Heterogeneity of astrocytic resting [Ca2+] – underlying mechanisms and functional consequences

脑中的非神经元胶质细胞大多是电不可兴奋的。相反，它们通常将传入信号转化为胞质Ca 2+浓度的变化。星形胶质细胞是神经胶质细胞的一种亚型，是一个重要的例子。它们在亚细胞和网络水平上显示具有非常复杂的时空模式的Ca2+信号。重要的是，它是坚定地建立，星形胶质细胞的Ca2+信号可以控制多个下游功能。后者包括谷氨酸和ATP等信号分子的释放，这反过来又可以深刻地改变神经元的兴奋性，突触传递及其可塑性。我们已经开始探索如何使用定量荧光寿命和比率成像的局部亚细胞静息[Ca2+]控制星形胶质细胞的Ca2+信号。我们发现，静息[Ca2+]，即，稳定的局部[Ca 2 +]在没有瞬时[Ca 2 +]信号的情况下显示出实质性的亚细胞异质性。重要的是，局部静息[Ca2+]动态地控制了体外局部[Ca2+]瞬变的规模，在麻醉和清醒的小鼠中，以及在海马和新皮质中。因此，局部静息[Ca2+]是跨脑区和生理相关实验条件的动态[Ca2+]信号传导的关键调节因子。1)什么机制产生的亚细胞和细胞间异质性的静息[Ca2+]在体外和体内？2)这种异质性对于控制重要的信号分子如ATP、谷氨酸和GABA的功能后果是什么？我们已经扩大了我们的实验曲目，以解决这些问题。例如，我们已经建立了额外的技术定量钙荧光显微镜，安装了一个专用的设置荧光显微镜在体内，并成功地获得了第一个钙成像数据从星形胶质细胞在体内。此外，我们已经成功地测试了细胞外ATP和GABA的光学传感器，这增加了我们现有的可视化细胞外谷氨酸的专业知识。这个新的工具集使我们现在能够1）建立急性脑切片中静息[Ca 2 +]的亚细胞和细胞间异质性的来源，2）揭示体内控制静息[Ca 2 +]的机制，3）可视化静息[Ca 2 +]及其变化如何与重要信号分子的细胞外水平相关。计划中的研究将提供新的，定量的，以及对星形胶质细胞中静息Ca 2+水平和Ca 2+信号调节的重要见解。此外，我们将直接可视化和量化其对重要信号分子的细胞外水平的影响。

Non-neuronal glial cells in the brain are mostly electrically non-excitable. Instead, they often transform incoming signals into cytosolic Ca2+ concentration changes. Astrocytes, a subtype of glial cell, are an important example. They display Ca2+ signals with remarkably complex spatiotemporal patterns on the subcellular and the network level. Importantly, it is firmly established that astrocytic Ca2+ signals can control multiple downstream functions. The latter include the release of signalling molecules like glutamate and ATP, which can in turn profoundly alter neuronal excitability, synaptic transmission and its plasticity.We have recently started to explore astrocytic Ca2+ signalling quantitatively, because there is a lack of information about the underlying biophysical mechanisms. We have started by exploring how the local subcellular resting [Ca2+] controls Ca2+ signals of astrocytes using quantitative fluorescence lifetime and ratiometric imaging. We found that the resting [Ca2+], i.e., the stable local [Ca2+] in the absence of transient [Ca2+] signals displayed a substantial subcellular heterogeneity. Importantly, the local resting [Ca2+] dynamically controlled the scale of local [Ca2+] transients in vitro, in anesthetized and awake mice, and in the hippocampus and neocortex. Thus, the local resting [Ca2+] is a key regulator of dynamic [Ca2+] signalling across brain regions and physiologically relevant experimental conditions.We here propose to build on our findings by answering two important questions. 1) What mechanisms generate the subcellular and intercellular heterogeneity of the resting [Ca2+] in vitro and in vivo? 2) What are the functional consequences of this heterogeneity for the control of important signalling molecules like ATP, glutamate and GABA. We have expanded our experimental repertoire to work on these questions. For instance, we have established additional techniques for quantitative Ca2+ fluorescence microscopy, installed a dedicated setup for fluorescence microscopy in vivo and successfully acquired first Ca2+ imaging data from astrocytes in vivo. Also, we have successfully tested optical sensors for extracellular ATP and GABA, which adds to our existing expertise in visualizing extracellular glutamate. This new tool set enables us now to 1) establish the source of the subcellular and intercellular heterogeneity of resting [Ca2+] in acute brain slices, 2) uncover mechanisms controlling resting [Ca2+] in vivo, and 3) to visualizee how resting [Ca2+] and its changes are related to the extracellular levels of the important signalling molecules.The planned research will provide new, quantitative, and important insights into the regulation of resting Ca2+ levels and of Ca2+ signals in astrocytes. In addition, we will directly visualize and quantify its impact on the extracellular levels of important signalling molecules.