OPTICAL IMAGING OF HIPPOCAMPAL-NEURONS WITH A CHLORIDE-SENSITIVE DYE - EARLY EFFECTS OF IN-VITRO ISCHEMIA

We determined if changes in intraneuronal Cl- occur early after ischem ia in the hippocampal slice. Slices from juvenile rats (14-19 days old ) were loaded with the cell-permeant form of 6-methoxy-N-ethylquinolin ium chloride (MEQ), a Cl--sensitive fluorescent dye. Real-time changes in intracellular chloride concentration ([Cl-](i)) were measured with UV laser scanning confocal microscopy in multiple neurons within each slice. In vitro ischemia (26-28 degrees C, 10 min) was confirmed by t he loss of synaptic transmission (evoked field excitatory postsynaptic potentials) from pyramidal cells in area CAI. After ischemia and reox ygenation (10 min), MEQ fluorescence decreased significantly in CA1 py ramidal cells and interneurons. The decreased fluorescence corresponde d to an ischemia-induced increase in [Cl-](i) of similar to 10 mM. Pre treatment with the GABA(A)-gated Cl- channel antagonist picrotoxin (10 0 mu M) blocked the ischemia-induced change in [Cl-](i). Analysis of t he superfusates indicated that ischemia also caused a transient amino acid (GABA, glutamate, and aspartate) release that was maximal at simi lar to 10 min, returning to baseline shortly thereafter. Recovery from ischemia was confirmed by the return of synaptic transmission in area CAI, the return toward baseline of the ischemia-induced decrease in M EQ fluorescence, and exclusion of propidium iodide from MEQ fluorescen t cells. Furthermore, pyramidal cells did not undergo cell swelling du ring this early phase of reoxygenation, as indicated by the volume-sen sitive dye calcein. Thus, mild ischemia induces the accumulation of [C l-](i) secondary to GABA(A) receptor activation, in the absence of cel lular swelling or death. In contrast, depolarization of the slice with K+ (50 mM) decreased MEQ fluorescence significantly but caused cell s welling. Picrotoxin did not prevent the K+-induced increase in [Cl-](i ). II is possible that an increased [Cl-]i, following either an ischem ic event or an episode of depolarization, would reduce the C- driving force and thereby limit synaptic transmission by GABA. To support this hypothesis, ischemia caused a reduction in the ability of the GABA ag onist muscimol to increase [Cl-](i) after 20-min reoxygenation.