Animals and chemicals
Male Wistar rats (10-15 weeks of age, Japan SLC, Hamamatsu, Japan) were housed under the standard laboratory conditions (23 ± 1°C, 55 ± 5% humidity) and had access to food and water freely. The present experiments were done according to the Guidelines for the Care and Use of Laboratory Animals of the University of Shizuoka, which refer to American Association for Laboratory Animals Science and the guidelines laid down by the NIH in the USA (NIH Guide for the Care and Use of Laboratory Animals). The ethics committee has approved all experimental protocols in the University of Shizuoka.
ZnAF-2DA (Sekisui Medical Co., LTD, Hachimantai, Japan), a membrane-permeable Zn2+ fluorescence probe, readily passes through the cell membrane and is hydrolyzed by esterase in the cytosol compartment followed by generation of ZnAF-2, which cannot permeate the cell membrane. ZnAF-2 is selectively bound to Zn2+, but not bound to other divalent cations such as Ca2+, Mg2+, Fe2+, and Cu2+. The probe was dissolved in dimethyl sulfoxide and then diluted to Ringer solution that consists of 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 1.0 mM NaH2PO4, 2.5 mM CaCl2, 26.2 mM NaHCO3, and 11 mM D-glucose (pH 7.3).
HYDROPTM, a membrane-permeable hydrogen peroxide (H2O2) fluorescence probe (Goryochemical, Sapporo, Japan) readily passes through the cell membrane and is hydrolyzed in the cytosolic compartment followed by generation of HYDROP-EXTM, which cannot permeate the cell membrane. HYDROP-EX is selectively bound to H2O2, but not bound to other ROS such as ・OH, O2-・, ClO-, 1O2, ・NO, and ONOO-. The probe was dissolved in N, N-dimethylformamide and then diluted to Ringer solution.
Surgical operation
The rats were anesthetized by intraperitoneal injection with chloral hydrate (400 mg/kg) and individually placed in a stereotaxic apparatus. The skull was exposed and a burr hole was drilled. An injection cannula (internal diameter, 0.15 mm; outer diameter, 0.35 mm) was carefully and slowly inserted into the right SNpc (5.3 mm posterior to the bregma, 2.0 mm lateral, 7.0 mm inferior to the dura) and the right striatum (0.48 mm anterior to the bregma, 4.1 mm lateral, 3.7 mm inferior to the dura) to prevent cellular damages. Thirty minutes after the surgical operation, 40 μM PQ, 40 μM PQ + 50 µM N-(p-amylcinnamoyl)anthranilic acid (ACA), a blocker of TRPM2 cation channels, 40 μM PQ + 10 mM 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors, or 2 µM GBR 13069 dihydrochloride (GBR), a dopamine reuptake inhibitor in saline were injected into the right SNpc via the cannula at the rate of 0.2 μl/min for 5 min. Ten minutes after injection, the injection cannula was carefully and slowly removed from the brain in approximately 10 min. In another experiment, 40 μM PQ in saline was injected into the right striatum in the same manner.
Tyrosine hydroxylase (TH) immunostaining
The rats were anesthetized with chloral hydrate 2 weeks after PQ injection and perfused with ice-cold 4% paraformaldehyde in PBS. The brain was removed from the rats followed by overnight fixation at 4°C in 4% paraformaldehyde in PBS. Fixed brains were cryopreserved in 30% sucrose in PBS for 2 day and frozen in Tissue-Tek Optimal Cutting Temperature embedding medium. Coronal brain slices (30 mm) were prepared at -20°C in a cryostat and picked up on slides followed by adhering at room temperature for 30 min. For TH immunostaining, the slides were incubated in blocking solution (3% BSA, 0.1% Triton X-100 in PBS) for 1 h and rinsed with PBS for 5 min followed by overnight incubating at 4°C with anti-tyrosine hydroxylase antibody (Abcam). The slides were rinsed with PBS for 5 min and incubated in blocking buffer containing Alexa Fluor 633 goat anti-rabbit secondary antibody (ThermoFisher) for 3 h at room temperature. The slides were rinsed with PBS for 5 min six times, mounted with Prolong Gold antifade reagent, and placed at 4°C for 24 h. Alexa Fluor 633 fluorescence was measured in the SNpc and the striatum using a confocal laser-scanning microscopic system.
In vivo imaging of intracellular H2O2
The rats anesthetized with chloral hydrate were treated in the same manner. Injection cannulae were carefully and slowly inserted into the both sides of the SNpc and the striatum to prevent cellular damages. Thirty minutes later, 40 μM PQ in saline containing 50 μM HYDROP was bilaterally injected into the SNpc and the striatum via cannulae at the rate of 0.2 μl/min for 5 min. Ten minutes later, the injection cannulae were slowly moved from the brain in approximately 3 min. The rats were decapitated. The brain was quickly excised from the rats and bathed in ice-cold choline-Ringer containing 124 mM choline chloride, 2.5 mM KCl, 2.5 mM MgCl2, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose (pH 7.3) to inhibit excessive neuronal excitation. Horizontal slices (400 μm) of the brains were prepared in an ice-cold choline-Ringer solution in a vibratome ZERO-1 (Dosaka Kyoto, Japan) and then bathed in an ice-cold choline-Ringer solution. The brain slices were transferred to a recording chamber filled with Ringer solution. Intracellular HYDROP-EX fluorescence was measured in the SNpc and the striatum with a confocal laser-scanning microscopic system. All solutions used in the experiments were continuously bubbled with 95% O2 and 5% CO2.
In vivo microdialysis
The rats anesthetized with chloral hydrate were treated in the same manner. A microdialysis probe (1-mm membrane, Eicom, Kyoto) was inserted into the right SNpc (5.3 mm posterior to the bregma, 2.0 mm lateral, 7.8 mm inferior to the dura) and the striatum (0.48 mm anterior to the bregma, 4.1 mm lateral, 4.2 mm inferior to the dura) of anesthetized rats. The SNpc and the striatum were preperfused with ACSF (127 mM NaCl, 2.5 mM KCl, 1.3 mM CaCl2, 0.9 mM MgCl2, 1.2 mM Na2HPO4, 21 mM NaHCO3 and 3.4 mM D-glucose, pH 7.3) at 2.0 µl/min for 180 min to stabilize the region, perfused with ACSF for 60 min in the same manner to determine the basal concentration of extracellular glutamate and then perfused with 40 μM PQ in ACSF, 40 μM PQ + 50 μM ACA in ACSF, or 40 μM PQ + 50 μM HYDROP in ACSF for 180 min.
The perfusate was collected for 15 min and analyzed for glutamate content by high-performance liquid chromatography (HPLC) [column, CAPCELL PAK C18 UG120A (1 mm x 150 mm) (Shiseido Co Ltd, Tokyo, Japan); mobile phase, 0.1 M potassium dihydrogen phosphate, 0.1 M di-sodium hydrogen phosphate, 10% acetonitrile, 0.5 mM EDTA-2Na, 3% tetrahydrofuran, pH 6.0] using the pre-column derivatization technique with o-phthaldialdehyde and a fluorescence detector (NANOSPACE SI-2, Shiseido Co Ltd). The basal levels and the levels during perfusion with PQ were averaged.
In vitro imaging of intracellular Zn2+
The brain was quickly excised from anesthetized rats and bathed in ice-cold choline-Ringer to inhibit excessive neuronal excitation. Horizontal brain slices (400 μm) were prepared in the same manner. The brain slices were immersed in 10 μM ZnAF-2DA in Ringer solution for 30 min, rinsed in choline-Ringer solution for 15 min, placed in a chamber filled with 40 μM PQ or 40 μM PQ + 50 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist in Ringer solution containing 10 nM ZnCl2 for 10 min, rinsed in choline-Ringer solution for 15 min, and transferred to a recording chamber filled with Ringer solution. Intracellular ZnAF-2 fluorescence (laser, 488.4 nm; emission, 500–550 nm) was observed in the SNpc and the striatum with a confocal laser-scanning microscopic system.
In vivo imaging of intracellular Zn2+
The rats were anesthetized with chloral hydrate and treated as described above. Injection cannulae were carefully and slowly inserted into the both sides of the SNpc to prevent cellular damages. Thirty minutes later, 40 μM PQ and 40 μM PQ + 10 mM NASPM in saline containing 100 μM ZnAF-2DA were bilaterally injected into the SNpc via cannulae at the rate of 0.2 μl/min for 5 min. Ten minutes later, the injection cannulas were slowly removed from the brain in about 3 min. The rats were decapitated 60 min after the start of the injection and the brain was quickly removed from the rats. Horizontal brain slices (400 μm) were prepared in the same manner and transferred to a recording chamber filled with Ringer solution. The fluorescence of intracellular ZnAF-2 was measured in the SNpc.
Data analysis
Student’s paired t-test was used for comparison of the means of paired data. For multiple comparisons, differences between treatments were assessed by one-way ANOVA followed by post hoc testing using the Tukey’s test (the statistical software, GraphPad Prism 5). A value of p < 0.05 was considered significant. Data were expressed as means ± standard error. The results of statistical analysis are described in each figure legend.