Prefrontal inhibition of neuronal Kv 7 channels enhances prepulse inhibition of acoustic startle reflex and resistance to hypofrontality

Abstract

Background and purpose: Dysfunction of the prefrontal cortex (PFC) is involved in the cognitive deficits in neuropsychiatric diseases, such as schizophrenia, characterized by deficient neurotransmission known as NMDA receptor hypofrontality. Thus, enhancing prefrontal activity may alleviate hypofrontality-induced cognitive deficits. To test this hypothesis, we investigated the effect of forebrain-specific suppression or pharmacological inhibition of native K_v 7/KCNQ/M-current on glutamatergic hypofrontality induced by the NMDA receptor antagonist MK-801.

Experimental approach: The forebrain-specific inhibition of native M-current was generated by transgenic expression, in mice, of a dominant-negative pore mutant G279S of K_v 7.2/KCNQ2 channels that suppresses channel function. A mouse model of cognitive impairment was established by single i.p. injection of 0.1 mg·kg^-1 MK-801. Mouse models of prepulse inhibition (PPI) of acoustic startle reflex and Y-maze spontaneous alternation test were used for evaluation of cognitive behaviour. Hippocampal brain slice recordings of LTP were used to assess synaptic plasticity. Hippocampus and cortex were dissected for detecting protein expression using western blot analysis.

Key results: Genetic suppression of K_v 7 channel function in the forebrain or pharmacological inhibition of K_v 7 channels by the specific blocker XE991 enhanced PPI and also alleviated MK-801 induced cognitive decline. XE991 also attenuated MK-801-induced LTP deficits and increased basal synaptic transmissions. Western blot analysis revealed that inhibiting K_v 7 channels resulted in elevation of pAkt1 and pGSK-3β expressions in both hippocampus and cortex.

Conclusions and implications: Both genetic and pharmacological inhibition of K_v 7 channels alleviated PPI and cognitive deficits. Mechanistically, inhibition of K_v 7 channels promotes synaptic transmission and activates Akt1/GSK-3β signalling.

Keywords: KCNQ2; Kv7; M-current; XE991; glutamatergic hypofrontality; prefrontal cortex; prepulse inhibition.

FIGURE 1

Genetic and pharmacological inhibition of K_v7 channels increased resistance to PPI impairment induced by the NMDA receptor antagonist MK‐801. (a) The time‐course of protocols to assay PPI of acoustic startle response in mice. (b) Enhancement of PPI in rQ2‐G279S Tg mice, compared to WT controls. ^* P < 0.05, significantly different as indicated. (c) Resistance to PPI deficits induced by MK‐801 (0.1 mg·kg⁻¹) in Tg mice compared with WT mice. ^* P < 0.05, significantly different from WT‐Vehicle, ^# P < 0.05, significantly different fromWT‐MK‐801 (0.1 mg·kg⁻¹). (d) Amelioration of MK‐801‐induced PPI deficits by administration of XE991 (1 mg·kg⁻¹, i.p.) or clozapine (1 mg·kg⁻¹, i.p.) as positive control. Retigabine (RTG; 10 mg·kg⁻¹, i.p.) was used to antagonize XE991‐mediated effect. Data shown are means ± SEM; n = 8–12. ^* P < 0.05, significantly different from Vehicle, ^# P < 0.05, significantly different from MK‐801 (0.1 mg·kg⁻¹), ^$ P < 0.05, significantly different from MK‐801 (0.1 mg·kg⁻¹) + XE991 (1 mg·kg⁻¹); two‐way ANOVA, followed by post hoc Bonferroni's test

FIGURE 2

Attenuation of MK‐801‐induced memory impairment by inhibition of K_v7 channel activity in Y‐maze test. (a) Time‐course of protocols for Y‐maze test in mice. (b) Left panels: Representative motion trajectories of mice in different groups during Y‐maze test. Right panels: A summary from left panels for the time spent in the novel arm and total distance travelled in three arms between the rQ2‐G279S Tg mice and WT mice. ^* P < 0.05, significantly different as indicated. (c) Left panels: Representative motion trajectories of mice in different groups of mice treated with MK‐801 (0.1 mg·kg⁻¹, i.p.). Clozapine (1 mg·kg⁻¹) and RTG (10 mg·kg⁻¹) were i.p. administered as controls. Right panels: A summary from left panels for the time spent in the novel arm and the total distance in three arms in different groups. ^* P < 0.05, significantly different from Vehicle, ^# P < 0.05, significantly different from MK‐801 (0.1 mg·g⁻¹), ^$ P < 0.05, significantly different from MK‐801 (0.1 mg·kg⁻¹) + XE991 (1 mg·kg⁻¹); one‐way ANOVA, followed by post hoc Bonferroni's test. Data shown are means ± SEM; n = 6–10

FIGURE 3

Amelioration of MK‐801‐induced deficits of hippocampal synaptic transmission by pharmacological inhibition of K_v7 channels. (a) Left panels: Representative traces of fEPSPs before (line 1) and after (line 2) LTP induction in mice treated i.p. with MK‐801 (0.1 mg·kg⁻¹) or in combination with XE991 (0.1 mg·kg⁻¹) or RTG (10 mg·kg⁻¹) and clozapine (1 mg·kg⁻¹). Right panels: The fEPSP slope for HFS‐induced LTP in CA1 area of the hippocampus and amelioration of 0.1 mg·kg⁻¹ MK‐801‐induced LTP impairment in response to XE991 (1 mg·kg⁻¹, i.p.). (b) Left panels: Representative traces of fEPSPs elicited a series of stimulus from 0.02 to 0.12 mA in different groups. Right panels: A plot of I/O curve for the fEPSP slope against the stimulus intensity. (c) Left panels: Representative traces of PPF by giving an interpulse interval of 40 ms. Right panels: The PPF in the hippocampal CA1 area in vehicle control mice and drug‐treated mice. Facilitation ratios were plotted against interpulse intervals. Data shown are means ± SEM; n = 10–12. ^* P < 0.05, significantly different from Vehicle, ^# P < 0.05, significantly different from MK‐801 (0.1 mg·kg⁻¹); two‐way ANOVA, followed by post hoc Bonferroni's test

FIGURE 4

Genetic suppression of K_v7 channels prevents MK‐801‐induced decrease of pAkt1 (Ser473) and pGSK‐3β (Ser9) in the hippocampus and cortex. (a) Left panels: Representative western blots of pAkt1 (Ser473), tAkt1, pGSK‐3β (Ser9), tGSK‐3β, and GAPDH levels from the hippocampi of WT and Tg mice treated i.p. with or without MK‐801 (0.1 mg·kg⁻¹). Right panels: Quantification of the relative changes in pAkt1 (Ser473), tAkt1, pGSK‐3β (Ser9), and tGSK‐3β expression from left panels. (b) Left panels: Representative western blots of pAkt1 (Ser473), tAkt1, pGSK‐3β (Ser9), tGSK‐3β, and GAPDH levels from the cortices. Right panels: Quantification of the relative changes in pAkt1 (Ser473), tAkt1, pGSK‐3β (Ser9), and tGSK‐3β expression from left panels. Data shown are means ± SEM; n = 6–8. ^* P < 0.05, significantly different as indicated; one‐way ANOVA, followed by post hoc Bonferroni's test

FIGURE 5

Pharmacological inhibition of K_v7 channels increases the phosphorylation of pAkt1 (Ser473) and pGSK‐3β (Ser9) in the hippocampus and cortex. (a) Left panels: Representative western blots of pAkt1 (Ser473), tAkt1, and GAPDH levels from the hippocampi (Hippo) of mice treated i.p. with MK‐801 (0.1 mg·kg⁻¹) in combination with XE991 (1 mg·kg⁻¹) or RTG (10 mg·kg⁻¹) or clozapine (1 mg·kg⁻¹). Right panels: Quantification of the relative changes in pAkt1 (Ser473) and tAkt1 expressions from left panels. (b) Left panels: Representative western blots of pGSK‐3β (Ser9), tGSK‐3β, β‐catenin, and GAPDH levels from the cortices in different groups. Right panels: Quantification of the relative changes in pGSK‐3β (Ser9), tGSK‐3β, and β‐catenin expressions from left panels. (c) Left panels: Representative western blots of pAkt1 (Ser473), tAkt1, and GAPDH levels from the hippocampi. Right panels: Quantification of the relative changes in pAkt1 (Ser473) and tAkt1 expression from left panels. (d) Left panels: Representative western blots of pGSK‐3β (Ser9), tGSK‐3β, β‐catenin, and GAPDH levels from the cortices. Right panels: Quantification of the relative changes in pGSK‐3β (Ser9), tGSK‐3β, and β‐catenin expressions from their left panels. Data shown are means ± SEM; n = 6‐8. ^# P < 0.05, significantly different from MK‐801, ^$ P < 0.05, significantly different from MK‐801 (0.1 mg·kg⁻¹) + XE991 (1 mg·kg⁻¹); one‐way ANOVA, followed by post hoc Bonferroni's test