Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice

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 µm 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 GABAA 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.