Selective Modulation of K+ Channel Kv7.4 Significantly Affects the Excitability of DRN 5-HT Neurons

Figures

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  Figure 1. Kv7.4 channels expressed in dorsal raphe nucleus (DRN) 5-HT neurons. (A) Confocal images showing co-expression of DAPI (blue), Kv7.4 (green) and TPH (red) represent 5-HT neurons (up-panel, scale bar, 100 μm). Lower panel represents a zoomed-in area of DRN (scale bar, 25 μm). (B) Single-cell PCR from DRN cells. “con” indicates negative control in which no template was added in the RT-PCR reaction. (C) Percentage of 5-HT neurons (with expression of TPH) and Kv7.4 positive neurons from 61 DRN neurons.

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  Figure 2. Fasudil potently augments Kv7.4 currents in DRN 5-HT neurons. (A1) Sample time course shows that fasudil (30 μM) augments Kv7 current, which is completely blocked by XE991 (3 μM) in neurons from WT mice. (A2) Voltage clamping protocol and the corresponding current traces recorded from (A1). (A3) Summarized data for the experiment shown in (A1) (Wilcoxon Singed Ranks Test, *P < 0.05, n = 8, N = 5). (A4) Single-cell PCR result shows that the recorded neuron is a 5-HT neuron with expression of Kv7.4 and 5-HT1A. (B1) Sample time course shows that fasudil (30 μM) has no effect on Kv7 current in Kv7.4−/− mice. (B2) Voltage clamping protocol and the corresponding current traces recorded from (B1). (B3) Summarized data for the experiment shown in (B1) (n = 10, N = 5). (B4) Single-cell PCR result shows that the recorded neuron is a 5-HT neuron with 5-HT1A expression and no Kv7.4 expression. **P < 0.01.

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  Figure 3. Kv7.4 activity modulates the excitability of the DRN 5-HT neurons. (A,B) Effects of fasudil on the elicited firing activity of DRN 5-HT neurons from WT mice and Kv7.4−/− mice. (A1) Fasudil (30 μM) significantly hyperpolarized resting membrane potential (RMP) and the effect completely reversed by XE991 (3 μM) in WT mice. (A2) Typical traces with firing activity elicited by depolarizing 250 pA current injection, recorded from a neuron of WT mice. (A3) Summary of RMP in response to fasudil (30 μM) in WT DRN 5-HT neurons (paired samples test, ***P < 0.001, n = 9, N = 6). (A4) Summary of the spike number in the response of fasudil (30 μM) in WT DRN 5-HT neurons (Wilcoxon Signed Ranks Test, *P < 0.05, n = 9, N = 6). (B1) Fasudil (30 μM) had no significant effect on RMP of neurons from Kv7.4−/− mice. (B2) Typical trace with firing activity elicited by depolarizing 250 pA current injection, recorded from a neuron of Kv7.4−/− mice. (B3) Summary of RMP in response to fasudil (30 μM) in Kv7.4−/− DRN 5-HT neurons (n = 10, N = 6). (B4) Summary of the spike number in response to fasudil (30 μM) in Kv7.4−/− DRN 5-HT neurons (n = 10, N = 6). (C) Cell-attach recording of spontaneous firing in…

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  Figure 4. 5-HT augments Kv7.4 currents in DRN 5-HT neurons. (A1) Sample time course shows that 5-HT (30 μM) induced a sharp augmentation of Kv7 current and the augmentation was completely reversed by XE991 (3 μM) in DRN 5-HT neurons of WT mice. (A2) Voltage clamping protocol and representative current traces recorded from (A1). (A3) Summarized data for experiments shown in (A1) (Wilcoxon Signed Ranks Test, ***P < 0.001, n = 14, N = 6). (B1) Sample time course shows that 5-HT did not affect Kv7 current in neurons from Kv7.4−/− mice. (B2) Voltage clamping protocol and representative current traces recorded from (B1). (B3) Summarized data for experiments shown in (B1) (n = 10, N = 6).

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  Figure 5. Kv7.4 is a regulation target of 5-HT1A auto-inhibition. (A1) 5-HT1A receptor agonist 8-OH-DPAT increased the Kv7 current in DRN 5-HT neurons of WT mice and the effect was reversed by XE991. (A2) Voltage clamping protocol and the corresponding traces recorded from (A1). (A3) Summarized data for experiments shown in (A1) (paired samples test, **P < 0.01, n = 8, N = 5). (B1) 5-HT1A receptor antagonist WAY-100635 abolished the 5-HT-induced augmentation of Kv7 current. (B2) Voltage clamping protocol and representative current traces recorded from (B1). (B3) Summarized data for experiments shown in (B1) (n = 10, N = 6).

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  Figure 6. A redox mechanism is involved in 5-HT-mediated activation of Kv7.4 currents in DRN 5-HT neurons. (A1) 5-HT activation of Kv7.4 current was reversed by a reducing agent dithiothreitol (DTT). (A2) Voltage clamping protocol and the corresponding traces recorded from (A1). (A3) Summarized data for experiments shown in (A1) (Wilcoxon Signed Ranks Test, *P < 0.05, **P < 0.01, n = 6, N = 4). (B1) Pretreatment with DTT (1 mM) had no effect on Kv7 currents but prevented the 5-HT-induced augmentation of the Kv7 currents. (B2) Voltage clamping protocol and representative current traces recorded from (B1). (B3) Summarized data for experiments shown in (B1) (Wilcoxon Signed Ranks Test, *P < 0.05, n = 11, N = 5).