Ortolani Domiziana, Manot-Saillet Blandine, Orduz David, Ortiz Fernando C, Angulo Maria Cecilia
INSERM U894, Institute of Psychiatry and Neuroscience of Paris, Paris, France.
INSERM U1128, Paris, France.
Front Cell Neurosci. 2018 Dec 6;12:477. doi: 10.3389/fncel.2018.00477. eCollection 2018.
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 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. 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 , 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.
光遗传学和药物遗传学技术已有效地用于分析神经元活动在控制幼年和成年行为小鼠少突胶质细胞谱系细胞中的作用。由于在出生后早期（PN）发育过程中少突胶质细胞动态发生重要变化，这类研究在出生后早期也备受关注。然而，在幼年时实施神经元操纵很困难，因为在新生小鼠中高水平的特异性蛋白表达不太可靠。在此，我们描述了一种方案，可对清醒幼鼠的神经元进行光遗传学刺激，目的是研究在PN早期阶段神经元活动对少突胶质细胞动态的影响。由于GABA能中间神经元在皮质发育过程中通过突触与少突胶质前体细胞（OPC）接触并与这些祖细胞保持密切关系，我们利用这个神经元 - 少突胶质细胞相互作用的相关例子来实施原理验证性光遗传学方法。首先，我们测试了Nkx2.1 - Cre和小白蛋白（PV） - Cre品系，以驱动光敏离子通道通道视紫红质 - 2（ChR2）在不同发育阶段的中间神经元亚群中表达。通过使用膜片钳记录和对急性体感皮质切片中ChR2阳性中间神经元的光刺激，我们分析了这些神经元中ChR2的功能表达水平。我们发现，在出生后第10天（PN10）的PV - Cre小鼠中ChR2表达不足，并且该通道需要从胚胎阶段开始表达（如在Nkx2.1 - Cre品系中），以便在幼鼠中实现可靠的光激活。然后，我们实施了立体定位手术，将一根微型光纤放置在皮质表面，以便在PN10时对ChR2阳性中间神经元进行光刺激。在第V层记录场电位，以验证光刺激是否到达皮质深层。最后，我们通过传统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。增加中间神经元活动既不影响OPC的总密度也不影响其增殖分数，这补充了先前关于GABA能突触活动对OPC增殖缺乏影响的研究结果。这里描述的方法应该为未来研究PN脑成熟过程中早期细胞相互作用的作用提供一个框架。
