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

• Applicant: Professor Dr. Christian Henneberger
• Subject Area: Molecular Biology and Physiology of Neurons and Glial Cells
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 464485552

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.

• DFG Programme: Research Grants
• Major Instrumentation: FLIM upgrade of in vivo setup
• Instrumentation Group: 5080 Optisches Mikroskopzubehör