ACTIVATION OF EXCITATORY AMINO-ACID RECEPTORS IN THE RAT HIPPOCAMPAL SLICE INCREASES INTRACELLULAR CL- AND CELL-VOLUME

The effects of glutamatergic excitotoxins on intracellular Cl- were in vestigated in the CA1 pyramidal cell layer of the hippocampal slice. H ippocampal slices from rats (14-19 days old) were loaded with 6-methox y-N-ethylquinolinium chloride (MEQ), a Cl--sensitive fluorescent probe with a fluorescence intensity that correlates inversely with intracel lular [Cl-]. Slices were exposed for at least 10 min at 26-28 degrees C to N-methyl-D-aspartate (NMDA; 100 mu M) or lpha-amino-3-hydroxy-5-m ethylisoxazole-4-propionic acid (AMPA; 50 mu M). A UV laser scanning c onfocal microscope was used to measure changes in MEQ fluorescence wit hin area CA1 pyramidal cell soma. Both glutamate receptor agonists pro duced a rapid decrease in MEQ fluorescence that persisted after washou t following a 10-min exposure. The effects of NMDA and AMPA were preve nted by the competitive antagonists 2-amino-5-phosphonopentanoic acid and 6,7-dinitroquinoxaline-2,3-dione, respectively. Neither tetrodotox in nor picrotoxin prevented the effect of NMDA or AMPA, indicating the lack of involvement of presynaptic mechanisms. The effects of NMDA an d AMPA on MEQ fluorescence were dependent on the levels of extracellul ar Cl-, but only NMDA responses were dependent on the levels of extrac ellular Na+. Removal of Ca2+ from the superfusion medium did not alter the effects of NMDA or AMPA on MEQ fluorescence. In addition, neither the Ca2+ ionophore ionomycin nor the L-type voltage-gated Ca2+ channe l agonist (Bay K 8644) decreased MEQ fluorescence. The effects of NMDA and AMPA on cell(somal) volume were also assessed with the fluorescen t probe calcein acetoxymethyl eater. Both NMDA and AMPA decreased calc ein fluorescence (indicating an increased cell volume), but this was p receded by the decrease in MEQ fluorescence (equivalent to an intracel lular accumulation of similar to 20 mM Cl-). Thus, excitotoxins may ca use Cl- influx via an anion channel other than the GABA(A) receptor an d/or reduce Cl- efflux mechanisms to produce cell swelling. Such anion ic shifts may promote neuronal excitability and cell death following a n excitotoxic insult to the hippocampal slice.