Shal/Kv4 Channels Are Required for Maintaining Excitability during Repetitive Firing and Normal Locomotion in Drosophila

BackgroundRhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K+ current (IA), encoded by the highly conserved Shal/Kv4 gene, in neuronal firing patterns and repetitive behaviors. While IA is present in nearly all neurons across species, elimination of IA has been complicated in mammals because of multiple genes underlying IA, and/or electrical remodeling that occurs in response to affecting one gene. Methodology/Principal FindingsIn Drosophila, the single Shal/Kv4 gene encodes the predominant IA current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNKv4), we show that IA is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNKv4 neurons display multiple defects during prolonged stimuli. DNKv4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/Kv4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/Kv4 function. Further, knock-out of Shal/Kv4 function specifically in motoneurons significantly affects the locomotion behaviors tested. Conclusions/SignificanceBased on our results, Shal/Kv4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/Kv4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors.背景 节律性行为（如行走与呼吸）依赖中枢神经系统（Central Nervous System, CNS）内中枢模式发生器的协同活动、周围神经系统（Peripheral Nervous System, PNS）的感觉反馈，最终经运动神经元输出以支配肌肉。解析这类细胞组分的内在电生理特性，是理解该协同活动的核心前提。本研究聚焦于由高度保守的Shal/Kv4基因编码的瞬时A型钾电流（transient A-type K+ current, I_A）在神经元放电模式与重复性行为中的功能意义。尽管I_A几乎存在于所有物种的各类神经元中，但在哺乳动物中靶向消除I_A颇具挑战：一方面I_A由多基因编码，另一方面干扰单个基因时常会引发代偿性电重构。 方法与主要结果 在果蝇（Drosophila）中，单个Shal/Kv4基因即可编码多数神经元胞体中的主要I_A电流。本研究通过转基因表达的显性负性亚基DNKv4，实现了神经元胞体中I_A的完全敲除，且未对其他离子电流产生影响。最为显著的是，DNKv4处理的神经元在持续性刺激下呈现多重放电缺陷：放电潜伏期缩短、重复放电阈值降低，且动作电位（Action Potential, AP）振幅会逐步衰减至适应状态。我们对已鉴定的运动神经元进行电生理记录，证实Shal/Kv4通道同样是维持重复放电过程中神经元兴奋性所必需的。随后我们检测了果蝇幼虫爬行、成虫攀爬与梳理行为——这些均依赖重复放电的节律性行为——结果显示，在Shal/Kv4功能缺失的个体中，所有行为均出现显著缺陷。进一步的特异性敲除实验证实，仅在运动神经元中敲除Shal/Kv4功能，即可显著干扰受试的运动行为。 结论与意义 本研究结果表明，Shal/Kv4通道可调控放电的起始过程，使神经元能够在持续性刺激下持续放电，并可影响放电频率。本研究证实，Shal/Kv4通道在持续性输入/输出过程中的重复放电神经元中发挥关键作用，提示其功能与调控机制对节律性行为至关重要。