In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups
In vivo Optogenetic Approach to Study Neuron-Oligodendroglia Interactions in Mouse Pups
复制标题
体内光遗传学方法研究小鼠幼崽神经元-少突神经胶质细胞的相互作用
DOI：
--
复制
发表时间：
2018
影响因子：
5.3
通讯作者：
M. C. Angulo
中科院分区：
文献类型：
--
作者：
Domiziana Ortolani;Blandine Manot;D. Orduz;F. Ortiz;M. C. Angulo
光遗传学和药物遗传学技术已有效地分析神经元活动的作用，在控制少突胶质细胞谱系细胞的行为少年和成年小鼠。这类研究也是高度关注的早期出生后（PN）的发展，因为重要的变化，少突胶质细胞的动力学发生在前两个PN周。然而，神经元操作难以在早期实施，因为高水平的特异性蛋白质表达在新生小鼠中不太可靠。在这里，我们描述了一个协议，允许在清醒的小鼠幼仔的神经元的光遗传学刺激的目的，调查在早期PN阶段的神经元活动对少突胶质细胞动力学的影响。由于GABA能中间神经元通过真正的突触接触少突胶质前体细胞（OPC），并在皮质发育期间与这些祖细胞保持密切关系，因此我们使用神经元-少突胶质细胞相互作用的这个相关实例来实施原理验证光遗传学方法。首先，我们测试了Nkx2.1-Cre和小白蛋白（PV）-Cre系，以驱动不同发育阶段的中间神经元亚群中光敏离子通道通道视紫红质-2（ChR 2）的表达。通过采用膜片钳记录和光刺激急性体感皮层切片中ChR 2阳性中间神经元，我们分析了ChR 2在这些神经元中的功能表达水平。我们发现PV-Cre小鼠在PN第10天（PN 10）ChR 2表达不足，并且该通道需要从胚胎阶段表达（如在Nkx2.1-Cre系中）以允许小鼠幼仔中可靠的光活化。然后，我们实施了立体定位手术，在皮质表面放置一根微型光纤，以在PN 10光刺激ChR 2阳性中间神经元。在层V中记录体内场电位以验证光刺激到达深层皮质层。最后，我们通过常规免疫染色分析了光刺激对第V层少突胶质细胞群体的影响。无论是总密度，也没有一个增殖部分的OPC的影响，增加体内的中间神经元活性，补充以前的研究结果表明缺乏GABA能突触活性OPC增殖的影响。这里描述的方法应该提供一个框架，为未来的调查PN脑成熟过程中的早期细胞相互作用的作用。
Optogenetic and pharmacogenetic techniques have been effective to analyze the role of neuronal activity in controlling oligodendroglia lineage cells in behaving juvenile and adult mice. This kind of studies is also of high interest during early postnatal (PN) development since important changes in oligodendroglia dynamics occur during the first two PN weeks. Yet, neuronal manipulation is difficult to implement at an early age because high-level, specific protein expression is less reliable in neonatal mice. Here, we describe a protocol allowing for an optogenetic stimulation of neurons in awake mouse pups with the purpose of investigating the effect of neuronal activity on oligodendroglia dynamics during early PN stages. Since GABAergic interneurons contact oligodendrocyte precursor cells (OPCs) through bona fide synapses and maintain a close relationship with these progenitors during cortical development, we used this relevant example of neuron-oligodendroglia interaction to implement a proof-of-principle optogenetic approach. First, we tested Nkx2.1-Cre and Parvalbumin (PV)-Cre lines to drive the expression of the photosensitive ion channel channelrhodopsin-2 (ChR2) in subpopulations of interneurons at different developmental stages. By using patch-clamp recordings and photostimulation of ChR2-positive interneurons in acute somatosensory cortical slices, we analyzed the level of functional expression of ChR2 in these neurons. We found that ChR2 expression was insufficient in PV-Cre mouse at PN day 10 (PN10) and that this channel needs to be expressed from embryonic stages (as in the Nkx2.1-Cre line) to allow for a reliable photoactivation in mouse pups. Then, we implemented a stereotaxic surgery to place a mini-optic fiber at the cortical surface in order to photostimulate ChR2-positive interneurons at PN10. In vivo field potentials were recorded in Layer V to verify that photostimulation reaches deep cortical layers. Finally, we analyzed the effect of the photostimulation on the layer V oligodendroglia population by conventional immunostainings. Neither the total density nor a proliferative fraction of OPCs were affected by increasing interneuron activity in vivo, complementing previous findings showing the lack of effect of GABAergic synaptic activity on OPC proliferation. The methodology described here should provide a framework for future investigation of the role of early cellular interactions during PN brain maturation.
登录
查看更多内容
影响因子：
3.4
作者：
Lin, John Y.;Lin, Michael Z.;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.
影响因子：
4.6
作者：
S. Anderson;O. Marín;Carrie Horn;Kelly Jennings;J. Rubenstein
通讯作者：
S. Anderson;O. Marín;Carrie Horn;Kelly Jennings;J. Rubenstein
影响因子：
25
作者：
Gunaydin, Lisa A.;Yizhar, Ofer;Hegemann, Peter
通讯作者：
Hegemann, Peter
影响因子：
64.8
作者：
Zhang, Feng;Wang, Li-Ping;Deisseroth, Karl
通讯作者：
Deisseroth, Karl