Animal care and experimental protocols followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0 (16) and were approved by the Health Products Regulatory Authority, Ireland and Animal Care and Use Committee of Zhengzhou University and Wannan Medical College, China. All efforts were made to minimize the number of animals used and their suffering.
Adult (250-350g, 8-11 weeks old) male Wistar and Lister Hooded rats were provided by the Comparative Medicine Unit of Trinity College Dublin and the Laboratory Animal Center of Zhengzhou University and Nanjing University. The animals were housed under a 12h light-dark cycle at room temperature (19-22oC). Prior to the acute experiments, animals were anesthetized with urethane (1.5-1.6 g/kg, i.p.). Lignocaine (10 mg, 1% adrenaline, s.c.) was injected over the area of the skull where electrodes and screws were to be implanted. The body temperature of the rats was maintained at 37-38 oC with a feedback-controlled heating blanket. For i.c.v. injection of synthetic Aβ, the animals were anaesthetized with ketamine (80 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.). The animals were monitored until full consciousness was regained and housed singly for one week or until wound healing had completed, after which they were housed in pairs with continuous access to food and water ad libitum.
Electrodes were made and implanted as described previously (17). Briefly, monopolar recording electrodes were constructed from Teflon-coated tungsten wires (75 μm inner core diameter, 112 μm external diameter) and twisted bipolar stimulating electrodes were constructed from Teflon-coated tungsten wires (50 μm inner core diameter, 75 μm external diameter) separately. Field excitatory postsynaptic potentials (EPSPs) were recorded from the stratum radiatum in the CA1 area of the right hippocampus in response to stimulation of the ipsilateral Schaffer collateral-commissural pathway. Electrode implantation sites were identified using stereotaxic coordinates relative to bregma, with the recording site located 3.4 mm posterior to bregma and 2.5 mm lateral to midline, and stimulating site 4.2 mm posterior to bregma and 3.8 mm lateral to midline. In some animals, another stimulating electrode was implanted at a site located 2.5 mm posterior to bregma and 2.2 mm lateral to the midline. The final placement of electrodes was optimized by using electrophysiological criteria and confirmed via postmortem analysis.
Test EPSPs were evoked by a single square wave pulse (0.2 ms duration) at a frequency of 0.033 Hz and an intensity that triggered a 50% maximum EPSP response. LTD was induced using 1 Hz low frequency stimulation (LFS) consisting of 900 pulses (0.2 ms duration). During the LFS the intensity was raised to trigger EPSPs of 95% maximum amplitude. A relatively weak LFS protocol, used to study the Aβ-mediated facilitation of LTD, consisted of 300 pulses (0.2 ms duration) at 1 Hz, with an intensity that evoked 95% maximum amplitude. None of the conditioning stimulation protocols elicited any detectible abnormal changes in background EEG, which was recorded from the hippocampus throughout the experiments.
A stainless-steel cannula (22 gauge, 0.7 mm outer diameter) was implanted above the right lateral ventricle (1 mm lateral to the midline and 4 mm below the surface of the dura). The solutions were injected in a 5 μL volume over a 3-min period or 10 μL volume over a 6-min period via an internal cannula (28 gauge, 0.36 mm outer diameter). Verification of the placement of cannula was performed postmortem by checking the spread of ink dye after i.c.v. injection.
Morris water maze
Two weeks after a single i.c.v. injection of soluble Aβ1-42 or reverse control Aß42-1 under recovery anaesthesia, the rats were trained in a water pool (150 cm diameter) with a hidden platform of 10 cm diameter. Animals were handled daily for 3 days before the experiment, and the training protocol consisted of four swimming trials per day. A relatively weak protocol consisted of one swimming trial per day. Each animal swam until it found the hidden platform or 120 s, when it was gently guided to the platform and stayed there for 10 s before being returned to the cage. Immediately after the swimming trial the animals were injected intraperitoneally with ISRIB (0.25 mg/kg in saline, 1% DMSO). For the probe test, the platform was removed and each animal was allowed to swim for 120 s, while its swimming trajectory was monitored with a video tracking system (Smart, PANLAB, Spain).
Animals were sacrificed with decapitation after finishing experiments. The whole brain was taken out and the hippocampus were separated from other parts and then all the brain tissues were immediately frozen in liquid nitrogen and stored at -80oC. The rat hippocampus was homogenized Tris-HCl buffer (10mM Tris-HCl, pH 7.5, 150mM NaCl, and 0.5% Triton X-100, 0.1mM PMSF) containing 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich,). Protein concentration was determined by the Bradford technique (Bio-Rad Laboratories), and equal amounts of protein from each sample were loaded on 10% Tris-glycine SDS-PAGE (Invitrogen) gels. The protein were transferred onto PVDF membranes (Millipore, IPVH00010 ), After transfer, membranes were blocked for at least 60 min at room temperature with blocking buffer (BB; 5% non-fat milk in TBS containing 0.1% Tween 20 (TBS-T)), then probed overnight at 4 °C using the following primary antibodies, rabbit anti-phospho-eIF2α (Ser51) (1: 500, ab32157, Abcam), rabbit anti-total eIF2α (1: 1000, A0764, ABclonal), rabbit anti-ATF4 (1: 1000, ab23760, Abcam) and rabbit anti-GAPDH (1: 1000, AC001, ABclonal), washed three times use TBST then incubated with the secondary antibody goat anti-rabbit (1:10,000, 111-035-144, Jackson ImmunoResearch) for 2h at room temp. And the proteins were visualized by the chemiluminescence reagents provided with the ECL kit (Affinity Biosciences) and then detected with a machine of ProteinSimple (FluorChem E, USA). And then used the ImageJ to quantify the intensities of the blot.
TBS Extract of Human Brain
The same human brain tissue was used as our previous report (17). Human brain tissue was used in accordance with local Ethics Committee guidelines. Briefly, tris-buffered saline (TBS) extracts of brain specimens were prepared, processed and analyzed as described previously. Frozen cortex (0.9 g) was allowed to thaw on ice, chopped into small pieces and homogenized in 4.5 ml of ice-cold 20 mM Tris-HCl, pH 7.4, containing 150 mM NaCl with 25 strokes of a Dounce homogenizer (Fisher, Ottawa, Ontario, Canada). Water-soluble Aβ was separated from membrane-bound and plaque Aβ by centrifugation at 91,000g and 4oC in a TLA 55 rotor (Beckman Coulter, Fullerton, CA, USA) for 78 minutes. To eliminate bioactive small molecules the supernatant was exchanged into ammonium acetate. Thereafter, extracts were divided into 2 parts: one aliquot was immunodepleted of Aβ by 3 rounds of 12-h incubations with the anti-Aβ antibody, AW8, and protein A at 4oC. The second portion was not manipulated in any way and is simply referred to as AD. Aliquots of samples were stored at -80oC .
Soluble Aβ1-42 and reverse control Aß42-1 (ChinaPeptides, Shanghai) was prepared as a stock solution of 75 µM in mild alkali (0.1% ammonium hydroxide) in milliQ water to avoid isoelectric precipitation, and then centrifuged at 100,000 x g for 3 h to remove any fibril aggregates. An aliquot of the supernatant was taken to estimate peptide concentration using the micro BCA protein assay (Thermo-Fisher Scientific Life Science Research Products, Rockford, IL) and the remaining supernatant was stored at -80 oC until required.
Emetine dihydrochloride hydrate (Emetine, Sigma, E2357) was prepared in ultra-clean water. Trans-N,N′-(Cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy) acetamide (ISRIB, Sigma, SML0843) was dissolved in DMSO with gentle warming and diluted in polyethylene glycol 400 (PEG400) or saline before injection; 1:1 DMSO and PEG400 or 1 % v/v solution of DMSO in saline was used as vehicle control.
Values are expressed as the mean ± s.e.m. For the electrophysiology experiments, the last 10 min prior to LFS was used to calculate the “Pre” –induction fEPSP amplitude. Unless otherwise stated the magnitude of LTD was measured over the last 10 min at 3 h after (“Post”) LFS. Control experiments were interleaved randomly throughout. To compare between two group with one time point, unpaired t test was used. To compare between groups of three or more, one-way ANOVA with Bonferroni multiple comparisons was used. A two-tailed paired Student’s t-test (paired t) was used to compare between “Pre” and “Post” within groups. For the Morris water maze test, two-way ANOVA followed by a post hoc Bonferroni multiple comparisons test was used for escape latency analysis and one-way ANOVA with Bonferroni multiple comparisons was used to analyze the results from the probe trials. For the Western blots, one-way ANOVA with Bonferroni multiple comparisons was used. A value of P < 0.05 was considered statistically significant.