Animals and housing conditions
Male Wistar rats weighing 120–140 g (about 5 weeks) were purchased from the Experimental Animal Centre of Shandong University. All experiments were approved by the Shandong University Animal Care and Use Committee and were conducted according to the International Guiding Principles for Animal Research provided by the International Organizations of the Medical Sciences Council. Rats were housed under a 12 h light/dark cycle and had free access to food and water. Ambient temperature was maintained at 22 ℃ ± 2 ℃, except when subjected to the conditions of specific experiments. All efforts were made to minimize the pain and numbers of the animals used in the experiments.
Rats in the CUS group were subjected to procedures described previously . Briefly, these rats were individually housed and subjected to a daily stress regime for 5 weeks consisting of: (1) 24 h of water and food deprivation, (2) swimming in cold (4 °C) water for 5 min, (3) damp sawdust for 24 h, (4) tail clamp for 3 min, (5) cage tilted 45°for 12 h, (6) restricted movement for 2 h and (7) horizontal oscillation for 5 min. One stressor was applied per day in a random order. Rats in the control group were housed under laboratory conditions.
All behavioral experiments were conducted in isolated behavioral testing rooms and performed by experimenters who were blind as to the identity of the experimental groups.
Forced swimming test
The forced swimming test (FST) was used to evaluate depression-like behaviors. After the 5-week period of stress administration, rats were placed individually in a water filled cylinder (height: 80 cm, diameter: 30 cm, 25 °C) for 15 min of forced swimming in the training session. At 24 h after this training session, the rats were again placed in the water filled cylinder for the 5 min test session. During this 5 min test session, immobility and horizontal movement times were recorded. Rats floating motionless without struggling or making minimal movements only necessary to maintain their heads above the water surface were considered to be immobile. Horizontal movement throughout the cylinder was defined as swimming while vertical movement against the wall of the cylinder was defined as climbing.
Sucrose preference test
The sucrose preference test (SPT) was performed 5 weeks after CUS treatment as a means of assessing anhedonia symptoms in rats. As described in the previous literature with minor modifications . Briefly, rats were placed individually in cages with two bottles of sucrose solution (1%, w/v) for the first 24 h period and then one bottle of sucrose solution was replaced with tap water for the second 24 h period. After this adaptation phase, rats were deprived of food and water for 24 h and then permitted access to two bottles for 3 h, one bottle containing 100 ml of 1% sucrose solution and the other containing 100 ml of tap water. Sucrose preference was defined as sucrose consumption/[water consumption + sucrose consumption] × 100% during the test phase.
Morris water maze
The Morris water maze (MWM) test was performed to evaluate spatial memory and learning ability in an aqueous environment. The water maze, a cylindrical pool (60 cm in radius, 50 cm in height), was visually separated into four quadrants. A platform with a diameter of 13 cm was placed 1 cm below the waterline in the center of one quadrant below the water surface. During the 5 days of training sessions, each rat was placed at one of four start locations facing the wall of the pool and trained to locate the platform in the fourth quadrant within 60 s and then remained on the platform for 30 s. If the rat failed to locate the platform within 60 s, it was guided to the platform and was required to remain there for 15 s. The sixth day was the test day. The platform was withdrawn, and the rat was placed in the opposite quadrant of the goal quadrant to swim freely for 60 s. Defines the platform as the target quadrant, and SMART records the time within the target quadrant and the number of times across the platform (SMART is a video tracking platform software).
Dual-luciferase reporter assay
The experimental plasmid was constructed by RiboBio Technology (Guangzhou). Luciferase reporter assays were performed according to the following description. First, the 3′-UTR sequences of wild-type (WT) or mutant-type (MUT) RGS12s were cloned into the luciferase reporter plasmid, and were co-transfected with the miR-204-5p mimic or miR-NC into HEK-293 T cells for 48 h. To analyze interactions between miR-204-5p and RGS12, firefly luciferase activities were measured after transfection using the Dual-Luciferase Reporter Assay System. All luciferase values were normalized to those of firefly luciferase and expressed as fold induction relative to basal activity.
miRNA library construction and sequencing
According to the manufacturer's instructions, total RNA was isolated from hippocampal DG region tissues using Trizol kit (Invitrogen) for the preparation and sequencing of miRNA libraries. The RNA integrity (RIN) was accessed using Agilent 2200 Tapestation (Agilent Technologies, USA). Small RNAs were reverse transcribed into cRNAs and amplified by PCR. The PCR products were sequenced using the HiSeq 2500 and HiSeq 3000 (Illumina, USA) platform at the RiboBio Company (Guangzhou, China). The up-regulated and down-regulated MicroRNAs with significant differences in expression ranked by fold changes in microarray are listed in Additional file 2: Table S2.
Differentially expressed microRNAs in the sequencing results were analyzed by Diana miRpath. MicroRNAs and pathways were classified according to their interaction levels, and these results were combined by combination and meta-analysis algorithms. Used three different databases, miRTARBASE, miRDB and TargetScan, to predict the possible target genes downstream of miR-204-5p, screened the same target genes predicted in the three databases. MiRNA target genes implicated in the pathway were investigated among genes with use of the KEGG database . A P < 0.05 was used as the criterion for statistical significance.
Stereotactic injection of the AAV virus
After completion of behavioral tests, rats in the CUS and control groups were randomly selected for virus injection. Different groups of rats were randomly selected by non-participants. The AAV9-virus was obtained from GENEchem (Shanghai, China). The AAV9-rno-miR-204-5p virus (primer sequence: UUCCCUUUGUCAUCCUAUGCCU) was constructed to overexpress miR-204-5p while the AAV9-rno-miR-204-5p-sponge virus (inverse complementary sequence: AGGCATAGGATGACAAAGGGAA) was used to block miR-204-5p in the DG region of the hippocampus. Rats were anesthetized with an intraperitoneal injection of 2.5% isoflurane as based on their body weights and then positioned within the stereotaxic apparatus (Stoelting, USA). Small burr holes were drilled on two sides of the skull (3.24 mm posterior to bregma and 1.8 mm lateral to the midline) to allow access to the hippocampal DG region for injection of the AAV virus (~ 1012 infection units per ml, a flow rate of 140 nl/min) at the depth of 3.5 mm. Behavioral tests or biochemical assays were performed at ≥ 14 days after injection. The injection sites were verified after behavioral tests and only rats with correct injection sites were used for analyses in the subsequent assays.
Brain anatomy and tissue preparation
Twenty-four hours after behavioral tests, rats from each group were anesthetized with sodium phenobarbital (30 mg/kg) and transcardially perfused with 300 ml 0.9% NaCl containing heparin sodium salt followed by fixation with 4% paraformaldehyde (PFA). Brains were removed and post-fixed in 4% PFA overnight at 4℃followed by a graded dehydration. Brain tissue was encased in optimum cutting temperature compound and frozen serial coronal sections were cut. Sections, consisting of prefrontal cortex and hippocampus, were selected for immunofluorescent staining.
Immunofluorescent staining and confocal microscopy
Frozen slices (40 μm) were incubated overnight at 4 ℃ with the following antibodies: rabbit anti-NeuN (24,307, Cell Signaling Technology), mouse anti-NeuN (ab104224, Abcam), rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1, 019-19741, Wako Pure Chemical Inc), mouse anti-RGS12 (sc-398545, Santa Cruz Biotechnology) or rabbit anti-MAP2 (4542, Cell Signaling Technology). Sections were then incubated with Alexa Fluor 488 or 546 conjugated (Abcam) or alexa-568 (Invitrogen), for 1 h at 37℃ in a thermostatic oscillator. DAPI (Beyotime) was used for nuclear staining. Before each step, slices were washed three times in PBS. Images were captured with use of a confocal microscope (LSM880, Carl Zeiss, Germany) and processed using ZEN software. The fluorescent intensity analysis was performed using ImageJ (NIH, Bethesda, MD, USA). The positive cells of Iba1 per 1 mm2 were counted by experimenters used ImageJ, blind to the treatment conditions. All experiments were conducted in a blinded manner, adhering to stereological principles.
Rats were anesthetized with an intraperitoneal injection of sodium phenobarbital (30 mg/kg) as based on their body weight. Inside the hippocampus, CA1 region is located on the dorsal side of the brain and DG is located on the ventral side of the brain. Under the stereomicroscope, CA1 and DG region could be divided along the hippocampal fissure between them in the ventral surface of the hippocampus. Bilateral DG tissue samples were isolated and lysed in RIPA buffer containing a cocktail of protease/phosphatase inhibitors. After centrifugation (20 min, 12,000 rpm, 4 ℃), cleared lysates containing the isolated proteins were harvested. Protein concentrations were determined using the Pierce BCA protein kit. Western blot was performed as described previously . Proteins (30 μg) from each tissue sample were loaded in each lane, electrophoretically separated on SDS-PAGE gels and then transferred onto PVDF membranes for primary antibody incubation at 4 °C for overnight. Membranes were incubated with the following antibodies: rabbit anti-β-actin (4970, Cell Signaling Technology), rabbit anti-NOX1 (DF8684, Affinity Biosciences), rabbit anti-NOX4 (bs60435, Bioworld), rabbit anti-Nrf2 (ab137550, Abcam), rabbit anti-HO-1 (ab13243, Abcam), mouse anti-RGS12 (sc-398545, Santa Cruz Biotechnology), rabbit anti-NF-κB p65 (bs90940, Bioworld) or mouse anti-NQO1 (ab2894, Abcam). Horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) were used as labelled secondary antibodies. Image-J software was used to perform pixel quantification of the images. Intra-run normalization against the internal actin control was performed for each sample.
Real-time quantitative PCR
Expression levels of miRNA were determined using quantitative real-time quantitative PCR with The All-in-OneTM miRNA qRT-PCR Detection Kit (GeneCopoeia, USA) and mRNA qRT-PCR detection kit (GeneCopoeia, USA) was used to determine mRNA expression level in real-time quantitative PCR. Real-time quantitative PCR analysis was performed on a Bio-Rad iCycler system (Bio-Rad, Hercules, CA). Rno-U6 served as a loading control for the sample to test for miRNA and GAPDH served as a loading control for the sample to test for mRNA, mRNA expression levels and miRNA expression levels were evaluated using the 2 − (ΔΔCt) method. Sequences of specific primers are listed in Additional file 1: Table S1.
Transmission electron microscopy (TEM)
The ultrastructure of DG neurons was observed with use of transmission electron microscopy (Philips Tecnai 20 U-Twin, Holland). Bilateral DG tissues were carefully dissected (1 × 1 × 1 mm), fixed with 1% osmium tetroxide for 2 h, and dehydrated with graded ethanol. The tissue was infiltrated overnight with a semi-epoxy-propane mixture and then embedded in resin. Tissues were cut into ultrathin sections (70 nm), stained with 4% uranyl acetate for 20 min and then stained with 0.5% lead citrate on the copper grid. At least 30 micrographs were randomly selected from each rat and analyzed using Image J analysis software (NIH, Scion Corporation, Frederick, MD).
Acute hippocampal slice preparation and electrophysiological analysis
Rats were anesthetized using 2.5% isoflurane and decapitated. The brain was extracted, blocked, placed on a vibrating slicer, and immersed in a cold solution containing (in mM) 119 choline chloride, 30 Glucose, 26 NaHCO3, 7 MgSO4, 2.5 KCl, 1 NaH2PO4, 1 CaCl2, 3 sodiumpyruvate, 1.3 sodium L-ascorbate, 1 kynurenicacid, and saturated with 95% O2/5% CO2. Slices were then transferred as quickly as possible to a recovery solution containing (in mM) 85 NaCl, 24 NaHCO3, 4 MgCl2, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 25 glucose and 50 sucrose and allowed to recover for 30 min at 30 °C. The glass micropipettes (4–6 MΩ) were filled with an internal solution containing (in mM) 130 CsMeSO4, 10 CsCl, 4 NaCl, 1 MgCl2, 5 MgATP, 5 EGTA, 10 HEPES, 0.5 Na3GTP, 10 phosphocreatine and 4 QX-314, with a pH of 7.35. During whole-cell clamp patch recordings, slices were continuously perfused with an artificial cerebral spinal fluid (ACSF) contained (in mM) 120 NaCl, 3.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose.
Oxidative stress measures
To measure the ROS production in tissue, frozen slices were incubated with 10 μM dihydroethidium (DHE) for 30 min at 37 ℃ and then stained with DAPI (Beyotime) for 10 min. Mitochondrial ROS levels were measured with use of 10 μM MitoSOX Red fluorescent dye for 15 min at 37 ℃ in a thermostatic oscillator, followed by staining with DAPI (ThermoFisher) for 10 min. Images were captured on a Zeiss LSM 880 scanning laser confocal microscope.
Antioxidant enzyme activities
Activity of antioxidant enzymes in CA1, DG and vmPFC tissue was measured using the superoxide dismutase (SOD, No. A001-3), lactic dehydrogenase (LDH, No. A020-2) and total antioxidant capacity (T-AOC, A015-2) activity assay kits according to the manufacturers’ guidelines. All assay kits were purchased from Jiancheng Inc. (Nanjing, China).
Assessment of DNA damage markers
Oxidative DNA damage in DG regions was then detected using an immunofluorescent assay. Frozen slices were incubated with the primary mouse anti-DNA/RNA Damage antibody (ab62623 Abcam) and rabbit anti-NeuN overnight at 4 ℃ followed by Alexa Fluor 488 or 546 conjugated (Abcam). DAPI (Beyotime) was used for nuclear staining.
Oxidative stress products
The lipid peroxidation product resulting from oxidative stress, 4-HNE, was measured using immunofluorescent staining with polyclonal goat anti-4-hidroxynonenal (4-HNE, ab46545, Abcam). In addition, CA1, DG and mPFC tissues were homogenized for determination of MDA content (No. A003-1) as performed using assay kits from Jiancheng Inc. (Nanjing, China). All samples were assayed in duplicate and results were normalized to total protein content.
All calculations were performed using GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA). All data were presented as the means ± standard errors of the mean. Statistical significance of differences in the groups was evaluated with use of either the Student's t test, two-way analysis of variance (ANOVA) or one-way analysis of variance (ANOVA) with the Tukey's multiple-comparison test used for post-hoc comparisons. A value of P < 0.05 was required for results to be considered statistically significant.