(A) Hippocampal slices were prepared 2 (n = 7), 6 (n = 12) and 24 h (n = 7) after i.p. injection of Zn-CQ (9.8 µmol/kg) in vehicle and stained with ZnAF-2DA. Hippocampal slices prepared 2 h after vehicle injection were used as the control (n = 14). Each right panel shows the magnified CA1 area that is shown by a representative red square. Bars; 50 µm. PLC, pyramidal cell layer; SR, stratum radiatum. (B). Each bar and line (mean±SEM) represents the rate (%) of the intensity of ZnAF-2 fluorescence after Zn-CQ injection to that after vehicle injection, which was represented as 100%. *, p<0.05, **, p<0.01, vs. control (vehicle).

To deliver high-frequency stimulation (HFS, 10 trains of 20 pulses at 200 Hz separated by 1 s) 2 h after i.p. injection of vehicle or Zn-CQ (9.8 µmol/kg) in vehicle, test stimuli (0.05 Hz) were delivered to the Schaffer collateral/commissural pathway or the perforant pathway of the anesthetized rats and population spike (PS) amplitudes were recorded in the CA1 (A and B) and dentate gyrus (C and D) (n = 5–6). HFS was delivered at time 0 min. Each point and line (the mean ± SEM) shows the mean of 120 s (6 points). Each bar and line (mean ± SEM) represents the averaged PS amplitude of the last 10 min (time 50–60 min). *, p<0.05, vs. control. Representative PS recordings at time −10 and 50 min are shown in the upper side.

Hippocampal slices stained with ZnAF-2DA were immersed in ACSF (control), Zn-CQ (4.4 µM) in ACSF, and ZnCl₂ (4.4 µM) in ACSF for 15 min (n = 5). ZnAF-2 fluorescence was measured in the CA1 (A and C) and the dentate gyrus (B and D). Each lower panel shows the magnified area that is shown by a representative red square. Bars; 50 µm. PLC, pyramidal cell layer; SR, stratum radiatum. GCL, granule cell layer. (C and D). Each bar and line (mean ± SEM) represents the rate (%) of the intensity of ZnAF-2 fluorescence in hippocampal slices immersed with Zn-CQ or ZnCl₂ to that immersed with ACSF, which was represented as 100%. *, p<0.05, vs. control (ACSF).

Hippocampal slices were prepared from the rats 2 (n = 6), 6 (n = 7) and 24 h (n = 7) after i.p. injection of Zn-CQ (9.8 µmol/kg) in vehicle. Hippocampal slices prepared 2 h after vehicle injection were used as the control (n = 15). (A) Hippocampal slices were perfused with ACSF for 60 min, tetanized at 100 Hz for 1 s, and perfused with ACSF for 60 min. Tetanic stimulation was delivered at time 0 min. Each point and line represents the mean ± SEM. (B) Each bar and line (mean ± SEM) represents the averaged fEPSP amplitude of the last 15 min (time 45–60 min). Representative fEPSP recordings at time −20 and 50 min are shown as insets (upper side). Calibration; 0.5 mV, 10 ms. **, p<0.01 vs. control. (C) Rats were placed for 10 min into an open field (n = 6). Twenty-four hours after open field exploration, rats were i.p. injected with vehicle (control) or Zn-CQ (9.8 µmol/kg) in vehicle. Two hours after injection, rats were subjected to a novel object recognition task. Each bar and line represent the mean ± SEM. ^#, p<0.05, vs. training; ***, p<0.001, vs. control.

Rats were placed for 10 min into an open field. Twenty-four hours after the open field exploration, vehicle (control), Zn-CQ (9.8 nmol/rat) in vehicle, and ZnCl₂ (9.8 nmol/rat) in vehicle were bilaterally injected via injection cannulae into the hippocampal CA1 or the dentate gyrus of the rats at the rate of 0.5 µl/min for 1 min (n = 8–9). Thirty minutes after injection, rats were trained for 5 min and then tested for 3 min in the novel object recognition task 1 h after training. In the case of ZnCl₂ injection into the CA1, 24 h after injection, rats were subjected to the test in the same manner. (A) The dots in the brain map show the representative points injected in the CA1 (orange) and the dentate gyrus (green). (B) Each bar and line represent the mean ± SEM. ^#, p<0.05, ^##, p<0.01, ^###, p<0.001, vs. training; *, p<0.05, control vs. Zn-CQ and ZnCl₂ 30 min vs. ZnCl₂ 24 h.

(A) The hippocampus was perfused with ACSF for 60 min to determine the basal concentration of extracellular zinc, and perfused with 100 mM KCl in ACSF for 40 min to determine the change in the extracellular zinc concentration by neuronal depolarization. The perfusate was collected every 20 min. The control represents the mean of 3 samples before perfusion with 100 mM KCl. The 100 mM KCl represents the mean of 2 samples during perfusion with 100 mM KCl. Each bar and line represents the mean ± SEM (n = 6). *, p<0.05, vs. control. (B) One week after implantation of guide cannulae as described in the method section, rats were placed for 10 min into an open field. Twenty-four hours after the open field exploration, vehicle (control, n = 9), 100 mM KCl in vehicle (n = 15), 100 mM KCl+1 mM CaEDTA in vehicle (n = 8), and 1 mM CaEDTA in vehicle (n = 7) were bilaterally injected via injection cannulae into the hippocampal CA1 of the rats at the rate of 0.5 µl/min for 2 min. Thirty minutes after injection, rats were trained for 5 min and then tested for 3 min in the novel object recognition task 1 h after training. In the case of 100 mM KCl injection into the CA1, 24 h after injection, rats were subjected to the test in the same manner. Each bar and line represents the mean ± SEM. ^#, p<0.05, ^##, p<0.01, vs. training; *, p<0.05, vs. control. (C) Hippocampal slices were prepared from rats, stained with ZnAF-2DA and immersed with ACSF (control,), 100 mM KCl and 100 mM KCl containing 1 mM CaEDTA for 15 min (n = 8). PLC, pyramidal cell layer. (D). Each bar and line (mean ± SEM) represents the rate (%) of the intensity of ZnAF-2 fluorescence in hippocampal slices immersed with 100 mM KCl or 100 mM KCl containing 1 mM CaEDTA to that immersed with ACSF, which was represented as 100%. **, p<0.01, vs. control; ^###, p<0.001, vs. KCl.