2.1. Reagents for slice culture
Slice medium contained 50% MEM (Nacalai tesque, Kyoto, Japan, 21442-25, 72mM Glucose, 2mM HEPES are added) 25% HBSS (Thermo Fisher Scientific, 24020-117,MA, USA ), and 25% Horse serum (Thermo Fisher Scientific, 16050-122, heat-inactivated at 56°C for 30 min). Dissection solution was ice-cold Gey’s BSS (137 mM NaCl, 5 mM KCl, 0.18 mM KH2PO4, 0.84 mM Na2HPO4 12H2O, 36 mM glucose, 1.5 mM CaCl2, 1 mM MgCl2, 0.32 mM MgSO4) saturated with O2. Phosphate-buffered saline (PBS). 4% Paraformaldehyde in PB (Nacalai 09154-85). Primary antibody: anti-NeuN antibody (a marker protein for neuronal nuclei; mouse monoclonal, A60, Chemicon, CA, USA, anti-GFAP antibody (a marker protein for astrocyte; mouse monoclonal, SMI22, BioLegend, CA, USA),anit-Iba1 antibody (a marker protein for microglia; rabbit, 019-19741, Wako Pure Chemical Industries, Osaka, Japan). Secondary antibody: Goat Anti-mouse IgG (Alexa Fluor 488) A-11001, Goat Anti-mouse IgG (Alexa Fluor 594) A-11032, Goat Anti-rabbit IgG (Alexa Fluor 488) A-11034, Goat Anti- rabbit IgG (Alexa Fluor 594) A-11037 (Thermo Fisher Scientific, MA, USA). RIPA buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 3 mM KCl with a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany)]. 1× Tris-buffered saline (TBS: 50 mM Tris, 138 mM NaCl, 2.7 mM KCl). TBST (1× TBS with 0.1% Tween 20). Mouse/rat amyloid β(1–40) assay kit (IBL, 27720), Mouse/rat amyloid β(1–42) assay kit (IBL, 27721), Mouse/Rat sAPPα (highly sensitive) Assay Kit (IBL, 27419 ), Mouse sAPPβ-w Assay Kit (IBL, 27416).
2.2. Hippocampal slice culture method
The experiments in this study were approved by the committees on animal experiments of Juntendo University. Animals were purchased from Nihon SLC (Hamamatsu, Japan) and treated according to our institutional guidelines. Rat or mouse hippocampal slice cultures were prepared following protocol. Whole brains were excised from anaesthetized Sprague-Dawley rats or ICR mice at postnatal day 7–8 and the hippocampi were isolated. Slices of 400-µm thickness were obtained from the central region of the hippocampi, using a tissue chopper (McIllwain). The slices were placed on a polytetrafluoroethylene membrane filter (Millicell-CM, Millipore,), and culture medium (see below for composition) was added up to the bottom surface of the filter. These prepared cultures were maintained at 37˚C with 5% CO2-entiched humidified atmosphere. The slice culture medium (SCM) contained 50% minimal essential medium based on Hanks' salts (Nacalai), 25% Hank's balanced salt solution (Nacalai), and 25% heat-inactivated horse serum (Life Technologies, Gaithersburg, MD). The culture medium was replaced twice a week with fresh medium during the entire culture period.
2.3. Protocol
1. Wipe the tissue chopper, a new blade, stereomicroscope, tissue chopper, and dissecting instruments with 70% ethanol solution. Sterilize them for 30 minutes with UV light in the clean bench.
2. Dispense GBSS into sterilized 100 mL bottle and place it on ice-box. Bubble the GBSS with 100% O2 for 20-30min. 40-50mL GBSS is required per culture session (2–4 pups).
3. Prepare the tissue chopper placing on clean bench and mounting a sterilized blade, and placing autoclaved overhead projector (OHP) sheet, cut into 5 cm squares, on cutting chamber.
4. Prepare six well plates. Add 1 mL slice culture media (SCM) per well and place culture inserts in each well. Make sure the filter membranes are thoroughly wet with no bubbles underneath. Place the plates with filter membrane cups maintained at 37˚C with 5% CO2-entiched humidified atmosphere until needed.
5. The rodent pups are deeply anaesthetized and then killed by decapitation. Cut the scalp and expose the skull. Remove the skull by cutting along the sagittal suture and then from rostral to caudal side. Scoop out the brain quickly with a micro medicine spoon and place it in ice-cold oxygenated GBSS. Place the brain on autoclaved filter paper immersed in ice-cold oxygenated GBSS on 100mm dish. Use a scalpel to separate the hemispheres leaving out the midbrain.
6. Under the stereomicroscope; after the gently scoop the thalamus, the hippocampi are then exposed on each hemisphere in ice-cold oxygenated GBSS. Then gently scoop the hippocampus out with cortex using micro spoon (Fig. 1C).
7. Transfer the hippocampus to the OHP sheet on the tissue chopper chamber. Align the hippocampi perpendicular to the blade to obtain coronal sections and drain excess of GBSS (Fig. 1D).
8. Slice the hippocampi every 400 µm using tissue chopper.
9. Transfer sliced hippocampi from the OHP sheet to 60 mm dish filled with ice-cold oxygenated GBSS. Since tissue chopper and hippocampi are placed at room temperature, quick operation is required in steps 6 to 8.
10. Under the stereomicroscope (Olympus, SZX7); separate well defined and undamaged slices from damaged slices, and pinch the cortex with precision tweezers and divide the hippocampal slices one by one. And then split into cortex and hippocampus by scalpels (Fig. 1E). It is important to complete the steps 5 to 10 within 45 minutes.
11. Incubate separated hippocampi in new ice-cold oxygenated GBSS for 30–60 min.
12. Transfer individual slices on to the filter membrane in the six-well plate with SCM. Place 1–6 slices per membrane, not to place the slices either close to the insert wall or close to each other (Fig. 1F).
13. Move dish back to incubator and prepared slices maintained at 37˚C with 5% CO2-entiched humidified atmosphere.
14. The culture medium was replaced twice a week with fresh medium during the entire culture period. Aspirate the medium in dish, and add 750µL of fresh pre-warmed medium per well. Approximate 250µL of medium would be remained in the culture dish and the filter membrane.
2.4. Immunohistochemical staining
For the immunohistochemical staining of presynaptic structures, the cultured slices were fixed with 4% paraformaldehyde in phosphate-buffered saline for 1 h at 4˚C. The fixed preparations were rinsed with phosphate-buffered saline (PBS) and then treated with PBS containing 0.1% Triton X-100 and 5% fetal bovine or horse serum at 24˚C for 30 min. The treated slices were incubated with a primary antibody against NeuN (1:200 dilution, 24 h, 4˚C), GFAP (1:200 dilution, 24 h, 4˚C), and Iba1 (1:100 dilution, 24 h). The primary antibody treated slices were incubated with a secondary antibody conjugated with Alexa Fluor 488 or 594 at 24˚C for 2–2.5 h. The slices were examined using a Leica SP5/TCS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) or an Olympus IX71 (Olympus, Tokyo, Japan) with Orca-ER cooled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).
2.5. Electrophoresis and Immunoblotting
Acute or cultured hippocampal slices were solubilized in ice-cold RIPA buffer with a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim, Germany). Samples were resolved by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (Immobilon-P, Millipore). Membranes were incubated in 5% skim milk in TBST at 25 ˚C for 30–60 min to block nonspecific binding. Membranes were incubated at 4 ˚C overnight or 25 ˚C for 2 h with primary antibodies against GFAP (BioLegend), Iba1 (Wako Pure Chemical Industries), anti-galectin-3 (mouse monoclonal, A3A12, Santa Cruz, CA, USA), or anti-β-actin (a loading control, mouse monoclonal; Wako Pure Chemical Industries). After four changes of 1× TBST and three 5-min washes at 25 ˚C, membranes were incubated in horseradish peroxidase-conjugated secondary antibodies. After four washes at 25 ˚C, membranes were incubated with ECL solution (Thermo Fisher Scientific, MA, USA). For quantification, chemiluminescence light signals in Super Signal Dura substrate (Thermo Fisher Scientific) were captured by a cooled charge-coupled device camera system (LAS-3000plus; Fuji Photo Film Company, Kanagawa, Japan) that ensured wide ranges of linearity. Densitometric quantification of synaptic protein expression normalized to GAPDH or β-actin. The protein content was estimated using bicinchoninic acid (BCA) reagent (Thermo Fisher Scientific).
2.6. Enzyme Immunoassay for Amyloid β and soluble APP.
The levels of Aβ40 were measured using a solid phase by sandwich ELISA kit following the supplier’s information (IBL, Gunma, Japan). The test samples and standards were added to the 96 well plate (precoated with anti-human Aβ (35−40) (1A10) mouse IgG) and incubated overnight at 4˚C. After several washes with EIA wash buffer, 100 µL of HRP-labeled anti-Aβ (1−16) rabbit IgG solution was added to each well (except the wells corresponding to reagent blanks) and incubated at 4˚C for 1 h. After a thorough wash, 100 µL of tetramethylbenzidine solution was added to each well and incubated in the dark at 24˚C for 30 min. The reaction was stopped by adding 100 µL of 1N sulfuric acid, and the readings were taken at 450 nm. The levels of Aβ in the test samples were calculated by incorporating the unknown values into the standard curve obtained in the assay. The levels of soluble amyloid precursor protein β (Aβ42) were measured using the IBL assay kit (IBL, 96 well plate precoated with anti-Aβ (38−42) rabbit IgG, HRP-labeled anti-Aβ (1−16) rabbit IgG Fab) as described above. The levels of soluble APPα and APPβ were measured using the assay kit (IBL, 96 well plate precoated with anti-mouse APP(599) rabbit IgG, HRP-labeled anti-mouse N-APP rabbit IgG Fab) or (IBL, 96 well plate precoated with anti-mouse sAPPβ-w rabbit IgG, HRP-labeled anti-mN-APP rabbit IgG Fab) as described above.
2.7. Statistical analysis
All quantitative data were presented as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analysis was performed using either Student’s t-test (two-group comparison) or ANOVA (more than two groups) followed by post hoc comparison. Statistical analyses were performed with Prism 5.0 (GraphPad Software, Inc., CA, USA). The level of significance was indicated by asterisks: *P < 0.05, **P < 0.01,***P < 0.001