title: Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice.
abstract: The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.
authors.affiliation: Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, Riken, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Slemmer
authors.forename: Shinichi
authors.initials: S
authors.lastname: Matsushita
authors.forename: Chris I
authors.initials: CI
authors.lastname: De Zeeuw
authors.forename: John T
authors.initials: JT
authors.lastname: Weber
authors.forename: Thomas
authors.initials: T
authors.lastname: Knöpfel
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001578:Benzopyrans; D001640:Bicuculline / Q000494:pharmacology; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology* / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008734:Methoxyhydroxyphenylglycol / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic / Q000235:genetics; D016202:N-Methylaspartate / Q000494:pharmacology; D009284:Naphthols; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D012235:Rhodamines; D013997:Time Factors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D001426:Bacterial Proteins; D001578:Benzopyrans; D002712:Chlorides; D018690:Excitatory Amino Acid Agonists; D018756:GABA Antagonists; D008164:Luminescent Proteins; D009284:Naphthols; D009579:Nitrobenzoates; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; C054562:yellow fluorescent protein, Bacteria; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; D018698:Glutamic Acid; D008734:Methoxyhydroxyphenylglycol; D016202:N-Methylaspartate; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D005665:Furosemide; D011188:Potassium; C010117:3,4-dihydroxyphenylglycol; D001640:Bicuculline