Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators
Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators
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DOI：
10.3791/51972
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发表时间：
2014-11-01
影响因子：
1.2
通讯作者：
Khakh, Baljit S.
中科院分区：
文献类型：
--
作者：
Jiang, Ruotian;Haustein, Martin D.;Khakh, Baljit S.
星形胶质细胞显示自发的细胞内Ca 2+浓度波动（[Ca 2 +]（i）），并在几种情况下响应神经元兴奋增强[Ca 2 +]（i）信号。已经提出星形胶质细胞反过来通过钙依赖性机制调节神经元和血管，例如释放信号分子。然而，[Ca2+]（i）成像在整个星形胶质细胞最近才成为可行的遗传编码的钙指标（GECIs），如GCaMP系列。现在，在星形胶质细胞中使用GECIs提供了在模型微电路（例如纹状体，其是基底神经节的最大核）内详细研究星形胶质细胞[Ca 2 +]（i）信号的机会。在本报告中，详细的手术方法表达GECIs在星形胶质细胞在体内，共聚焦成像方法记录[Ca 2 +]（i）信号在纹状体星形胶质细胞原位，进行了描述。我们强调预防措施，必要的控制和测试，以确定GECI表达是否是选择性的星形胶质细胞和评估的迹象明显的星形胶质细胞反应。我们还详细描述了脑切片和成像条件，允许可靠的[Ca2+]（i）在纹状体星形胶质细胞原位成像。这些方法的使用揭示了单个纹状体星形胶质细胞的整个区域和它们的胞体、分支和小枝内的自发[Ca 2 +]（i）信号。这些方法在纹状体中的进一步使用和扩展将允许详细研究纹状体微电路中的星形胶质细胞[Ca 2 +]（i）信号。
Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+](i)) and in several settings respond to neuronal excitation with enhanced [Ca2+](i) signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+](i) imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+](i) signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+](i) signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+](i) imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+](i) signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+](i) signals in the striatal microcircuitry.