Fig. 11CThe astrocytic Ttyh1/2/3 is necessary and sufficient for RVD in the hippocampus. (A) Schematic diagram of intrinsic optical signal (IOS) imaging experiment in SR of the hippocampus. (B) Representative bright-field image for IOS recording in SR after stimulating the Schaffer-collateral pathway. A white arrowhead indicates an electrical stimulation electrode in the Schaffer-collateral pathway. A white dotted lined circle indicates regions of interest (ROI). (C) (Left) Schematic diagram for electrical stimulation protocol for inducing RVD in SR of the hippocampus. (Right) The representative trace of IOS change by prolonged 1Hz for 30 min electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. The percentage of RVD is calculated with a portion of decreased transmittance (a) from the peak transmittance (b) during 1Hz, 30min stimulation. (D) Upper panels for the representative traces for IOS change indicated as astrocytic volume change before and after treatment of DCPIB (grey line) by brief 20Hz for 1 sec electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. Lower representative trace for IOS change by 100Hz for 1 sec electrical stimulation on the Schaffer-collateral pathway in CA1 hippocampus. (E) Representative traces of IOS induced by low-frequency stimulation (LFS) for 30 min in control (black), with 10 mM DCPIB (green) and with 50 mM Genistein (pink). The ‘a’ indicates the difference between the peak of response and the value of end of stimulation. The ‘b’ indicates the value of the peak for calculating RVD % (as described in , a/b*100). (F) Averaged traces for normalized transmittance by the peak of each trace. (G, L) Averaged bar graph of RVD percentage (%). RVD% is calculated by a/b*100. Data are represented as mean ± SEM (n=12, 7 and 7 for control and after DCPIB and Genistein treatment; *<0.05, **<0.01, Kruskal-Wallis test; n=10, 10 and 9 for control and Ttyh1/2/3 shRNA injected C57BL/6J mice and pSicoR Ttyh1/2/3 shRNA injected in tamoxifen-treated hGFAP-creERT2 mouse, respectively; *<0.05, **<0.01, NS>0.05, Kruskal-Wallis test) (H) Timeline from tamoxifen injection to IOS imaging. Tamoxifen was into hGFAP-CreERT2 by intraperitoneal (i.p.) injection for 6 days. On 7 day, lentivirus containing pSicoR Ttyh1/2/3 shRNA were injected into CA1 stratum radiatum of the hippocampus. IOS imaging was done 2 weeks after virus injection. (I) In the presence of tamoxifen, the inactivated Cre (CreER) were converted to the activated form of Cre (CreERT), which were translocated into Nucleus. CreERT recognized loxP sites in pSicoR Ttyh1/2/3 shRNA construct and cleaved the shRNA sequence. (J) Representative fluorescence images of lenti-viral carrying control or Ttyh1/2/3 shRNA injected into SR of CA1 hippocampus (upper panel). Representative traces of 30 min LFS-induced IOS recording from control shRNA (black), Ttyh1/2/3 shRNAs (red) injected C57BL/6J mice and glial rescue condition by pSicoR Ttyh1/2/3 shRNAs injected in tamoxifen-treated hGFAP-creERT2 mouse (blue) (bottom panel). (K) Averaged traces for 30 min LFS-induced IOS recording.
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