The experimental protocol was approved by the Fu Jen Institutional Animal Care and Utilization Committee (code A11009). The animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals. The minimum number of animals needed to obtain consistent data was employed.
HFP034 was synthesized by one of the authors (Pei-Wen Hsieh) . dl-threo-β-benzyloxyaspartate (dl-TBOA), bafilomycin A1, dantrolene, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157), and bisindolylmaleimide I (GF109203X) were purchased from Tocris (Bristol, UK). 3,3,3-dipropylthiadicarbocyanine iodide [DiSC3(5)] and fura-2-acetoxymethyl ester (Fura-2-AM) were purchased from Thermo (Waltham, USA). ω-conotoxin GVIA (ω-CgTX GVIA) and ω-agatoxin IVA (ω-Aga IVA) were purchased from the Alomone lab (Jerusalem, Israel). 4-aminopyridine (4-AP), dimethylsulfoxide (DMSO), kainic acid (KA) and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Adult male Sprague–Dawley rats (n = 42, 150-200 g) were purchased from BioLASCO (Taipei, Taiwan).
Rats (n = 18) were sacrificed via cervical dislocation and the cerebral cortex was removed immediately. The brain tissue was homogenized in 320 mM sucrose solution and centrifuged at 3000 g for 10 min. The supernatant was stratiﬁed on Percoll discontinuous gradients and centrifuged at 32,500 g for 7 min. The synaptosomal fraction was collected and centrifuged for 10 min at 27,000 g. The protein concentration was determined using the Bradford assay. Synaptosomes were centrifuged in a final wash to obtain synaptosomal pellets with 0.5 mg protein, as previously described [19-21].
Glutamate release analysis
For the glutamate release experiments, the synaptosomal pellet (0.5 mg protein) was resuspended in hepes-buffered solution, and glutamate release was assayed by online fluorimetry . CaCl2 (1.2 mM), glutamate dehydrogenase (GDH, 50 units/ml) and NADP+ (2 mM) were added at the start of the incubation. Glutamate release was induced with 4-AP (1 mM) and monitored by measuring the increase in fluorescence (excitation and emission wavelengths of 340 and 460 nm, respectively) resulting from NADPH being produced by oxidative deamination of released glutamate by GDH. The amount of released glutamate was calibrated against a standard of exogenous glutamate (5 nmol) and expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg).
Intrasynaptosomal Ca2+ concentration ([Ca2+]i)
Synaptosomes (0.5 mg protein) were incubated in hepes-buffered solution containing Fura 2-AM (5 μM) and CaCl2 (0.1 mM) for 30 min at 37 °C. Samples were centrifuged for 1 min at 3000 g, and pellets were resuspended in hepes-buffered medium containing CaCl2 (1.2 mM). Fura-2-Ca fluorescence was monitored at 5 s intervals for 5 min. [Ca2+]i (nM) was calculated using previously described calibration procedures and equations .
The synaptosomal membrane potential was assayed with the positively charged membrane potential-sensitive carbocyanine dye DiSC3(5). DiSC3(5) fluorescence was monitored at 2 s intervals, and the data are expressed in fluorescence units .
The rats (n = 24) were divided into four experimental groups: the DMSO-treated group (control), KA-treated group, HFP034 10 mg/kg + KA group, and HFP034 30 mg/kg + KA group. HFP034 was dissolved in a saline solution containing 1% DMSO and administered (i.p.) 30 min before KA injection (15 mg/kg in 0.9% NaCl, pH 7.0, i.p.). For Nissl staining, rats (n = 3 per group) were euthanized 72 h after KA injection by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) under inhalational anesthesia with 2–3% isofurane. The brains were removed, fixed overnight with 4% paraformaldehyde solution, and cryoprotected in sucrose phosphate buffer at 4 °C. The brains were cut into 30 µm coronal sections, mounted on gelatinized slides, air-dried and stained with 0.1% aqueous cresyl violet stain (Sigma Chemicals, St. Louis, MO, USA) for 20 min. Then, the slides were washed in distilled water, differentiated in 70% ethyl alcohol, dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and mounted with DPX (Sigma Chemicals, St. Louis, MO, USA). For immunofluorescence staining, the brain sections were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min and then incubated overnight at 4 °C with the primary antibodies anti-NeuN (1:500, Abcam, Cambridge, UK), anti-OX42 (1:500, Merck Millipore,Burlington, USA), and anti-GFAP (1:1000, Cell Signaling, MA, USA). The sections were incubated for 90 min at room temperature with the corresponding secondary antibodies (1:1000, Alexa Fluor 488, DyLight 594, Invitrogen, CA, USA), mounted on gelatin-coated slides and coverslipped with VectaShield medium (Vector Labs, Burlingame, CA). Cells were stained with the nuclear staining dye DAPI (1 µg/ml, Sigma–Aldrich) for 20 sec. Images were captured with an upright fluorescence microscope (Zeiss Axioskop 40, Goettingen, Germany) using ´ 4 (aperture is 0.1) and ×10 (aperture is 0.25) objectives. The numbers of living neurons and NeuN+, OX42+, and GFAP+ cells were counted in a 255 μm × 255 μm area of the hippocampal CA1 and CA3 using ImageJ software (Synoptics, Cambridge, UK).
High-performance liquid chromatography
Determination of glutamate concentrations in brain tissue was performed with a high-performance liquid chromatography (HPLC) system with electrochemical detection (HTEC-500). Briefly, frozen hippocampal tissue was prepared by homogenization in 5 ml of hepes-buffered medium. The homogenate was centrifuged at 1500 g at 4 °C for 10 min, and then the supernatant was filtered through 0.22 µm filters before injection into the HPLC system. The relative free glutamate concentration was determined using peak areas with an external standard method. Serial dilutions of the standards were injected, and their peak areas were determined. A linear standard curve was constructed by plotting the peak areas versus corresponding concentrations of each standard .
Synaptosomes and hippocampal tissue were lysed in ice-cold Tris–HCl buffer solution and centrifuged for 10 min at 13000 g at 4 °C. The protein concentration in the supernatant was measured using the Bradford protein assay (Bio–Rad laboratories, Hercules, CA, USA). Equal amounts (30 µg) of protein were loaded into each lane on a 10% polyacrylamide gel and then transferred to a polyvinylidenedifluoride (PVDF) membrane in a semidry system (Bio–Rad, Hercules, USA) for 120 min. Transferred membranes were blocked for 1 h in 5% nonfat dry milk in TBST (25 mM Tris-HCl, pH 7.5, 125 mM NaCl, and 0.05% Tween 20) and incubated overnight at 4 °C with specific primary antibodies [anti-protein kinase C (PKC), 1:700, Abcam, Cambridge, UK); phospho-PKC (1:2000, Cell Signaling, MA, USA); PKCa (1:600, Cell Signaling, MA, USA), phospho-PKCa (1:2000, Abcam, Cambridge, UK); phospho-MARCKS (1:250, Cell Signaling, MA, USA); calpain 1 (1:2000, Abcam, Cambridge, UK), calpain 2 (1:800, Millipore); caspase 12 (1:3000, Abcam, Cambridge, UK); C/EBP homologous protein (CHOP) (1:300, Santa Cruz,Texas, USA); glucose-regulated protein 78 (GRP 78) (1:1500, Abcam, Cambridge, UK); and β-actin (1:8000, Cell Signaling, MA, USA]. Membranes were washed with TBST for 15 min and incubated with horseradish peroxidase-coupled secondary antibodies (1:16000, GeneTex, CA, USA) for 1 h at room temperature. Then, the specific protein bands were visualized using film exposure with a chemiluminescence detection system (GeneTex, CA, USA) and quantified using ImageJ software (Synoptics, Cambridge, UK).
The results are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analysis was performed using GraphPad Prism-8 software (GraphPad Inc., San Diego, CA). When testing the significance of the effect of HFP034 versus the control, Student's t test was used. When comparing the effect of HFP034 in different experimental conditions, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. p < 0.05 was considered to indicate a statistically significant difference between groups.