title: Transient Oxygen/Glucose Deprivation Causes a Delayed Loss of Mitochondria and Increases Spontaneous Calcium Signaling in Astrocytic Processes.
abstract: Recently, mitochondria have been localized to astrocytic processes where they shape Ca(2+) signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor (iGluR) antagonists, tetrodotoxin, ziconotide (Ca(2+) channel blocker), two inhibitors of reversed Na(+)/Ca(2+) exchange (KB-R7943, YM-244769), or two inhibitors of calcineurin (cyclosporin-A, FK506). The effects of OGD were mimicked by NMDA. The glutamate uptake inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]amino]phenyl]methoxy]-l-aspartate increased neuronal loss after OGD or NMDA, and blocked the loss of astrocytic mitochondria. Exogenous glutamate in the presence of iGluR antagonists caused a loss of mitochondria without a decrease in neurons in area CA1. Using the genetic Ca(2+) indicator Lck-GCaMP-6S, we observed two types of Ca(2+) signals: (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ∼2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca(2+) through reversed Na(+)/Ca(2+) exchange triggers mitochondrial loss and dramatic increases in Ca(2+) signaling in astrocytic processes.
  
  
  
  Astrocytes, the most abundant cell type in the brain, are vital integrators of signaling and metabolism. Each astrocyte consists of many long, thin branches, called processes, which ensheathe vasculature and thousands of synapses. Mitochondria occupy the majority of each process. This occupancy is decreased by ∼50% 24 h after an in vitro model of ischemia/reperfusion injury, due to delayed fragmentation and mitophagy. The mechanism appears to be independent of neuropathology, instead involving an extended period of high glutamate uptake into astrocytes. Our data suggest that mitochondria serve as spatial buffers, and possibly even as a source of calcium signals in astrocytic processes. Loss of mitochondria resulted in drastically altered calcium signaling that could disrupt neurovascular coupling and gliotransmission.
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: John C
authors.identifier: 0000-0001-8101-3783
authors.initials: JC
authors.lastname: O'Donnell
authors.affiliation: Children's Hospital of Philadelphia Research Institute.
authors.forename: Joshua G
authors.identifier: 0000-0003-4166-9213
authors.initials: JG
authors.lastname: Jackson
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Department of Pediatrics, and Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Robinson@mail.med.upenn.edu.
authors.forename: Michael B
authors.identifier: 0000-0003-0162-2218
authors.initials: MB
authors.lastname: Robinson
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019922:GAP-43 Protein / Q000235:genetics / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000172:deficiency*; D006624:Hippocampus / Q000473:pathology*; D000860:Hypoxia / Q000473:pathology*; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000473:pathology*; D009924:Organ Culture Techniques; D051381:Rats; D055647:Rats, Transgenic; D026941:Sodium Channel Blockers / Q000494:pharmacology; D016559:Tacrolimus / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors
chemical_list: D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D019922:GAP-43 Protein; D005904:Glial Fibrillary Acidic Protein; D005947:Glucose; D008869:Microtubule-Associated Proteins; D026941:Sodium Channel Blockers; D016559:Tacrolimus; D013779:Tetrodotoxin; C526109:Anxa3 protein, rat
keywords: astrocytes; calcium; glutamate transport; mitochondria; mitophagy; oxygen/glucose deprivation