Fig. 8
Knocking down the expression of HCN2 decreases the activity of HCNs, increases the excitability of dCA1s and impairs cognitive functions in WT mice. (A) Representative images showing the expression of the AAV virus microinjected into the dCA1 of WT mice. Scale bar, 500 μm. (B, C) Western blot analysis to confirm the reduced expression of HCN2 in the hippocampus of WT mice. (B), representative protein bands of HCN2; (C), quantification of the relative HCN2 levels in the hippocampus (EGFP, n = 6; HCN2-shRNA, n = 5). Unpaired t-test: t (9) = 4.842, p = 0.0009. ***p < 0.001. (D) Representative traces elicited by a series of negative current injections in the dCA1 PNs of WT mice with (brown) or without (black) knocking down the expression of HCN2. (E) The sag ratios detected in the dCA1 PNs of WT mice with or without knocking down the expression of HCN2, n = 10 (EGFP) or 8 (HCN2-shRNA). Unpaired t-test: t (16) = 5.086, p = 0.0001. ***p < 0.001. (F) Sag amplitudes of dCA1 PNs in WT mice with or without knocking down the expression of HCN2 at − 200 to 0-pA current injections. Two-way ANOVA: virus (HCN2-shRNA), F (1, 16) = 13.86, p = 0.0018; current step, F (1.994, 31.90) = 330.7, p < 0.0001; interaction, F (4, 64) = 3.772, p = 0.0081; *p < 0.05, **p < 0.01 with Bonferroni’s post-hoc test. (G) Representative traces of the HCN-conducted Ih currents in dCA1 PNs of WT mice with or without knocking down the expression of HCN2. (H) Quantification of the Ih currents (EGFP, n = 8 cells from 5 mice; HCN2-shRNA, n = 8 cells from 5 mice). Two-way ANOVA: virus (HCN2-shRNA), F (1, 14) = 27.95, p = 0.0001; voltage step, F (1.281, 17.94) = 533.1, p < 0.0001; interaction, F (6, 84) = 26.51, p < 0.0001; **p < 0.01, ***p < 0.001 with Bonferroni’s post-hoc test. (I) Representative traces of sAP of dCA1 PNs in WT mice with or without knocking down the expression of HCN2. (J) Quantification of the sAP frequencies in the dCA1 PNs of WT mice with or without knocking down HCN2 (EGFP, n = 27 cells from 10 mice; HCN2-shRNA, n = 28 cells from 10 mice). Unpaired t-test: t (53) = 2.081, p = 0.0423. *p < 0.05. (K) Representative traces showing LTP in CA1 of WT mice with or without knocking down the expression of HCN2. (L) Quantification of the last 15 min of the fEPSP recordings (EGFP, n = 9 cells from 5 mice; HCN2-shRNA, n = 8 cells from 5 mice). Unpaired t-test: t (15) = 1.757, p = 0.0993. (M) The percentage of the alternations in Y-maze tests of WT mice with or without knocking down the expression of HCN2 (WT/EGFP: n = 10; WT/HCN2-shRNA: n = 11). Unpaired t-test: t (19) = 4.659, p = 0.0002. ***p < 0.001. (N) The discrimination index of novel location recognition (NLR) test of WT mice with or without knocking down the expression of HCN2 (WT/EGFP: n = 10; WT/HCN2-shRNA: n = 11). Unpaired t-test: t (19) = 3.588, p = 0.0020. **p < 0.01