Rats (Sprague-Dawley) were kept in standard conditions at room temperature (23 $\pm$ 1 ℃) and a 12:12 h light/dark cycle with light (7:00 AM–7:00 PM). Ad libitum access to food and water was provided, except during preparation for the SPT. Ten male rats were mated at a ratio of 1:3 with 30 female rats (230–300 g weight) at 20:00–22:00 PM. At 8:00 AM next morning, female rats underwent vaginal smear testing. Gestational day (GD) zero was designated as the day at which the first positive detection of sperm in the vagina was made. Pregnant female rats were raised separately and were divided randomly into control and experimental groups. Pregnant dams in the experimental group (n = 21) underwent exposure to PS as described previously [23]. Briefly, PS dams were placed head-first into well-ventilated bottles with an adjustable cap. This procedure was carried out three times daily and for 45 min each time between GD14 to GD20 inclusive. The interval between procedures was never 2 h or $>$4 h. Pregnant dams from the control group (n = 8) were placed in identical new cages but without exposure to the PS procedure. Approximately 30 days after birth, male offspring were selected at random for the subsequent experiments. Not more than two male siblings were used in the same experimental group. To avoid “litter effects”, the number of rats selected from each litter was between 6 and 12. The animal experiments received approval from the Animal Ethics Committee of Xi’an Jiaotong University (No. 2020-449) and were performed according to standard guidelines and protocols.

NVP-AAM077 (Cat# 459836-30-7) was purchased from Millipore Sigma (St. Louis, MO, USA) and prepared in a saline solution. PS pregnant dams (n = 8) were treated once daily for 8 days (GD7-GD14) with NVP-AAM077 (10 mg/kg, intraperitoneal administration) at 30 min prior to PS exposure and at a volume of 2 mL/kg of body weight [24]. Control animals (n = 8) were administered an appropriate vehicle.

Rat hippocampal neurons (Cat# CM-R107, Wuhan, Hubei, China) were purchased from Procell Life Science & Technology and identified by immunofluorescence for $\beta$-Tubulin-Ⅲ. The degree of purity was $>$90% and there was no evidence of HIV-1, hepatitis B virus (HBV), hepatitis C virus (HCV), mycoplasma, bacteria, yeast or fungi. Cells for subsequent experiments were grown at 37 ℃ according to instructions from the supplier.

The effect of corticosterone on neuronal cell viability was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described below. First, rat hippocampal neurons were added to 96-well plates and cultured overnight at 37 ℃. These were divided into 6 groups and treated with increasing concentrations of corticosterone (0, 0.01, 0.1, 1, 10 and 100 µM) for 24 hours (Table 1). Ten µL of MTT was added and the cells cultured for another 4 h at 37 ℃. The culture medium was then aspirated, 150 µL of DMSO added, and the cells shaken for 10 min. Measurement of cell viability was performed using the MTT assay (Biofroxx, Cat# 3580MG250, Jiangsu, Nanjing, China) as recommended by the manufacturer. The absorbance value of all wells was measured with a microplate reader (Elx 800, Bio-TEK Instruments, Beijing, China). Data for the experimental groups were expressed as a percentage value relative to the controls. For drug treatment experiments, hippocampal neurons were treated with NVP-AAM077 (5 µM) for 12 h prior to treatment with NMDA. Appropriate vehicle was administered to the controls.

Rats were weaned on day 21 after birth. Prepubertal, 30-day-old male offspring were used for the depression-related behavioral studies. Because of the different stress levels caused by the various behavioral tests, the SPT was conducted first, followed by the forced swim test. The two behavioral tests were conducted with a one-day interval between them. As one week was required for the completion of all three behavioral experiments, the rats were almost 40 days old when sacrificed. Experiments were conducted blind to the treatment groups.

For the SPT, rats were selected at random to determine their susceptibility or resistance to PS. They were divided into an experimental group (n = 121) and a control group (n = 16). The SPT was carried out as described earlier to quantify anhedonia and depression symptoms [25]. Prior to the SPT, rats were habituated for 24 h with 1% sucrose solution and then deprived of food and water for a further 24 h. The SPT was conducted between 8: AM to 9: AM the next day. The animals were concurrently offered normal tap water and a 1% sucrose solution. The drinking bottles were weighed one hour later to measure the consumption of water and sucrose, with the preference for sucrose being calculated as: sucrose consumption/(sucrose consumption + water consumption) $\times$ 100%. The PS-S group was defined as PS offspring that showed a $>$30% decrease in sucrose preference relative to controls. The PS resistant (PS-R) group showed a 10% decrease in sucrose preference, while the PS medium (PS-M) group showed a 30% but $>$10% decrease.

Following the SPT, 8 rats from each group (PS-S, PS-R, PS-M) were used for the FST. This test was carried out as reported earlier and is used to assess depressive-like behavior [26]. Briefly, rats were put into a circular glass tank (20 cm diameter, 50 cm height) containing 30 cm deep water at 30 °C. The room temperature was 25 °C and fluorescent lighting used throughout. To start the experiment, each rat was put in the tank and allowed to swim for 15 min. It was then taken out, dried, and returned to its cage. After 24 h, the rat was placed in the tank again for 5 min and the immobile time was measured. This was defined as the time spent not struggling, or with minimal movement only to keep its head above water.

Eight animals from each group were euthanized and underwent cardiac perfusion with ice-cold PBS followed by overnight fixation at 4 °C in paraformaldehyde (4% w/v). A vibratome (VT 1000S, Leica, Shanghai, China) was then used to prepare coronal tissue sections of 4 µm thickness. Antigen retrieval was achieved by incubating for 20 min in a sodium citrate solution (Baxter, Deerfield, IL, C532382) containing Tween (0.05% v/v). The sections were washed thrice with PBS containing Triton X-100 (PBST, 0.1% v/v) for 10 min and then permeabilized at room temperature for 30 min with PBST containing goat serum (10% v/v). The primary antibodies used for immunofluorescence were human monoclonal anti-MBP (1:1000; ab209328, Abcam, Eugene, OR, USA) and mouse monoclonal anti-$\beta$-Tubulin-Ⅲ (1:100; ab78078, Abcam). Tissue sections were incubated overnight at 4 °C with primary antibody, rinsed four times with PBST for 3 min each, and then incubated at 37 °C for 1 h with FITC-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (1:100; BA1101, BOSTER Biological Technology, Pleasanton, CA, USA). After blocking with goat serum, tissue sections were incubated overnight at 4 °C with antibody, washed four times with PBST for 3 min each, and then incubated for 1 h at 37 °C with Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:100; BA1032, BOSTER Biological Technology). The sections were then incubated for 5 min with DNA stain 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, C1002, Beyotime Biotech, Shanghai, China), washed four times with PBST for 3 min each, and mounted in glycerol (50% v/v). Fluorescence microscopy was used to view slides (BX53, Olympus, Tokyo, Japan) and the fluorescence intensity measured with Image-Pro Plus (Version 6.0, Media Cybernetics, Rockville, MD, USA).

Following completion of the behavioral experiments, 8 animals from each group were euthanized, the hippocampus sectioned, and the tissue homogenized on ice. TRIzol (Aidlab Biotechnologies, Beijing, China) was used to extract total RNA and the concentration measured at 260 nm with a spectrophotometer (ND-100, NanoDrop Technologies, Wilmington, DE, USA). Ominiscript® Reverse Transcriptase kit (Vazyme Biotech, Nanjing, China) was used to reverse transcribe RNA into cDNA. qRT‒PCR was then performed with ABI 7900HT or QuantStudio 6 System (Applied Biosystems, Grand Island, NY) instruments using the following primer sequences: GluN2A, 5${}^{\prime }$-ACATTGCAGAAGCTGCCTTT-3${}^{\prime }$ (F), 5${}^{\prime }$-TTCTGTGACCAGTCCTGCTG-3${}^{\prime }$ (R); MBP, 5${}^{\prime }$-CACACAGCAGACCCAAAGAA-3${}^{\prime }$ (F), 5${}^{\prime }$-GTCGCTGTGAGGGTCTCTTC-3${}^{\prime }$ (R); CaMKII, 5${}^{\prime }$- AACTGGCAGACTTCGGCTTA-3${}^{\prime }$ (F), 5${}^{\prime }$-ATCCCAGAAGGGTGGGTATC-3${}^{\prime }$ (R); GAPDH, 5${}^{\prime }$-ACAGCAACAGGGTGGTGGAC-3${}^{\prime }$ (F), 5${}^{\prime }$-TTTGAGGGTGCAGCGAACTT-3${}^{\prime }$ (R). The 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method was used to quantify relative expression, and GAPDH was used as a control to normalize for input. All experiments were carried out in triplicate.

Eight rats from each group were euthanized, the hippocampus sectioned, and the tissue homogenized on ice with RIPA lysis buffer containing a cocktail of phosphatase enzyme inhibitors (Beyotime Ins. Biotec, Shanghai, China). Lysates were centrifuged at 12,000 $\times$g for 5 min and the protein concentration in the supernatant quantified using a BCA kit (Beyotime Ins. Biotec). Proteins in the lysates (20 mg) were separated using 5% or 10% SD–SPAGE and then transferred to porous polyvinylidene fluoride (PVDF) membranes (Millipore). Primary antibodies for Western blot were: rabbit monoclonal anti-GluN2A (1:1000; ab124913, Abcam), rabbit monoclonal anti-CaMKII (1:2000; ab52476, Abcam), rabbit monoclonal anti-p-CAMKII (1:1000; I2716, CST Annoron, Beijing, China), and human monoclonal anti-MBP (1:1000; ab209328, Abcam). GAPDH (1:1000; AB-P-R001, Hangzhou Goodhere Biotechnology, Hangzhou, China) was detected as the internal loading control. Detection was achieved using SuperSignal® West Dura Extended Duration Substrate ((Pierce Bio- technology, Rockford, IL, USA) and X-ray Film (Eastman Kodak, Rochester, NY), while signal intensities were measured with Bandscan 5.0 software (Funglyn Biotech, Richmond Hill, Ontario, Canada).

Six rats from each group were euthanized. The hippocampus tissue was thawed and placed into a glass homogenizer. Frozen formic acid (1 mol/L, 2 mL) was added and the tissue was fully homogenized manually on an ice bath. Homogenates were centrifuged at 4 ℃ for 30 min and 7000 r/min, and the supernatant stored at –20 ℃. Homogenate supernatant (1 mL) was mixed with 4% sodium bicarbonate solution (0.75 mL), centrifuged at 4 ℃ for 5 min at 3000 r/min and the supernatant collected. This was passed through a 0.45 µm filter membrane (Sigmaaldrich, St. Louis, MO, USA), and then pack. To the dispensing solution (24 µL) in the sample bottle was added derivative reagent (12 µL) and sodium tetraborate buffer (960 µL, pH 9.18). This was mixed well and stood for 3 min at –20 ℃ then performed to determine the hippocampus glutamate concentration.

Results are shown as the mean $\pm$ SD. Kolmogorov–Smirnov test was used to assess whether the data was normally distributed. Student’s t-test was used to analyze for significant differences in pairwise comparisons. One-way ANOVA was used to compare more than two groups. Bonferroni or Tukey methods were used as appropriate for post-hoc analyses. Statistical significance was assumed at p 0.05. All analyses were conducted using Prism version 5.0 (GraphPad Software Inc., Boston, MA, USA).

One-way ANOVA found there were significant differences between pre-defined PS groups for behavioral test results (Fig. 1). Offspring from PS-S and PS-M groups (as defined in the Methods and Methods) showed significantly less sucrose preference in the SPT (Fig. 1A). These groups subsequently showed longer immobility time in the FST (Fig. 1B) compared to controls. The PS-R group showed no significant differences compared to controls (Fig. 1A,B).

Fig. 1.

Results from the behavioral tests. (A) Sucrose preference results for the four groups in the sucrose preference test (SPT). (B) Immobility time results for the four groups in the forced swim test (FST). All results shown are mean $\pm$ standard deviation (SD) (n = 8 to n = 51 for the different groups). * p 0.05 vs. control. CON, control group; PS-S, PS susceptible group; PS-M, PS medium group; PS-R, PS resistant group.

Glutamate levels in the hippocampus of PS-S rats were significantly higher (p 0.05) than in controls (Fig. 2A). Immunostaining showed the optical density of MBP staining in the PS-S group was lower than in controls (Fig. 2B). Moreover, expression levels for GluN2A and MBP protein were lower in PS-S offspring than in controls (Fig. 2C,E), whereas p-CaMKII protein expression was higher (Fig. 2D). Similarly, GluN2A and MBP mRNA levels in PS-S offspring were also lower than in controls (Fig. 2F,H), whereas CaMKII mRNA levels were higher (Fig. 2G).

Fig. 2.

Effect of PS on the glutamate level and on GluN2A, CaMKII, p-CaMKII and Myelin basic protein (MBP) expression levels in the hippocampus. (A) Glutamate level. (B) MBP staining visualized by immunofluorescence (400$\times$). (C) GluN2A protein expression. (D) CaMKII and p-CaMKII protein expression. (E) MBP protein expression. (F) GluN2A mRNA level. (G) CaMKII mRNA level. (H) MBP mRNA level. Results shown are the mean $\pm$ SD for 6 to 9 rats in each group. * p 0.05 vs. CON. CON, control group; PS-S, PS susceptible group; PS, prenatal stress; MBP, myelin basic protein.

Compared to the controls, treatment of PS-S rats with the GluN2A receptor antagonist NVP-AAM077 increased immobility time in the FST (Fig. 3A). The optical density for MBP staining in the NVP group was also increased compared to the controls (Fig. 3B). The PS-S group showed significantly increased CaMKII mRNA expression compared to controls, but this was attenuated by NVP-AAM077 (Fig. 3C). Similarly, the PS-S group showed significantly elevated CaMKII and p-CaMKII protein expression levels compared to controls that was decreased by NVP-AAM077 (Fig. 3D). The PS-S group showed a significantly lower MBP mRNA level compared to controls, but this was increased by NVP-AAM077 (Fig. 3E). Similarly, the MBP protein expression level was lower in the PS-S group, but this was significantly increased by NVP-AAM077 (Fig. 3F).

Fig. 3.

The effect of the GluN2A receptor antagonist NVP-AAM077 on depressive-like behavior and on mRNA and protein levels for CaMKII and MBP in the hippocampus. (A) Immobility time in the FST. (B) MBP staining visualized by immunofluorescence (400$\times$). (C) CaMKII mRNA levels. (D) CaMKII and p-CaMKII protein expression levels. (E) MBP mRNA levels. (F) MBP protein expression levels. Results shown are the mean $\pm$ SD for 6 to 9 rats in each group. * p 0.05 vs. CON, # p 0.05 vs. PS+NS. CON, control group; PS-S, PS susceptible group; PS-S+NS, PS susceptible + normal saline group; PS-S+NVP, PS susceptible + NVP-AAM077 group.

Isolated rat hippocampal neurons were identified by $\beta$-Tubulin-Ⅲ immunofluorescence, with the degree of purity being $>$90% (Fig. 4A). Immunostaining revealed the optical density of Ca${}^{2+}$ staining was significantly higher in the corticosterone treatment group than the controls, and this elevated level was not reduced by NVP-AAM077 (Fig. 4B). The expression of GluN2A protein in the corticosterone treatment group was significantly less than in controls, but was increased by NVP-AAM077 (Fig. 4C). The levels of CaMKII and p-CaMKII protein expression in the corticosterone treatment group were both higher than in controls, but were attenuated by NVP-AAM077 (Fig. 4D). The GluN2A mRNA level in the corticosterone treatment group was significantly lower than controls, but was increased by NVP-AAM077 (Fig. 4E). The CaMKII mRNA level was higher in the corticosterone treatment group than controls, but this was decreased by NVP-AAM077 (Fig. 4F).

Fig. 4.

Effect of the GluN2A receptor antagonist NVP-AAM077 on hippocampal neurons. (A) Identification of hippocampal neurons by immunofluorescence of $\beta$-Tubulin-Ⅲ (400$\times$). (B) Ca${}^{2+}$ staining visualized by immunofluorescence (400$\times$). (C) GluN2A protein expression. (D) CaMKII and p-CaMKII protein expression. (E) GluN2A mRNA level. (F) CaMKII mRNA level. The results shown are the mean $\pm$ SD for 6 to 9 rats. * p 0.05 vs. CON, # p 0.05 vs. Cor+NS. CON, control group; Cor, corticosterone group; Cor+NS, corticosterone + normal saline group; Cor+NVP, corticosterone + NVP-AAM077 group.