Neurons in nucleus gigantocellularis (NGC) have been shown by many lines of evidence to be important for regulating generalized CNS arousal. Our previous study on mouse pups suggested that the development of NGC neurons’ capability to fire action potential (AP) trains may both lead to the development of behavioral arousal and may itself depend on an increase in delayed rectifier currents. Here with whole-cell patch clamp we studied delayed rectifier currents in two stages. First, primary cultured neurons isolated from E12.5 embryonic hindbrain (HB), a dissection which contains all of NGC, were used to take advantage of studying neurons in vitro over using neurons in situ or in brain slices. HB neurons were tested with Guangxitoxin-1E and Resveratrol, two inhibitors of Kv2 channels which mediate the main bulk of delayed rectifier currents. Both inhibitors depressed delayed rectifier currents, but differentially: Resveratrol, but not Guangxitoxin-1E, reduced or abolished action potentials in AP trains. Since Resveratrol affects the Kv2.2 subtype, the development of the delayed rectifier mediated through Kv2.2 channels may lead to the development of HB neurons’ capability to generate AP trains. Stage Two in this work found that electrophysiological properties of the primary HB neurons recorded are essentially the same as those of NGC neurons. Thus, from the two stages combined, we propose that currents mediated through Kv2.2 are crucial for generating AP trains which, in turn, lead to the development of mouse pup behavioral arousal.

许多证据表明巨细胞核 (NGC) 中的神经元对于调节全身中枢神经系统唤醒非常重要。我们之前对小鼠幼崽的研究表明，NGC 神经元激发动作电位 (AP) 序列的能力的发展可能会导致行为唤醒的发展，而且本身可能取决于延迟整流电流的增加。在这里，我们利用全细胞膜片钳研究了两个阶段的延迟整流器电流。首先，使用从 E12.5 胚胎后脑 (HB)（包含所有 NGC 的解剖结构）分离的原代培养神经元，与使用原位或脑切片中的神经元相比，利用体外研究神经元的优势。 HB 神经元用广西毒素-1E 和白藜芦醇进行测试，这两种 Kv2 通道抑制剂介导大部分延迟整流电流。两种抑制剂均抑制延迟整流电流，但有所不同：白藜芦醇（而非广西毒素-1E）降低或消除了 AP 序列中的动作电位。由于白藜芦醇影响 Kv2.2 亚型，因此通过 Kv2.2 通道介导的延迟整流器的发展可能导致 HB 神经元产生 AP 序列的能力的发展。这项工作的第二阶段发现，记录的初级 HB 神经元的电生理特性与 NGC 神经元的电生理特性基本相同。因此，从这两个阶段的结合来看，我们认为通过 Kv2.2 介导的电流对于生成 AP 序列至关重要，而 AP 序列反过来又导致小鼠幼崽行为唤醒的发展。