Illuminating glia in the intact brain

The project aimed at elucidating neuron-glial-vascular signaling pathways in the intact brain. In particular we focused on the starshaped glia, the astrocytes. We employed 2-photon imaging and genetically encoded sensors for intracellular Ca2+ and extracellular glutamate. The sensors were delivered by a viral approach and targeted expression was achieved by using cell-specific promoters to drive the expression. Vascular diameter changes and perfusion were revealed using an intravascular dye. In anesthetized mice subjected to cortical spreading depression (CSD) we revealed the sequence of the intracellular and extracellular ionic shifts, by combining 2-photon calcium and glutamate imaging with extracellular K+ measurements using ion-sensitive electrodes. Notably, it had for seven decades been an enigma whether the CSD wave was carried by extracellular glutamate, potassium or intracellular Ca2+ signals. Having demonstrated that the CSD wave was carried by extracellular potassium rather than glutamate and Ca2+ signals, we showed that the long-lasting vascular uncoupling known to occur following CSD was not due to Ca2+ elevations in perivascular astrocytic endfeet. However, Ca2+ signals in endfeet were associated with the initial CSD-induced vasoconstriction. Next, we showed that abnormal Ca2+ signals occurred in perivascular endfeet in a mouse model for temporal lobe epilepsy with hippocampal sclerosis. Signaling mechanisms were also studied in a more physiological setting. We used the hippocampal slice model to characterize the glial response to neuronal action potentials. When the Schaffer collaterals in the stratum radiatum were stimulated we detected a robust astrocytic Ca2+ respons. The activity-induced Ca2+ signals were seen in all cellular compartments, including the endfoot processes around blood vessel. However, the signals occurred first and were of highest amplitude in the perisynaptic processes. Using a pharmacological tools we demonstrated that the glial response was dependent on glutamate and ATP and relied on Ca2+ release from internal stores. In a yet unpublished follow-up study, we shoved that activity-dependent astrocytic Ca2+ signals in hippocampus depended on IP3R2-receptors. Furthermore, we showed that the latency of these signals was shortened when using tetanic stimuli, probably due to the elevated extracellular glutamate and potassium. Finally, we showed by using the extracellular glutamate sensor, that activity-dependent glial Ca2+ signals were not associated with detectable glutamate release. During the last year of the project we successfully established protocols for awake animal two-photon imaging and dual color imaging. We found that astrocytic Ca2+ signals are much more frequent in awake animals than during anesthesia. The Ca2+ signals in awake mice are either localized, transient and asynchronous or synchronized, longer-lasting and wide-spread, the latter typically elicited by startle or running. We have also confirmed that the CSD-associated ionic shifts in awake mice mimics those in anesthetized mice. An important difference is faster CSD wave propagation in awake mice. We also provided evidence that glial cells release glutamate in CSD, likely due to swelling and/or Ca2+ signaling. In conclusion, our project has provided new insight into the intricate mechanisms underlying neuron-glia-vascular signaling. Our data may pawe the way for new treatment strategies for epilepsy, migraine and other neurological disorders with pathological signaling.

A number of neurological diseases for which current therapies are nonexisting or inadequate involve deficiencies in glial cell function. Examples are Alzheimer's disease and stroke. The Nobel-recognized discovery of aquaporins - the channels that mediate rapid and regulated transport of water across plasma membranes - opened up a new field in molecular medicine. The applicant pioneered the research on aquaporins in the central nervous system and disclosed that aquaporin-4 (AQP4), the predominant brain aqu aporin, is concentrated in specialized glial membranes at the brain-blood interface. Despite a decade of intense research the roles of glial water channels are an enigma. The project utilizes emerging technologies, two-photon microscopy and optogenetics, to unravel novel roles of AQP4 and perivascular glia. The concepts launched are that perivascular glial membranes serve as a diffusion barrier for solutes entering and leaving the brain, and that Ca2+ signals in glial microdomains govern the activity of c ontractile cells in blood vessels and thus cerebral perfusion. Light-induced manipulation of select brain cell types will be utilized to crack the code of neuronal-glial-vascular communication. The project will take advantage of optimized genetically enco ded Ca2+ indicators delivered into the brain of living animals by viruses. The project is multidisciplinary and draws on the expertise of a national and international competence network including the EMBL Partnership for Molecular Medicine, Centre for Mol ecular Biology and Neuroscience, one group at Max-Planck Institute for Medical Research, Heidelberg, Germany, and two US groups.