Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.
Bulk loading of calcium indicator dyes to study astrocyte physiology: key limitations and improvements using morphological maps.
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DOI：
10.1523/jneurosci.0127-11.2011
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发表时间：
2011-06-22
期刊：
影响因子：
--
通讯作者：
Khakh BS
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文献类型：
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作者：
Reeves AM;Shigetomi E;Khakh BS
使用大量加载钙指示剂染料在星形胶质细胞细胞体中研究了钙信号传导，并且已知星形胶质细胞显示细胞内钙瞬变。最近有关体细胞星形胶质细胞钙瞬变对神经元影响的数据的一个假设是，大量负载反映了相关星形胶质细胞区室（例如过程）中的信号传导。我们使用装载了常见钙指示剂染料的星形胶质细胞的 Sholl 分析来评估批量加载，并将这些数据与星形胶质细胞形态的 Sholl 分析进行比较。在大鼠海马的 CA1 区域，我们发现大量加载的钙指示剂染料仅报告胞体内和最近端突起中的钙信号，而星形胶质细胞的约 90% 的区域及其广泛的突起未采样。通过在成像后采用形态学重建作为“地图”，我们提出了简单的程序来弥补这些缺陷，并允许可靠地检测远端星形胶质细胞过程中的钙瞬变。因此，这些数据揭示了解释通过批量加载收集的星形胶质细胞钙成像数据的局限性，并提供了改进以尽量减少这些缺点。
Calcium signalling has been studied in astrocyte cell bodies using bulk loading of calcium indicator dyes and astrocytes are known to display intracellular calcium transients. An assumption in recent data on the neuronal impact of somatic astrocyte calcium transients has been that bulk loading reflects signalling in relevant astrocyte compartments such as processes. We assessed bulk loading using Sholl analysis of astrocytes loaded with common calcium indicator dyes and compared these data to Sholl analysis of astrocyte morphology. In the CA1 region of the hippocampus from rats, we found that bulk loading of calcium indicator dyes only reports on calcium signals within the soma and in the most proximal processes, leaving ~90% of an astrocyte’s area and its extensive processes unsampled. By employing morphological reconstructions as “maps” after the imaging session, we present simple procedures that remedy these shortfalls and permit reliable detection of calcium transients in distal astrocyte processes. The data thus reveal limitations in the interpretation of astrocyte calcium imaging data gathered with bulk loading and provide refinements to minimize these shortcomings.