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. 2021 May 29;29(10):3072–3092. doi: 10.1016/j.ymthe.2021.05.023

Figure 4.

Figure 4

Neuro-specific hippocampal AAV9 delivery reduced NKCC1 expression in Ts65Dn mice

(A) Left: schematic representation of the experimental timeline: 2–3 month-old WT and Ts65Dn mice were stereotaxically injected in the dorsal hippocampal CA1 region with AAV9 vectors and analyzed by immunohistochemistry 6 WPIs. Right: low-magnification images showing the expression of EGFP (green) in the dorsal CA1 hippocampal region in WT and Ts65Dn mice. Nuclei are counterstained with Hoechst (blue). Scale bar: 250 μm. (B) Top-left: representative confocal z stack projection images of AAV-transduced WT and Ts65Dn hippocampi expressing EGFP (green) and immunostained for the neuronal marker NeuN (red); nuclei are counterstained with Hoechst (blue). EGFP expression was restricted to NeuN+ neurons. Scale bar: 50 μm. Bottom-left: representative confocal z stack projection images showing EGFP expression (green) in the dorsal hippocampal CA1 region of AAV9-injected WT and Ts65Dn mice at 6 WPIs. Sections were immunostained with the astrocyte marker GFAP (blue) and the microglia marker Iba1 (red). EGFP expression was virtually absent in both astrocytes and microglia cells. Scale bar: 50 μm. Right: quantification of the percentage of EGFP and NeuN-double-positive (NeuN+-EGFP+) cells over the total EGFP+ cells in the hippocampal CA1 pyramidal layer of AAV9-injected WT and Ts65Dn mice. (C) Top: schematic representation of the experimental timeline: 2–3 month-old WT and Ts65Dn mice were stereotaxically injected in the dorsal hippocampal CA1 region with AAV9 vectors and analyzed by immunoblot at 4 WPIs. Bottom: representative immunoblot for NKCC1 in protein extracts from AAV9-transduced WT and Ts65Dn dorsal CA1 regions. (D) Quantification of NKCC1 protein (expressed as the percentage of WT neurons transduced with control amiR) showed increased NKCC1 expression in Ts65Dn compared with that of WT samples. Expression of amiRs 1 and 2 induced significant NKCC1 knockdown in the Ts65Dn dorsal CA1 region. (E) Representative immunoblot for KCC2 in protein extracts from AAV9-transduced WT and Ts65Dn dorsal CA1 regions. (F) Quantification of KCC2 protein, in the same samples as in (D), showed no significant difference in KCC2 expression in any of the experimental groups. Actin was used as an internal standard. Data in (B), (D), and (F) are means ± SEM, and dots indicate values of individual samples. ∗p < 0.05, ∗∗∗p < 0.001, Tukey post hoc test after two-way ANOVA.