Abstract：
Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 μM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 μM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10 μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.
摘要：
脆性X综合征（FXS），最常见的单基因形式的智力残疾，是由FMR1基因的转录沉默引起的，这可能使神经元过度兴奋。在这里，我们显示海马背侧CA1区域的锥体细胞（PC）在幼年Fmr1敲除（KO）小鼠中响应于阈值刺激而引起的动作电位（AP）数量比野生型（WT）小鼠更大。因为Kv7/M通道调节大鼠CA1PC兴奋性，我们调查了它们的功能障碍是否在Fmr1KO小鼠中产生神经元过度兴奋。免疫组织化学和蛋白质印迹分析显示，基因型之间Kv7.2和Kv7.3通道亚基的表达没有差异；然而，Kv7/M通道介导的电流在Fmr1KO小鼠中降低。在两种基因型中，XE991（10μM）的浴应用，Kv7/M通道的阻断剂：产生了增加的AP数，产生了增加的输入电阻，通过增加平均速度，产生了降低的AP电压阈值，并形成了AP介质后超极化。雷替加宾(10μM),Kv7/M频道的开瓶器，产生与XE991相反的效果。XE991和瑞替加滨都消除了在基因型之间的对照条件中发现的所有这些参数的差异。此外，低浓度的瑞替加滨（2.5μM）使Fmr1KO小鼠的CA1PC兴奋性正常化。最后,在Fmr1KO小鼠中，背侧CA1区4-氨基吡啶（200μM）诱发的离体癫痫样事件更为频繁，并被瑞替加滨（5-10μM）废除。我们得出结论，Fmr1KO小鼠的CA1PCs表现出过度兴奋，由Kv7/M通道功能障碍引起，癫痫样活动增加，被瑞替加宾废除了。关键点：Fmr1基因敲除小鼠海马CA1区的背侧锥体细胞表现出过度兴奋。Kv7/M通道活动，但不是表达，在Fmr1敲除小鼠的海马CA1区的锥体细胞中减少。Kv7/M通道功能障碍通过增加输入阻力导致Fmr1敲除小鼠海马CA1区锥体细胞过度兴奋，降低AP电压阈值和超极化后整形介质。Kv7/M通道开放剂使Fmr1敲除小鼠海马CA1区锥体细胞的神经元兴奋性正常化。在Fmr1KO小鼠中，背侧CA1区诱发的离体癫痫样事件更为频繁，根据药物浓度，Kv7/M通道开放剂消除了这种癫痫样活性。Kv7/M通道可以代表用于治疗脆性X综合征中与海马改变相关的症状的治疗靶标。
