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 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.