Fig. 10. The ICl,swell is mediated by TTYH family in ex-vivo hippocampal astrocytes. (A) Upper panels in the hippocampus show immunostaining for TTYH1 expression in GFAP-positive (arrow) and NeuN-positive cells (arrowhead) in the hippocampal CA1 region. The lower panel in the hippocampus shows higher magnification images of TTYH1 expression in GFAP-positive (left) and NeuN-positive (right) cells. Upper panels in the cortex show immunostaining for TTYH1 expression in GFAP-positive (arrow) and NeuN-positive (arrowhead) in the cortex. Lower panel in the cortex show higher magnification images of TTYH1 expression in GFAP-positive (right) and NeuN-positive (arrowhead) cells. (B) Immunostaining images from the hippocampus of C57BL/6J mouse injected with AAV-control shRNA (top) or AAV-Ttyh1 shRNA (bottom) stained with DAPI (blue), TTYH1 (green), GFAP (red) and merged images by confocal microscope. Inset: High magnification images focusing on single GFAP-positive cells from the mouse injected with AAV-control shRNA or Ttyh1 shRNA stained with DAPI (blue), TTYH1 (green), GFAP (red) and merged images. (C) Quantification of TTYH1 intensity in astrocytes (GFAP-positive cells) from hippocampal CA1 stratum radiatum (SR) injected with AAV-virus containing control or all Ttyh1/2/3 shRNAs. Data are represented as mean±SEM (n=112 and 94 for control and Ttyh1/2/3 shRNAs; ****<0.0001, unpaired t-test). (D) Top: schematic diagram of whole-cell patch-clamp recordings in SR (left) and representative images for bright-field and fluorescence images showing Ttyh1/2/3 shRNAs–expressing astrocytes (right). Bottom: representative traces of ICl,swell in naïve, control and Ttyh1/2/3 shRNAs expressing astrocytes in SR. (E) Averaged I~V curves for ICl,swell in naive (black), control shRNA (blue) and Ttyh1/2/3 shRNAs (red). (F) Summary bar graph showing the maximal amplitude of ICl,swell from +100 mV to −100 mV in the naïve, control and Ttyh1/2/3 shRNAs. Data are represented as mean±SEM (n=10, 10 and 12 for naïve, control shRNA and Ttyh1/2/3 shRNA; **<0.01, unpaired t-test).
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