Timm’s staining in the hippocampus.

<p>Hippocampal slices were prepared from Wistar rats (A) and NER (B) (n = 10). The lower panel show the density of Timm’ stain, which was measured by using Multi Gauge V3.1, in the stratum lucidum where mossy fiber terminals exist. As a representative sample (circle) in Fig. 1A, five regions of interest per slice were set in the stratum lucidum and the densities measured were averaged. Each bar and line (mean ± SEM) represents the rate (%) of the density of Timm’ stain of Wistar rats to that of NER, which was represented as 100%.</p

Enhanced exocytosis in mossy fibers after CQ injection.

<p>Hippocampal slices (400- µm thickness) were prepared 2 h after i.p. injection of CQ (30 mg/kg). To measure the decrease in FM4-64 fluorescence intensity in mossy fiber terminals (boutons), mossy fiber terminals were doubly stained with ZnAF-2 and FM4-64. ZnAF-2 staining was done under an optimal condition to stain mossy fiber terminals clearly, unlike the ZnAF-2 staining condition of Fig. 4. The terminals strongly stained with ZnAF-2 were determined as region of interest. To observe presynaptic activity, tetanic stimulation at 10 Hz for 30 s, which was shown by a shaded bar, was delivered to mossy fibers and then single strong stimulation at 100 Hz for 18 s was delivered to the same position. The activity-dependent component of FM4-64 signal was measured for each punctum (1 s) by subtracting its residual fluorescence intensity measured by the strong electrical stimulation. The data (mean ± SEM) represent the ratio (%) of each FM4-64 intensity to the basal FM4-64 intensity before tetanic stimulation at 10 Hz for 30 s, which was averaged and expressed as 100% (vehicle, n = 10; CQ, n = 12). SL, stratum lucidum; PCL, CA3 pyramidal cell layer. *, p<0.05, vs. vehicle.</p

Suppression of CQ-induced seizures in NER by enhancing GABAergic activity.

<p>AOAA (5, 10, 20 mg/kg) or phenobarbital (PHB, 20 mg/kg) was i.p. co-injected with CQ (30 mg/kg) into NER, and then the behavior was observed for 6 h in home cage. The incidence represents the rate of seized rats, which exhibited tonic-clonic convulsion, to the total rats (vehicle, n = 22; CQ 30 mg/kg, n = 38; CQ+AOAA (5 mg/kg), n = 10; CQ+AOAA (10 mg/kg), n = 10; CQ+AOAA (20 mg/kg), n = 20; CQ+PHB (20 mg/kg), n = 18). Each bar and line represents the mean ± SEM (A). ***, p<0.001, vs. vehicle; ^#, p<0.05, ^##, p<0.01, ^###, p<0.001, vs. CQ. To check the effect of AOAA on locomotor activity, NER were subjected to the open-field test 2 h after i.p. injection of AOAA (n = 10). Each bar and line represents the mean ± SEM (B). *, p<0.05, vs. vehicle.</p

Decrease in extracellular Zn²⁺ in the hippocampus by zinc chelators.

<p>Hippocampal slices were prepared 2 h after i.p. injection of vehicle (A, n = 7) or CQ (30 mg/kg, B, n = 5) into NER, and 3 h after i.p. injection of vehicle (C, n = 7) or TPEN (1 mg/kg, D, n = 8) into NER. The hippocampal slices were imaged with ZnAF-2 (left side). To evaluate the effects of CQ (the left image of A and B, vehicle; the right image of A and B, CQ) and TPEN (he left image of C and D, vehicle; the right image of C and D, TPEN), CQ-injected (A and B) and TPEN-injected (C and D) groups were separately measured under the exactly same condition. The measurement condition was slightly different between CQ and TPEN groups for the optimal imaging of the decrease in Zn²⁺ by fluorescence intensity. Region of interest was set the area surrounding the abbreviated letters, i.e., ML and SL, where zincergic (glutamatergic) synapses exist. ML, the molecular layer of the dentate gyrus (A and C). SL, the stratum lucidum of the CA3 (B and D). The data (mean ± SEM) represent the rate of ZnAF-2 intensity after zinc chelator injection to that after vehicle injection, which was expressed as 100% (right side).</p

Ictal electroencephalogram recorded in CQ-injected NER.

<p>A recording electrode was implanted into the hippocamal CA3 region of NER. Two days later, vehicle (A) or clioquinol (30 mg/kg) in vehicle (B) was i.p. injected into NER and then the behavior was observed in the home cage until seizures were observed. Note that no seizure was observed in vehicle-treated NER. The basal activity before seizures (1) and the stages of seizures were observed as follows: beginning of aberrant activity on electroencephalogram without behavioral changes (2); asymmetric wave with myoclonus (3); high-voltage wave with tonic-clonic convulsion (4); low-voltage wave with postictal flaccidness (5). The component of γ-wave (7–9 Hz) (2, 6.9%; 3, 19.4%; 4, 15.4%; 5, 10.0%) became rhythmic during seizures as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071372#pone.0071372-Noda1" target="_blank">[26]</a>.</p

Zn-CQ injection attenuates in vivo CA1 LTP, but not DG LTP.

<p>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.</p

Transient increase in intracellular Zn²⁺ in the CA1 by delivery with Zn-CQ.

<p>(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).</p

Transient attenuation of CA1 LTP and object recognition memory deficit.

<p>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.</p

Transient object recognition memory deficit and increase in intracellular Zn²⁺ after CA1 excitation.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028615#s4" target="_blank">method</a> 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.</p

Increase in intracellular zinc after application of Zn-CQ and ZnCl₂ to the hippocampus.

<p>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).</p