Impaired pattern separation in Tg2576 mice is associated with hyperexcitable dentate gyrus caused by Kv4.1 downregulation

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory loss. Most AD researches have focused on neurodegeneration mechanisms. Considering that neurodegenerative changes are not reversible, understanding early functional changes before neurodegeneration is critical to develop new strategies for early detection and treatment of AD. We found that Tg2576 mice exhibited impaired pattern separation at the early preclinical stage. Based on previous studies suggesting a critical role of dentate gyrus (DG) in pattern separation, we investigated functional changes in DG of Tg2576 mice. We found that granule cells in DG (DG-GCs) in Tg2576 mice showed increased action potential firing in response to long depolarizations and reduced 4-AP sensitive K⁺-currents compared to DG-GCs in wild-type (WT) mice. Among Kv4 family channels, Kv4.1 mRNA expression in DG was significantly lower in Tg2576 mice. We confirmed that Kv4.1 protein expression was reduced in Tg2576, and this reduction was restored by antioxidant treatment. Hyperexcitable DG and impaired pattern separation in Tg2576 mice were also recovered by antioxidant treatment. These results highlight the hyperexcitability of DG-GCs as a pathophysiologic mechanism underlying early cognitive deficits in AD and Kv4.1 as a new target for AD pathogenesis in relation to increased oxidative stress.

Keywords: Alzheimer’s disease; Dentate gyrus; Intrinsic excitability; Kv4.1; Tg2676.

Fig. 1

Impaired pattern separation in Tg2576 mice. A Experimental procedure for pattern separation in 10-week-old WT and Tg2576 mice. Ba On day 4 to 5, the kinetics of freezing across the 5 min test in context A. Bb Percentage of freezing in context A (filled bar) and context B (open bar) during day 4–5. WT (blue, n = 8) and Tg2576 (green, n = 9) mice displayed equal amounts of freezing in both contexts A and B. Ca On day 6 to 13, time course of the discrimination ratio in WT (blue, n = 8) and Tg2576 (green, n = 7) mice. Statistical significance for each day was tested using ANOVA (*P < 0.05; **P < 0.01; *** P < 0.001). Cb Percentage of freezing in context A (filled bar) and context B (open bar) for WT (blue, n = 8) and Tg2576 (green, n = 7) mice on day 11. Freezing levels were compared between the two contexts for each group (N.S., no statistical significance; *P < 0.05). Da Experimental procedure for one-trial contextual fear conditioning between WT (n = 8) and Tg2576 (n = 7) mice. Db The percentage of freezing in context A (filled bar) and context C (open bar, distinct object) for WT (n = 8) and Tg2576 (n = 7) mice

Fig. 2

Electrophysiological analysis for the excitability of WT and Tg2576 mice. Aa Representative traces in the current-clamp recording from the WT (blue) and Tg2576 (green) dentate GCs in response to prolonged (1 s) depolarizing current injection (200 pA). Ab Number of spike evoked during 1 s current injections with varying amplitudes (from 100 to 600 pA, 100 pA increment). At all amplitudes, the number of spikes is significantly higher in Tg2576 GCs (green closed circle, n = 14) compared to WT GCs (blue closed circle, n = 15). Mean ± S.E.M. **P < 0.01; *** P < 0.001 B Expanded view of overlaid 1st APs evoked by 300 pA current injection to show the shortening of AP onset in Tg2576 GCs compared to WT GCs. Mean ± S.E.M. *** P < 0.001 C The mean values of the resting membrane potential (WT: − 84.5 ± 1.3 mV, Tg2576: − 81.8 ± 1.0 mV), input resistance (WT: 161.6 ± 6.9 MΩ, Tg2576: 158.8 ± 9.5 MΩ), and rheobase (WT: 181.1 ± 6.7 pA, Tg2576: 173.5 ± 7.7) from the WT (n = 15) and Tg2576 GCs (n = 14). Mean ± S.E.M. N.S., not significantly different. Da Representative traces of current-clamp recordings from CA1 of WT (blue) and Tg2576 mice (green). A train of APs were evoked by 1 s depolarizing current pulse injection (100 pA). Db The F-I curve for CA1 from WT (n = 4) and Tg2576 (n = 4) mice

Fig. 3

Voltage-dependent K⁺ currents in the WT and Tg2576. Aa Superimposed current traces evoked by various steps of depolarization (− 60 mV to + 30 mV in 10 mV step) for 1 s from the holding potential of − 70 mV in the WT (blue) and Tg2576 (green) dentate GCs. Ab Amplitudes of peak currents (I_peak) and steady state currents (I_ss) measured at the end of depolarization are plotted as a function of the given potential (V). WT (closed blue circle, n = 9) and Tg2576 (closed green circle, n = 7). Ba The difference in currents before and after applying 5 mM 4-AP (I_4-AP) in the WT (blue) and Tg2576 (green) dentate GCs. Bb I_peak and I_ss for I_4-AP are plotted as a function of the given potential (V). WT (closed blue circle, n = 9) and Tg2576 (closed green circle, n = 7). Ca I_4-AP traces obtained at + 30 mV are averaged (WT, blue, n = 9; Tg2576, green, n = 7) and superimposed. Cb I_4-AP recorded at + 30 mV were fitted to double-exponential functions. Amplitude and time constant for the fast component (A_fast, τ_fast) and the slow component (A_slow, τ_slow). Mean ± S.E.M., **, P < 0.01, ***, P < 0.001. N.S. not significantly different, P > 0.05

Fig. 4

Downregulation of Kv4.1 expression in Tg2576 causes hyperexcitability of Tg2576 GCs. A mRNA expression levels for Kv4 family channels in the WT (blue) and Tg2576 (green) DG detected by quantitative RT-PCR. B Western blot analysis for Kv4.1 in DG of 8-week-old mice. For a loading control, β-actin was used. Right, Kv4.1expression in Tg2576 was significantly smaller than WT mice. *, P < 0.05. Ca Example traces of APs evoked by 200 pA current injection immediate (3 min, blue for WT and green for Tg2576) and 15 min (red) after brake-in of patch pipettes containing Kv4.1 antibody. Cb Firing frequency at 200 pA is compared in each conditions

Fig. 5

Effect of pre-treatment with Trolox in the Tg2576 mice. Aa Representative trace in the current-clamp recording of pre-treatment with Trolox for one hour (orange) and 1 week (purple) in Tg2576 in response to depolarizing current injection (200 pA with 1 s duration). Ab F-I curve (from 100 to 600 pA, 100 pA increment) obtained from GCs of Tg2576 mice treated with Trolox for 1 week (Tg + Trolox, purple, n = 8) was compared with the F-I curve obtained from Tg2576 GCs (green dashed line, n = 14) shown in Fig. 2Ab. *, P < 0.05, ***, P < 0.001. B Western blot analysis for Kv4.1 in DG of 8-week-old Tg2576 mice and those treated with Trolox (20 mg/kg) for 1 week. For a loading control, β-actin was used. Right, Kv4.1expression in Tg2576 was significantly increased after Trolox treatment. **, P < 0.01. Ca On day 4–5, the kinetics of freezing across the 5 min test in context A obtained from Tg2576 mice treated with Trolox for 1 week (Tg + Trolox, purple, n = 10) was compared with the that obtained from control Tg2576 GCs (green, n = 7) shown in Fig. 1Ba. Cb Percentage of freezing in context A (filled bar) and context B (open bar) during day 4 to 5. Tg2567 (green, n = 7) and Tg + Trolox (purple, n = 10) mice displayed equal amounts of freezing in both contexts A and B. Da On day 6 to 12, time course of the discrimination ratio in Tg + Trolox mice (purple, n = 10) was compared with that obtained from control Tg2576 mice shown in Fig. 1Ca (green, n = 7) mice. Statistical significance for each day was tested using unpaired t-test (*P < 0.05; **P < 0.01; ***P < 0.001). Db Percentage of freezing in context A (filled bar) and context B (open bar) for Tg2576 (green, n = 7) and Tg + Trolox (purple, n = 10) mice on day 11. Freezing levels were compared between the two contexts for each group. Tg2567 (green, n = 7) mice displayed equal amounts of freezing in both contexts A and B, while and Tg + Trolox (purple, n = 10) mice displayed more freezing in context A compared to context B