Kinoshita-Kawada Mariko, Hasegawa Hiroshi, Hongu Tsunaki, Yanagi Shigeru, Kanaho Yasunori, Masai Ichiro, Mishima Takayasu, Chen Xiaoping, Tsuboi Yoshio, Rao Yi, Yuasa-Kawada Junichi, Wu Jane Y
Department of Neurology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan.
Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan.
Bio Protoc. 2019 Sep 20;9(18):e3373. doi: 10.21769/BioProtoc.3373.
Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.
在与靶细胞形成突触的过程中，正在发育的轴突对导向线索的反应性会发生变化。轴突在腹侧中线处交叉，可作为研究轴突如何实现这种反应转换的模型。尽管原代神经元培养一直是阐明各种发育机制的通用技术，但神经元的许多特性，如长轴突延伸能力和轴突区室，并未得到充分保留。在植块培养中，分化神经元的这些特性和组织结构得以维持。为了研究中线排斥分子Slit如何在轴突穿过中线时调节脊髓连合轴突中Robo受体的分布，以及Robo转运机制是否是中线交叉的决定因素，我们开发了新的植块培养系统。我们将“翻开式”脊髓植块方法与用于视网膜平铺标本的方法相结合。在此，我们展示了胚胎小鼠脊髓植块培养的方案，该方案允许灵活操控实验条件、对延伸轴突进行免疫染色以及对单个轴突进行定量分析。此外，我们还展示了一种结合电穿孔和“闭合式”脊髓植块培养的改良方法。这些培养系统通过采用基因敲低、敲除和基因组编辑技术，为详细分析轴突导向提供了新的平台。
