Watanabe Tadayoshi, Kiyomoto Takahiro, Tadokoro Ryosuke, Takase Yuta, Takahashi Yoshiko
Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
AMED Core Research for Evolutional Science and Technology (AMED-CREST), Japan Agency for Medical Research and Development (AMED), Chiyoda-ku, Tokyo, 100-0004, Japan.
Dev Growth Differ. 2017 Dec;59(9):677-687. doi: 10.1111/dgd.12406. Epub 2017 Oct 25.
The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.
自主神经系统由交感神经和副交感神经组成，它们在功能上相互拮抗，以控制器官的生理功能和内环境稳态。然而，自主神经系统在发育过程中是如何形成的，在很大程度上仍未得到探索。特别是，由于副交感神经网络复杂的解剖结构，其早期形成仍然不清楚。为了区分副交感神经（胆碱能）和交感神经（肾上腺素能）神经节，已知与乙酰胆碱合成相关的囊泡乙酰胆碱转运体（VAChT）和胆碱O-乙酰转移酶（ChAT）蛋白是有用的标记物。虽然针对这些蛋白的市售抗体广泛用于包括小鼠和大鼠在内的哺乳动物标本，但这些抗体在鸡中不能令人满意地发挥作用，尽管鸡是研究自主神经系统的优秀模型。在这里，我们新制备了针对鸡VAChT和ChAT蛋白的抗体。获得了一种针对VAChT的单克隆抗体和三种多克隆抗体，以及一种针对ChAT的多克隆抗体，可以用于蛋白质印迹分析和免疫组织化学。使用这些经过验证的抗体，我们在鸡E13胚胎消化道背侧形成的自主神经系统的Remak神经节中检测到了胆碱能细胞。本研究中获得的抗体可用于可视化包括副交感神经节在内的胆碱能神经元。
