophoresis method with tetramethylammonium (TMA(+)) to measure three diffusion parameters: ECS volume fraction (alpha), tortuosity (lambda), and TMA(+) loss (k'). In vivo measurements were performed in the somatosensory cortex of AQP4-deficient (AQP4(-/-)) mice and wild-type controls with matched age. Mice lacking AQP4 showed a 28% increase in alpha (0.23 +/- 0.007 vs 0.18 +/- 0.003) with no differences in lambda (1.62 +/- 0.04 vs 1.61 +/- 0.02) and k' (0.0045 +/- 0.0001 vs 0.0031 +/- 0.0009 s(-1)). Additional recordings in brain slices showed similarly elevated alpha in AQP4(-/-) mice, and no differences in lambda and k' between the two genotypes. This is the first direct comparison of ECS properties in adult mice lacking AQP4 water channels with wild-type animals and demonstrates a significant enlargement of the volume fraction but no difference in hindrance to TMA(+) diffusion, expressed as tortuosity. These findings provide direct evidence for involvement of AQP4 in modulation of the ECS volume fraction and provide a basis for future modeling of water and ion transport in the CNS