Enlarged extracellular space of aquaporin-4-deficient mice does not enhance diffusion of Alexa Fluor 488 or dextran polymers

Abstract

Aquaporin-4 (AQP4) water channels expressed on glia have been implicated in maintaining the volume of extracellular space (ECS). A previous diffusion study employing small cation tetramethylammonium and a real-time iontophoretic (RTI) method demonstrated an increase of about 25% in the ECS volume fraction (alpha) in the neocortex of AQP4(-/-) mice compared to AQP4(+/+) mice but no change in the hindrance imposed to diffusing molecules (tortuosity lambda). In contrast, other diffusion studies employing large molecules (dextran polymers) and a fluorescence recovery after photobleaching (FRAP) method measured a decrease of about 10%-20% in lambda in the neocortex of AQP4(-/-) mice. These conflicting findings on lambda would imply that large molecules diffuse more readily in the enlarged ECS of AQP4(-/-) mice than in wild type but small molecules do not. To test this hypothesis, we used integrative optical imaging (IOI) to measure tortuosity with a small Alexa Fluor 488 (molecular weight [MW] 547, lambda(AF)) and two large dextran polymers (MW 3000, lambda(dex3) and MW 75,000, lambda(dex75)) in the in vitro neocortex of AQP4(+/+) and AQP4(-/-) mice. We found that lambda(AF)=1.59, lambda(dex3)=1.76 and lambda(dex75)=2.30 obtained in AQP4(-/-) mice were not significantly different from lambda(AF)=1.61, lambda(dex3)=1.76, and lambda(dex75)=2.33 in AQP4(+/+) mice. These IOI results demonstrate that lambda measured with small and large molecules each remain unchanged in the enlarged ECS of AQP4(-/-) mice compared to values in AQP4(+/+) mice. Further analysis suggests that the FRAP method yields diffusion parameters not directly comparable with those obtained by IOI or RTI methods. Our findings have implications for the role of glial AQP4 in maintaining the ECS structure.

Figure 1

Diffusion of AF in in vitro somatosensory neocortex of AQP4^+/+ and AQP4^−/− mice. (a) Images from AF time sequence obtained in AQP4^+/+ mouse. White dashed line in the first image taken after the dye release (labeled as t = 0 s) represents the horizontal axis used to extract intensity profiles. The scale bar is 100 µm. (b) Intensity profiles (red) fitted with theoretical Gaussian curves (black). (c) A time series of γ²/4 values with a linear regression line recorded in AQP4^+/+ (grey circles) and AQP4^−/− (white circles) mice. D^* (34°C) was 20.25×10⁻⁷ cm²s⁻¹ in AQP4^+/+ mice and 20.24×10⁻⁷ cm²s⁻¹ in AQP4^−/− mice. The λ_AF was 1.60 in both genotypes. (d) The tortuosities (mean and SD) obtained with AF from all experiments.

Figure 2

Diffusion of dextran polymers in in vitro somatosensory neocortex of AQP4^+/+ and AQP4^−/− mice. (a) A time series of γ²/4 values recorded with dex3 and dex75 in AQP4^+/+ (grey circles) and AQP4^−/− (white circles) mice. For dex3, D^* (34°C) was 7.45×10⁻⁷ cm²s⁻¹ in AQP4^+/+ mice and 7.46×10⁻⁷ cm²s⁻¹ in AQP4^−/− mice. The λ_dex3 was 1.73 in both genotypes. For dex75, D^* (34°C) was 0.81×10⁻⁷ cm²s⁻¹ in AQP4^+/+ mice and 0.85×10⁻⁷ cm²s⁻¹ in AQP4^−/− mice. The λ_dex75 was 2.42 in AQP4^+/+ mice and 2.36 in AQP4^−/− mice. (b) The tortuosities (mean and SD) obtained with dex3 and dex75 from all experiments.

Figure 3

Quantile plot of λ values measured with AF and dextran polymers. The values of λ_AF, λ_dex3 and λ_dex75 obtained in AQP4^+/+ (black symbols) and AQP4^−/− (white symbols) mice sorted in ascending order were plotted as a fraction of the total number of measurements normalized to 1.

Figure 4

Simulation of extracellular diffusion in AQP4^+/+ and AQP4^−/− mice. Molecules (dex75) were released from a point source (black circle; pulse duration 10 ms) into the ECS where α is 0.19 (AQP4^+/+) or 0.23 (AQP4^−/−) as measured in the mice neocortical slices (Yao et al., 2008). In AQP4^+/+ genotype, λ obtained in this study (2.33) was used. In AQP4^−/− genotype, either unchanged λ (2.30) or λ decreased by 15% (10–20% was reported; Binder et al., 2004b; Papadopoulos and Verkman, 2005; Zador et al., 2008) was used. The concentration profiles obtained at the distance 30 µm from the release site are shown. In AQP4^−/− genotype (dashed and dotted lines), the amplitude of diffusion curves is smaller than in AQP4^+/+ genotype (solid line) reflecting larger dilution of molecules released in enlarged ECS (discussed by Yao et al., 2008). In AQP4^−/− genotype with decreased λ (dashed line), the diffusion curve peaks earlier than when λ is unchanged (dotted line) demonstrating a faster spread of molecules. We note that the result would hold qualitatively also for smaller molecules and ions.