title: Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.
abstract: Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl-]intto be controlled by two opposing transport processes: chloride is actively accumulated by the Na+-K+-2Cl-cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl-]intto values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl-]intin adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl-]int. Other tested chloride channels or chloride transporters do not contribute to [Cl-]intunder our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K+-Cl-cotransporter change resting Bergmann glial [Cl-]intin acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.
authors.affiliation: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
authors.forename: Verena
authors.initials: V
authors.lastname: Untiet
authors.affiliation: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Kovermann
authors.affiliation: Institute of Neurobiology, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: Niklas J
authors.initials: NJ
authors.lastname: Gerkau
authors.affiliation: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Gensch
authors.affiliation: Institute of Neurobiology, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: Christine R
authors.initials: CR
authors.lastname: Rose
authors.affiliation: Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Fahlke
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002531:Cerebellum / Q000166:cytology; D002712:Chlorides / Q000378:metabolism*; D027341:Excitatory Amino Acid Transporter 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D007192:Indenes / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D009415:Nerve Net / Q000502:physiology; D009457:Neuroglia / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels / Q000378:metabolism
chemical_list: C553295:2-amino-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile; D000085:Acetates; D001578:Benzopyrans; D002712:Chlorides; D027341:Excitatory Amino Acid Transporter 1; D005904:Glial Fibrillary Acidic Protein; D007192:Indenes; C493393:Slc1a3 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D064506:Solute Carrier Family 12, Member 2; D050994:Voltage-Dependent Anion Channels; C120673:benzyloxyaspartate; D002034:Bumetanide; C095651:((2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy)acetic acid; D001224:Aspartic Acid
keywords: Bergmann glia; EAAT; fluorescence lifetime imaging; glial chloride switch