The axon initial segment (AIS) is involved in action potential initiation. Structural and biophysical characteristics of the AIS differ among cell types and/or brain regions, but the underlying mechanisms remain elusive. Using immunofluorescence and electrophysiological methods, combined with super-resolution imaging, we show in the developing nucleus magnocellularis of the chicken in both sexes that the AIS is refined in a tonotopic region-dependent manner. This process of AIS refinement differs among cells tuned to different frequencies. At hearing onset, the AIS was ~50 µm long with few voltage-gated sodium channels regardless of tonotopic region. However, after hatching, the AIS matured and displayed an ~20-µm-long structure with a significant enrichment of sodium channels responsible for an increase in sodium current and a decrease in spike threshold. Moreover, the shortening was more pronounced, while the accumulation of channels was not, in neurons tuned to higher frequency, creating tonotopic differences in the AIS. We conclude that AIS shortening is mediated by disassembly of the cytoskeleton at the distal end of the AIS, despite intact periodicity of the submembranous cytoskeleton across the AIS. Importantly, deprivation of afferent input diminished the shortening in neurons tuned to a higher frequency to a larger extent in posthatch animals, with little effect on the accumulation of sodium channels. Thus, cytoskeletal reorganization and sodium channel enrichment at the AIS are differentially regulated depending on tonotopic region, but work synergistically to optimize neuronal output in the auditory nucleus.

SIGNIFICANCE STATEMENT The axon initial segment (AIS) plays fundamental roles in determining neuronal output. The AIS varies structurally and molecularly across tonotopic regions in avian cochlear nucleus. However, the mechanism underlying these variations remains unclear. The AIS is immature around hearing onset, but becomes shorter and accumulates more sodium channels during maturation, with a pronounced shortening and a moderate channel accumulation at higher tonotopic regions. Afferent input adjusts sodium conductance at the AIS by augmenting AIS shortening (via disassembly of cytoskeletons at its distal end) specifically at higher-frequency regions. However, this had little effect on channel accumulation. Thus, cytoskeletal structure and sodium channel accumulation at the AIS are regulated differentially but work synergistically to optimize the neuronal output.

轴突初始节（AIS）参与动作电位的启动。AIS的结构和生物物理特征在细胞类型和/或大脑区域之间有所不同，但是潜在的机制仍然难以捉摸。使用免疫荧光和电生理学方法，与超分辨率成像相结合，我们在发育中的两性鸡的巨细胞核中都表明，AIS以与toontopic区域相关的方式被提纯。在调谐到不同频率的小区之间，AIS改进的过程有所不同。听力发作时，AIS的长度约为50 µm，几乎没有电压门控性钠通道，而与tonotopic区域无关。但是，孵化后 AIS逐渐成熟并显示出约20 µm长的结构，其中钠通道显着富集，这导致钠电流增加和尖峰阈值降低。此外，在调谐到更高频率的神经元中，缩短更明显，而通道的积累却没有，从而在AIS中产生了色调差异。我们得出结论，尽管跨整个AIS的膜下细胞骨架完整无缺，但AIS缩短是由AIS远端细胞骨架的拆卸介导的。重要的是，剥夺传入输入减少了在孵化后动物中较大频率调谐的神经元的缩短，对钠通道的积累几乎没有影响。从而，

重要性声明轴突初始节（AIS）在确定神经元输出中起基本作用。AIS在禽耳蜗核的整个鼻息肉区域的结构和分子上都不同。但是，这些变化的潜在机制仍不清楚。AIS在听力发作时尚不成熟，但在成熟过程中会变短，并积累更多的钠通道，在较高的Tonotopic区域明显缩短并有中等程度的通道积聚。传入输入通过增强AIS缩短（通过在远端分解细胞骨架）来增强AIS处的钠电导，特别是在高频区域。但是，这对信道累积影响很小。因此，AIS的细胞骨架结构和钠通道积累受到不同的调节，但可以协同工作以优化神经元输出。