(a) Representative heat maps of glutamate biosensor FRET change (ΔFRET) for regions of interest adjacent to stimulating electrodes in the Schaffer collaterals overlayed on a bright-field image. Baseline signal (top panel) was obtained during an initial period of 0.2Hz stimulation at both electrodes. After the left electrode was subjected to iHFS protocol (bottom panel) there was a marked decrease in the signal, while there was little change in the signal at control electrode stimulated continuously at 0.2 Hz (right electrode). Scale bars=200μm. (b) Representative time course of evoked FRET signal change by iHFS stimulated electrode in aCSF (upper) and in glutamine (lower) at baseline (black) and after iHFS (red); scale bars=100ms, 0.005 ΔFRET. Summary of the reduction in evoked peak ΔFRET 5min after iHFS with and without glutamine (n=4 aCSF, n=7 glutamine, 1-way ANOVA, **p=0.002). There were no significant differences at the control electrode stimulated with continuous 0.2 Hz in presence and absence of glutamine (data not shown). (c–e) Effect of low-affinity, fast equilibrating AMPA receptor antagonist γ-DGG. Example traces and corresponding paired sample data (analyzed by student's t-test) showing the effect of 1 mM γ-DGG applied for 6 min (indicated as black bars in traces) before and after iHFS (left traces) compared to control electrode (right traces) in the same slice in (c) aCSF, (d) MSO pretreated slices, and (e) MSO pretreated slices to which glutamine was added. The bottom number represents the evoked amplitude 5min after addition of γ-DGG, and the top number represents the evoked amplitude in the absence of γ-DGG, derived from the evoked amplitudes immediately before and after γ-DGG wash-in. R is the ratio of the amplitude of γ-DGG effect to the baseline amplitude (equal to the bottom number divided by the top number). The change in effect of γ-DGG was calculated by determining the change in ratio of amplitude reduction caused by γ-DGG before and after iHFS.