Background

The hyperpolarizing activity of γ-aminobutyric acid A (GABA_A) receptors depends on the intracellular chloride gradient that is developmentally regulated by the activity of the chloride extruder potassium (K) chloride (Cl) cotransporter 2 (KCC2). In humans and rodents, KCC2 expression can be detected at birth. In rodents, KCC2 expression progressively increases and reaches adult-like levels by the second postnatal week of life. Several studies report changes in KCC2 expression levels in response to early-life injuries. However, the functional contribution of KCC2 in maintaining the excitation-inhibition balance in the neonatal brain is not clear. In the current study, we examined the effect of KCC2 antagonism on the neonatal brain activity under hyperexcitable conditions ex vivo and in vivo.

Methods

Ex vivo electrophysiology experiments were performed on hippocampal slices prepared from 7 to 9 days-old (P7-P9) male rats. Excitability of CA1 pyramidal neurons bathed in zero-Mg²⁺ buffer was measured using single-unit extracellular (loose) or cell-attach protocol before and after application of VU0463271, a specific antagonist of KCC2. To examine the functional role of KCC2 in vivo, the effect of VU0463271 on hypoxia-ischemia (HI)-induced ictal (seizures and brief runs of epileptiform discharges - BREDs), and inter-ictal spike and sharp-wave activity was measured in P7 male rats. A highly sensitive LC–MS/MS method was used to determine the distribution and the concentration of VU0463271 in the brain.

Results

Ex vivo blockade of KCC2 by VU0463271 significantly increased the frequency of zero-Mg²⁺-triggered spiking in CA1 pyramidal neurons. Similarly, in vivo administration of VU0463271 significantly increased the number of ictal events, BREDs duration, and spike and sharp-wave activity in HI rats. LC–MS/MS data revealed that following systemic administration, VU0463271 rapidly reached brain tissues and distributed well among different brain regions.

Conclusion

The results suggest that KCC2 plays a critical functional role in maintaining the balance of excitation-inhibition in the neonatal brain, and thus it can be used as a therapeutic target to ameliorate injury associated with hyperexcitability in newborns.

中文翻译：

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KCC2 在新生儿大脑过度兴奋中的作用。

 背景

γ-氨基丁酸 A (GABA _A ) 受体的超极化活性取决于细胞内氯离子梯度，该梯度受氯离子挤出机氯化钾 (K) 氯化物 (Cl) 协同转运蛋白 2 (KCC2) 的活性调节。在人类和啮齿类动物中，KCC2 的表达可以在出生时检测到。在啮齿动物中，KCC2 表达逐渐增加，并在出生后第二周达到成人样水平。几项研究报告了 KCC2 表达水平因早期伤害而发生的变化。然而，KCC2 在维持新生儿大脑兴奋-抑制平衡方面的功能贡献尚不清楚。在当前的研究中，我们在离体和体内超兴奋条件下检查了 KCC2 拮抗作用对新生儿大脑活动的影响。

 方法

在7至9日龄（P7-P9）雄性大鼠的海马切片上进行离体电生理学实验。在应用 VU0463271（一种 KCC2 特异性拮抗剂）之前和之后，使用单单位细胞外（松散）或细胞附着方案测量沐浴在零 Mg ²⁺缓冲液中的 CA1 锥体神经元的兴奋性。为了检查 KCC2在体内的功能作用，VU0463271 对缺氧缺血 (HI) 诱发的发作（癫痫发作和短暂的癫痫样放电 - BRED）的影响，以及在 P7 中测量了发作间期尖波和尖波活动雄性大鼠。采用高灵敏度 LC-MS/MS 方法测定 VU0463271 在大脑中的分布和浓度。

 结果

VU0463271 对 KCC2 的体外阻断显着增加了 CA1 锥体神经元中零 Mg ²⁺触发的尖峰频率。同样，体内施用 VU0463271 显着增加 HI 大鼠的发作事件数量、BRED 持续时间以及尖波和尖波活动。 LC-MS/MS数据显示，全身给药后，VU0463271迅速到达脑组织并在不同脑区分布良好。

 结论

结果表明，KCC2在维持新生儿大脑兴奋-抑制平衡方面发挥着关键的功能作用，因此它可以作为改善新生儿过度兴奋相关损伤的治疗靶点。