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Glutamate‐induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP‐expressing mice
Author(s) -
Slemmer Jennifer E.,
Matsushita Shinichi,
De Zeeuw Chris I.,
Weber John T.,
Knöpfel Thomas
Publication year - 2004
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/j.0953-816x.2004.03422.x
Subject(s) - biophysics , ionotropic effect , glutamate receptor , niflumic acid , reversal potential , depolarization , chemistry , bumetanide , kainate receptor , yellow fluorescent protein , dids , membrane potential , channel blocker , nmda receptor , cnqx , biochemistry , patch clamp , ampa receptor , biology , receptor , ion transporter , membrane , organic chemistry , gene , calcium
Abstract The homeostasis of intracellular Cl − concentration ([Cl − ] i ) is critical for neuronal function, including γ‐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 µ m 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.