Köhidi Tímea, Jády Attila G, Markó Károly, Papp Noémi, Andrási Tibor, Környei Zsuzsanna, Madarász Emília
Laboratory of Cellular and Developmental Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.
Roska Tamás Doctoral School of Sciences and Technology, Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary.
Front Cell Neurosci. 2017 Dec 19;11:401. doi: 10.3389/fncel.2017.00401. eCollection 2017.
During neural tissue genesis, neural stem/progenitor cells are exposed to bioelectric stimuli well before synaptogenesis and neural circuit formation. Fluctuations in the electrochemical potential in the vicinity of developing cells influence the genesis, migration and maturation of neuronal precursors. The complexity of the environment and the coexistence of various progenitor populations hinder the understanding of the significance of ionic/bioelectric stimuli in the early phases of neuronal differentiation. Using optogenetic stimulation, we investigated the motility responses of radial glia-like neural stem/progenitor populations to ionic stimuli. Radial glia-like neural stem cells were isolated from transgenic mouse embryos. After transfection with Cre-recombinase, ChR2(channelrhodopsin-2)-expressing and non-expressing cells were separated by eYFP fluorescence. Expression of light-gated ion channels were checked by patch clamp and fluorescence intensity assays. Neurogenesis by ChR2-expressing and non-expressing cells was induced by withdrawal of EGF from the medium. Cells in different (stem cell, migrating progenitor and maturing precursor) stages of development were illuminated with laser light (λ = 488 nm; 1.3 mW/mm; 300 ms) in every 5 min for 12 h. The displacement of the cells was analyzed on images taken at the end of each light pulse. Results demonstrated that the migratory activity decreased with the advancement of neuronal differentiation regardless of stimulation. Light-sensitive cells, however, responded on a differentiation-dependent way. In non-differentiated ChR2-expressing stem cell populations, the motility did not change significantly in response to light-stimulation. The displacement activity of migrating progenitors was enhanced, while the motility of differentiating neuronal precursors was markedly reduced by illumination.
在神经组织发生过程中，神经干/祖细胞在突触形成和神经回路形成之前就已暴露于生物电刺激之下。发育中细胞附近的电化学电位波动会影响神经元前体的发生、迁移和成熟。环境的复杂性以及各种祖细胞群体的共存阻碍了我们对离子/生物电刺激在神经元分化早期阶段重要性的理解。我们利用光遗传学刺激，研究了放射状胶质样神经干/祖细胞群体对离子刺激的运动反应。从转基因小鼠胚胎中分离出放射状胶质样神经干细胞。用Cre重组酶转染后，通过eYFP荧光分离表达和不表达ChR2（通道视紫红质-2）的细胞。通过膜片钳和荧光强度测定法检查光门控离子通道的表达。通过从培养基中撤除表皮生长因子（EGF）来诱导表达和不表达ChR2的细胞进行神经发生。每隔5分钟用激光（λ = 488 nm；1.3 mW/mm；300 ms）照射处于不同发育阶段（干细胞、迁移祖细胞和成熟前体）的细胞，持续12小时。在每个光脉冲结束时拍摄的图像上分析细胞的位移。结果表明，无论是否受到刺激，随着神经元分化的推进，迁移活性都会降低。然而，光敏感细胞的反应具有分化依赖性。在未分化的表达ChR2的干细胞群体中，光刺激后运动性没有显著变化。迁移祖细胞的位移活性增强，而光照则使分化中的神经元前体的运动性明显降低。
