Figure 7. ECS is reduced in the CA1 stratum pyramidale in Has3−/− mice. A, Transit of extracellular marker molecules through the CA1 stratum pyramidale. Fluorescently labeled extracellular marker dex3 was released by a short puff from a glass micropipette (black square denotes tip) placed in the stratum radiatum close to the border with the stratum pyramidale. Overlay of bright field and fluorescence images at 80 s after release. Distribution of dex3 molecules showed an asymmetry, which was much more prominent in the Has3−/− mice than in the WT mice. The asymmetry arose at the border between the stratum radiatum and the stratum pyramidale. SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. B–D, Quantitative analysis of ECS in the CA1 hippocampus by the RTI method. B, Representative examples of diffusion records from the stratum oriens and the stratum radiatum of WT and Has3−/− mice. TMA curves (data) fitted with traditional model curves (t-model) are shown, together with corresponding values of α and θ. For stratum oriens, κ was 0.0050 s−1 in WT and 0.0059 s−1 in Has3−/−. For stratum radiatum, κ was 0.0092 s−1 in WT and 0.0014 s−1 in Has3−/−. Microelectrode spacing was 80 and 124 μm for the stratum oriens records and the stratum radiatum records, respectively, and nt was 0.31 for both records taken in the two locations. C, Representative examples of multilayer diffusion curves in WT and Has3−/− mice. TMA curves (WT, Has3−/−) fitted with multilayer model curves (m-model) are shown, and a diagram indicates the placement of TMA-selective (detector) and iontophoretic (source) microelectrodes. Multilayer analysis was used to obtain ECS parameters for the CA1 stratum pyramidale: in WT, α = 0.129, θ = 0.308, and κ = 0.0039 s−1; in Has3−/−, α = 0.073, θ = 0.297, and κ = 0.0070 s−1. Microelectrode spacing was 120 μm and nt was 0.33 for both records taken in the two genotypes. D, Summary of ECS volume fraction and diffusion permeability values in the stratum pyramidale of WT and Has3−/− mice. ECS volume fraction was reduced by 40% in Has3−/− mice, whereas diffusion permeability was not changed (Table 3 shows statistical analysis and values of κ). Data are expressed as mean ± SD.