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

大脑中的非神经元神经胶质细胞大多是电不能兴奋的。相反，它们经常将传入的信号转换为细胞内钙离子浓度的变化。星形胶质细胞是神经胶质细胞的一个亚型，是一个重要的例子。它们在亚细胞和网络层面上显示具有非常复杂的时空模式的钙信号。重要的是，星形胶质细胞的钙信号可以控制多种下游功能，这一点已经得到证实。后者包括谷氨酸和三磷酸腺苷等信号分子的释放，这些分子可以深刻地改变神经元的兴奋性、突触传递及其可塑性。由于缺乏关于潜在生物物理机制的信息，我们最近开始对星形胶质细胞中的钙信号进行定量研究。我们从探索局部亚细胞静息[Ca~(2+)]如何利用定量荧光寿命和比率成像来控制星形胶质细胞的Ca~(2+)信号开始。我们发现，静息的[Ca~(2+)]，即在没有瞬时[Ca~(2+)]信号的情况下，稳定的局部[Ca~(2+)]表现出显著的亚细胞异质性。重要的是，在体外，在麻醉和清醒的小鼠，以及在海马区和大脑皮层，局部静息的[Ca~(2+)]动态地控制局部[Ca~(2+)]瞬变的规模。因此，局部静息[Ca~(2+)]是跨大脑区域和生理相关实验条件的动态[Ca~(2+)]信号的关键调节器。我们在这里建议通过回答两个重要问题来建立我们的发现。1)在体外和体内，是什么机制导致静息[Ca~(2+)]的亚细胞和细胞间的异质性？2)这种异质性对于控制重要的信号分子如ATP、谷氨酸和GABA有什么功能后果？我们已经扩展了我们的实验曲目，以解决这些问题。例如，我们已经建立了额外的钙离子定量荧光显微镜技术，安装了专门的体内荧光显微镜装置，并成功地从体内星形胶质细胞获得了第一个钙离子成像数据。此外，我们还成功地测试了细胞外ATP和GABA的光学传感器，这增加了我们在可视化细胞外谷氨酸方面的现有专业知识。这一新的工具使我们现在能够1)建立急性脑片静息[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，以及3)可视化静息[Ca~(2+)]及其变化与重要信号分子的细胞外水平的关系。计划中的研究将为静息[Ca~(2+)]水平和星形胶质细胞中的Ca~(2+)信号的调节提供新的、定量的和重要的见解。此外，我们将直接可视化和量化其对重要信号分子的胞外水平的影响。

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.