New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.
New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.
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DOI：
10.1016/j.neuron.2016.11.030
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发表时间：
2016-12-21
期刊：
影响因子：
16.2
通讯作者：
Khakh BS
中科院分区：
文献类型：
--
作者：
Srinivasan R;Lu TY;Chai H;Xu J;Huang BS;Golshani P;Coppola G;Khakh BS
星形胶质细胞存在于整个神经系统中，并被认为影响神经回路和行为。然而，研究星形胶质细胞已被证明是困难的，因为缺乏允许星形胶质细胞选择性遗传操作的工具。在这里，我们报告的代Aldh 1 l1-Cre/ERT 2转基因小鼠选择性地在体内靶向星形胶质细胞。我们使用成像，免疫组织化学，AAV-FLEX-GFP显微注射和与RiboTag，Ai 95和我们也产生的新的Cre依赖性膜栓系Lck-GCaMP 6 f敲入小鼠杂交来表征Aldh 1 l1-Cre/ERT 2小鼠。在他莫昔芬诱导后两到三周，Aldh 1 l1-Cre/ERT 2选择性地靶向基本上所有的成年（P80）脑星形胶质细胞，没有可检测到的神经元污染，导致胞质和Lck-GCaMP 6 f的表达，并允许在体内惊吓反应期间进行亚细胞星形胶质细胞钙成像。与RiboTag小鼠的杂交允许对活跃翻译的mRNA进行测序并确定成年皮质星形胶质细胞转录组。因此，我们提供了良好的特点，易于使用的资源，选择性地研究星形胶质细胞在原位和体内在多个实验方案。Khakh实验室开发并表征了新的光学和遗传试剂，以探索神经回路中的星形胶质细胞功能。该方法允许在体内诱导星形胶质细胞的遗传靶向和星形胶质细胞过程中的钙信号的选择性监测。
Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterised Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections and crosses to RiboTag, Ai95 and new Cre-dependent membrane tethered Lck-GCaMP6f knock-in mice that we also generated. Two-to-three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well characterised, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios. The Khakh laboratory developed and characterized new optical and genetic reagents to explore astrocyte functions in neural circuits. The approaches permit inducible genetic targeting of astrocytes in vivo and the selective monitoring of calcium signals in astrocyte processes.