Fig. 7
Overexpressing HCN2 enhances the activity of HCNs, decreases the excitability of dCA1s, increases the CA1 LTP and improves cognitive functions in hAPP-J20 mice. (A) Representative images showing the expression of the virus microinjected in the dCA1 of hAPP-J20 mice. Scale bar, 500 μm. (B) Representative bands of HCN2 in western blottings of the hippocampal tissues from hAPP-J20 mice with or without overexpressing HCN2. (C) Quantification of the relative HCN2 levels in the hippocampus (EGFP, n = 7; HCN2, n = 7). Unpaired t-test: t (12) = 2.718, p = 0.0187. *p < 0.05. (D) Representative traces elicited by a series of negative current injections of dCA1 PNs in hAPP-J20 mice with (HCN2) or without (EGFP) overexpression of HCN2. (E) The sag ratios detected in dCA1 PNs of hAPP-J20 mice with HCN2 overexpression (n = 8) or without HCN2 overexpression (n = 8). Unpaired t-test: t (15) = 2.182, p = 0.0454. *p < 0.05. (F) Sag amplitudes of dCA1 PNs in hAPP-J20 mice with or without overexpressing HCN2 at − 200 to 0-pA current injections. Two-way ANOVA: virus (HCN2), F (1, 14) = 4.448, p = 0.0534; current step, F (1.612, 22.57) = 150.8, p < 0.0001; interaction, F (4, 56) = 13.14, p < 0.0001; *p < 0.05, ***p < 0.001 with Bonferroni’s post-hoc test. (H) Representative traces of the HCN-conducted Ih currents in the dCA1 PNs with or without overexpressing HCN2. (I) Quantification of the Ih currents (EGFP, n = 8 cells from 4 mice; HCN2, n = 8 cells from 4 mice). Two-way ANOVA: virus (HCN2), F (1, 15) = 6.178, p = 0.0252; voltage step, F (1.314, 19.71) = 533.1, p < 0.0001; interaction, F (6, 90) = 23.46, p < 0.0001; *p < 0.05 with Bonferroni’s post-hoc test. (J) The input resistance was significantly decreased in dCA1 PNs of hAPP-J20 mice after overexpressing HCN2 (EGFP, n = 8 cells from 4 mice; HCN2, n = 8 cells from 4 mice). Unpaired t-test: t (14) = 2.249, p = 0.0411. *p < 0.05. (K) Sample traces of action potentials in dCA1 PNs of hAPP-J20 mice with or without HCN2 overexpression obtained at the 150 pA current injection. (L) Quantifications of the spike numbers of dCA1 PNs in response to current injections (EGFP, n = 8 cells from 4 mice; HCN2, n = 8 cells from 4 mice). Two-way ANOVA: virus (HCN2), F (1, 14) = 6.597, p = 0.0223; current step, F (2.272, 31.80) = 107.9, p < 0.0001; interaction, F (12, 168) = 3.584, p < 0.0001; *p < 0.05 with Bonferroni’s post-hoc test. (M) Representative traces of sAP of dCA1 PNs in hAPP-J20 with or without HCN2 overexpression. (N) Frequency of sAP in the dCA1 PNs of hAPP-J20 mice with or without HCN2 overexpression (EGFP, n = 20 cells from 8 mice; HCN2, n = 15 cells from 7 mice). Unpaired t-test: t (33) = 2.047, p = 0.0487. *p < 0.05. (O) Representative traces showing LTP in CA1 of hAPP-J20 with or without HCN2 overexpression. (P) Quantification of the last 15 min of the fEPSP recordings (EGFP, n = 9 cells from 5 mice; HCN2, n = 12 cells from 5 mice). Unpaired t-test: t (19) = 2.683, p = 0.0147. *p < 0.05. (Q) The percentage of alternations in Y-maze test from WT and hAPP-J20 mice with or without overexpressing HCN2 (WT/EGFP: n = 9; WT/HCN2: n = 9; J20/EGFP: n = 8; J20/HCN2: n = 8). Two-way ANOVA: genotype (hAPP), F (1,30) = 2.938, p = 0.0969; virus (HCN2), F (1,30) = 0.4145, p = 0.5246; interaction, F (1,30) = 22.76, p < 0.0001; *p < 0.05, **p < 0.01, ***p < 0.001 with Bonferroni’s post-hoc test. (R) The total arm entries in the Y-maze test from WT and hAPP-J20 mice with or without overexpressing HCN2 (WT/EGFP: n = 9; WT/HCN2: n = 9; J20/EGFP: n = 8; J20/HCN2: n = 8). Two-way ANOVA: genotype (hAPP), F (1,30) = 21.32, p < 0.0001; virus (HCN2), F (1,30) = 0.4806, p = 0.4935; interaction, F (1,30) = 0.1246, p = 0.7266; *p < 0.05, **p < 0.01 with Bonferroni’s post-hoc test