title: Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses.
abstract: Intracellular Ca(2+) transients are considered a primary signal by which astrocytes interact with neurons and blood vessels. With existing commonly used methods, Ca(2+) has been studied only within astrocyte somata and thick branches, leaving the distal fine branchlets and endfeet that are most proximate to neuronal synapses and blood vessels largely unexplored. Here, using cytosolic and membrane-tethered forms of genetically encoded Ca(2+) indicators (GECIs; cyto-GCaMP3 and Lck-GCaMP3), we report well-characterized approaches that overcome these limitations. We used in vivo microinjections of adeno-associated viruses to express GECIs in astrocytes and studied Ca(2+) signals in acute hippocampal slices in vitro from adult mice (aged ∼P80) two weeks after infection. Our data reveal a sparkling panorama of unexpectedly numerous, frequent, equivalently scaled, and highly localized Ca(2+) microdomains within entire astrocyte territories in situ within acute hippocampal slices, consistent with the distribution of perisynaptic branchlets described using electron microscopy. Signals from endfeet were revealed with particular clarity. The tools and experimental approaches we describe in detail allow for the systematic study of Ca(2+) signals within entire astrocytes, including within fine perisynaptic branchlets and vessel-associated endfeet, permitting rigorous evaluation of how astrocytes contribute to brain function.
authors.affiliation: Department of Physiology and 2 Department of Neurobiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Eiji
authors.initials: E
authors.lastname: Shigetomi
authors.forename: Eric A
authors.initials: EA
authors.lastname: Bushong
authors.forename: Martin D
authors.initials: MD
authors.lastname: Haustein
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Tong
authors.forename: Olan
authors.initials: O
authors.lastname: Jackson-Weaver
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kracun
authors.forename: Ji
authors.initials: J
authors.lastname: Xu
authors.forename: Michael V
authors.initials: MV
authors.lastname: Sofroniew
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.forename: Baljit S
authors.initials: BS
authors.lastname: Khakh
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D003600:Cytosol / Q000378:metabolism / Q000502:physiology; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism; D005260:Female; D006624:Hippocampus / Q000378:metabolism / Q000502:physiology; D008297:Male; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections / Q000379:methods
chemical_list: D002118:Calcium