ISSN:
1460-9568
Quelle:
Blackwell Publishing Journal Backfiles 1879-2005
Thema:
Medizin
Notizen:
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.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1111/j.0953-816X.2004.03422.x
