Duration-dependent and bidirectional effect of transient chemical ischemia on synaptic transmission.

(A) Schematic of experimental design. Extracellular field potentials (Field) were recorded in the CA1 region in response to Schaffer collateral stimulation (Stim; paired pulses, 50 ms interstimulus interval, every 20 s). Arrow indicates the time point of application of a modified ACSF, inducing acute chemical ischemia, with 0 mM glucose, 2 mM deoxyglucose (2-DG), 5 mM sodium azide (NaN3) for 2-5 min. (B-C) Example traces for 2 min chemical ischemia (B) and 5 min chemical ischemia (C) (black, baseline; red, during stress; blue, end of the experiment at 60 min). (D) Relative change of the axonal fiber volley amplitude compared to baseline (0-10 min) for 2 min of chemical ischemia (green) and 5 min of chemical ischemia (orange). Arrow indicates time point of application of the modified ACSF for chemical ischemia induction. (E) Same as in (D) but for the fEPSP slope. (F) Example traces of extracellular [K+] recordings during for 2 and 5 min of chemical ischemia. (G) Quantification of the relative change of the axonal fiber volley amplitude in the last 10 min of recordings (50-60 min) relative to baseline for 2, 3, 4 and 5 min (n = 9, 8, 6, and 7, respectively) of chemical ischemia. (H) Same experiments as in (G) but analysis of the fEPSP slope. Graphs in H and G include results from recording shown in B-E. Persistent failure of synaptic transmission highlighted in orange. Filled circles correspond to recordings, in which astrocytes expressed iGluSnFR (see Fig. 2 and text). (I) Change of fEPSP in the last 10 min of recordings (50-60 min) relative to the baseline plotted against maximum [K+] increase during chemical ischemia. Data are expressed as mean ± s.e.m.

Bidirectional dysregulation of glutamate release by transient chemical ischemia.

(A) Schematic of experimental design. Combined recording of extracellular field potentials (Field) in the CA1 region in response to Schaffer collateral stimulation (Stim; paired pulses, interstimulus interval of 50 ms, every 20 s) and two-photon excitation fluorescence line scan imaging (910 nm, 6x every 5 min) of the glutamate indicator iGluSnFR. (B) Top: example of virally induced GFAP-iGluSnFR expression by astrocytes in the stratum radiatum of the CA1 region. Bottom: single iGluSnFR-expressing astrocyte and representative location of line scan (red line). (C-E) Left, field excitatory postsynaptic potential (fEPSP) slope, axonal fiber volley, iGluSnFR (iGlu) ΔF/F0 and iGlu resting fluorescence (F0) relative to baseline (0-10 min) for control (n = 8) (C), chemical ischemia (n = 11) (D) and chemical ischemia followed by postsynaptic failure of synaptic transmission (n = 6) (E). Arrow indicates start of chemical ischemia. Right, example traces of fEPSP (top) and iGluSnFR ΔF/F0 (bottom, average of 6 scans) at the beginning and the end of the recording. The electrophysiological results in D and E are a subset from Fig. 1. (F-H) Summary of parameters analyzed in C-E in the last 10 min of recording (60-70/80-90 min) compared to baseline (Bsl). (F) Control: fEPSP slope, p = 0.894; fiber volley, p = 0.012; iGlu ΔF/F0, p = 0.310; iGlu F0, p = 0.006; n = 8, paired Student’s t-test. (G) Chemical ischemia: fEPSP slope, p = 0.013; fiber volley, p = 0.003; iGlu ΔF/F0, p = 0.024; iGlu F0, p = 0.001; n = 11, paired Student’s t-test. (H) Chemical ischemia with postsynaptic failure: fEPSP slope, p < 0.0001; fiber volley, p = 0.134; iGlu ΔF/F0, p = 0.073; iGlu F0, p = 0.0004; n = 6, paired Student’s t-test. Data are expressed as mean ± s.e.m.

Glutamate clearance is not affected by transient chemical ischemia.

(A) Example traces of iGluSnFR ΔF/F0 in response to paired-pulse stimulation (interstimulus interval 50 ms) during baseline (black) and last 10 min (60-70/80-90 min) of control (left, gray), chemical ischemia (middle, green) and chemical ischemia with postsynaptic failure (right, orange) recordings. Black line indicates exponential fit for the analysis of iGluSnFR decay time. (B) Quantification of iGluSnFR fluorescence decay time of the 2nd pulse of the paired-pulse stimulation during the last 10 min of recording (60-70/80-90 min) compared to baseline (Bsl) for control, chemical ischemia, and chemical ischemia with postsynaptic failure. Control: p = 0.860, n = 9; chemical ischemia, p = 0.062, n = 12; chemical ischemia with postsynaptic failure, p = 0.648, n = 6; paired Student’s t-tests. (C) Schematic of experimental design. Combined recording of extracellular field potentials (Field) in the CA1 region in response to Schaffer collateral stimulation (Stim; paired pulse (50 ms), 0.05 Hz) and two-photon excitation fluorescence line scan imaging (910 nm) of iGluSnFR. After 70 min, D-AP5 (50 µM), NBQX (20 µM), LY341495 (50 µM) and MPEP (10 µM) were added to the recording solution and iGluSnFR fluorescence changes (ΔF/F0) were recorded in 4-5 cells in response to 10x 50 Hz and 10x 100 Hz stimulation before and after application of DL-TBOA (100 µM). (D) Example traces of iGluSnFR ΔF/F0 line scan recordings (average of 6 scans) in response to 10x 50 Hz stimulation (top row) after control (left), chemical ischemia (middle) and chemical ischemia with postsynaptic failure recordings (right). The same cells were tested again after block of glutamate transporters by DL-TBOA (bottom row, red traces together with ‘before’ traces from upper row on different time scale for comparison). (E) Quantification of iGluSnFR fluorescence decay time in response to 10x 100 Hz and 10x 50 Hz stimulation after control, chemical ischemia recording without and with postsynaptic failure. 100 Hz: p = 0.060, 50 Hz: p = 0.149; n = 25, 29, and 25 cells for control/chemical ischemia/chemical ischemia with postsynaptic failure from 5, 6, and 5 independent experiments, respectively; Kruskal-Wallis ANOVA. (F) Quantification of iGluSnFR fluorescence decay time in response to 10x 100 Hz stimulation in the presence and absence of DL-TBOA (same cells as in E). p < 0.0001 for control, chemical ischemia, and chemical ischemia with postsynaptic failure, paired sample Wilcoxon Signed Ranks tests. (G) As in F but for 10x 50 Hz stimulation. p < 0.0001 for control, chemical ischemia, and chemical ischemia with postsynaptic failure, paired sample Wilcoxon Signed Ranks tests. Data are expressed as mean ± s.e.m.

Chemical ischemia increases synaptic glutamate release.

(A) Schematic of experimental design. Extracellular field potentials (Field) were recorded in the CA1 region in response to Schaffer collateral stimulation (Stim; paired pulse (50 ms), 0.05 Hz). After 60 min, a whole-cell patch clamp recording from a CA1 pyramidal neuron was started. After recording baseline excitatory postsynaptic current (EPSC) responses for 15 min, γDGG (1 mM) was added to the extracellular solution. (B) Example traces of EPSCs (top) and fEPSPs (bottom) before (black) and after γDGG application for control (left, gray) and chemical ischemia (right, green) recordings. (C) Quantification of the remaining EPSC amplitude (left) and fEPSP slope (right) (in % of the baseline value) after γDGG application for control (n = 9) and chemical ischemia (n = 8) recordings. EPSC, p < 0.0001; fEPSP, p = 0.014; paired two-sample Student’s t-test. Data are expressed as mean ± s.e.m. (D) Example of axonal fiber volleys from experiments shown in Fig. 3C-E. Stimulus artefact removed for clarity. (E) Comparison of the fiber volley amplitude (left, p = 0.57), fiber volley half width at half maximum (middle, p = 0.026) and fiber volley delay (time between onset of the stimulus and fiber volley peak, right, p = 0.39). n = 5 (control) and 6 (chemical ischemia), unpaired Student’s t-tests.