Aquaporin-4-deficient mice have increased extracellular space without tortuosity change

Abstract

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.

Figure 1.

Diffusion of TMA⁺ in the somatosensory neocortex of AQP4^+/+ and AQP4^−/− mice in vivo. A, Photograph of recording arrangement for RTI-TMA diffusion measurements. The mouse head was immobilized in a stereotaxic frame. A micromanipulator positioned the microelectrodes that were glued with dental cement into an array with fixed intertip distance. The grounding electrode and the temperature probe were positioned nearby. See Materials and Methods for details. B, Sequence of diffusion curves recorded in dilute agarose gel and neocortex of AQP4^+/+ mice. Several records were obtained in agarose before and after brain measurements. See Results for details. C, Examples of TMA⁺ diffusion curves in AQP4^+/+ and AQP4^−/− mice. The measurements were done at 37°C, where D₃₇ = 1.31 × 10⁻⁵ cm²/s. TMA⁺ pulse was applied for 50 s (horizontal bar), r was 144 and 124 μm for AQP4^+/+ and AQP4^−/−, respectively, and n_t was 0.33 for both records. Recorded curves (red and blue) are superimposed with corresponding theoretical curves obtained from fitting procedure (dashed and dotted). D, Diffusion curves obtained in AQP4^+/+ compared with those in AQP4^−/− mice by generating theoretical curves based on the average α, λ, and k′ values (Table 1 and E) with the following parameters: temperature, 37°C; bias current, + 20 nA; main current, +120 nA for 50 s; r = 130 μm; n_t = 0.4. The AQP4^−/− theoretical curve had smaller amplitude than that for AQP4^+/+ animals (left), reflecting a larger α, but both curves had similar shapes (right), indicating similar values of λ. E, Scatter plots of whole datasets. Each small circle represents the average from one animal. Large circles are mean ± SEM values; *p < 0.001.

Figure 2.

Diffusion of TMA⁺ in the somatosensory neocortex (S1) of AQP4^+/+ and AQP4^−/− mice in vitro. A, A schematic of coronal brain slice with two independent microelectrodes positioned in S1 [Franklin and Paxinos (2008), their Figure 43, modified with permission]. B, Representative TMA⁺ diffusion curves in AQP4^+/+ (top) and AQP4^−/− (bottom) mice. The measurements were done at 34 and 32°C in AQP4^+/+ and AQP4^−/− mice, respectively; D₃₄ = 1.24 × 10⁻⁵ cm²/s and D₃₂ = 1.19 × 10⁻⁵ cm²/s. TMA⁺ pulse was applied for 50 s (horizontal bar), r was 130 μm, and n_t was 0.39 and 0.48 for AQP4^+/+ and AQP4^−/− mice, respectively. Actual AQP4^+/+ record was taken with 60 nA current but scaled to 120 nA for consistency. In both panels, recorded curves (solid red or blue lines) are superimposed with corresponding theoretical curves obtained from fitting procedure (dashed and dotted). C, As in Figure 1, to compare the diffusion properties in AQP4^+/+ and AQP4^−/− mice, two theoretical curves were generated from average α, λ, and k′ values (Table 1 and D) with the same parameters as in Figure 1, except temperature was 34°C. As in Figure 1, TMA⁺ diffusion curve in AQP4^−/− mice has smaller amplitude than AQP4^+/+ but similar shape (inset). D, Scatter plots of entire datasets showing a significant increase in the volume fraction in AQP4^−/− (top) but no change in the tortuosity (bottom). Each small circle represents the average from a single slice. Large circles are mean ± SEM values; *p < 0.001.