c57BL/6J mice (8 weeks old) were purchased from Beijing Vital River Laboratory Animal Device Co., Ltd. Before the experiment, the animals were acclimatized for 7 days under standard laboratory conditions [ambient temperature: 22–25°C, relative humidity: 50 ± 5%, 12-h/12-h light/dark cycle (lights on at 08:00 h)]. Mating was carried out in a cage at a male: female ratio of 1:2 at 21:00 h when it was conducive to mating due to the mice being in active phase and low sleep tension. At the same time, we did not turn on the lights during the dark period, we mated under a red-light source with a brightness of 30 lx. Fertilization was confirmed by the presence of a vaginal plug and was marked as GD0. Pregnant mice were housed individually in cages and were provided with standard rodent feed and purified water ad libitum. On GD15, the pregnant mice were intraperitoneally injected with LPS (Abcam LPS, Shanghai Universal Biotech Co., Ltd., Shanghai, China) at a dose of 50 μg/kg or saline and were subjected or not to sleep deprivation for 6 h daily until GD21 (Yan et al., 2022). The day of delivery was designated as postnatal day 0 (PND 0). After weaning, the offspring of the pregnant mice were randomly assigned to the following four groups (n = 8 per group), one offspring mouse was randomly selected from each litter: a control group, a maternal intraperitoneal injection of LPS (MLPS) group, a maternal sleep deprivation (MSD) group, and a MLPS + MSD group. The experiments were performed when the offspring reached 3 months of age. All behavioral experiments were conducted from 13:00 to 18:00. All experimental procedures involving animals were approved by the Laboratory Animal Committee of Anhui Medical University (Figure 1).

For sleep deprivation, pregnant mice were placed in a sleep deprivation machine (BW-NSD404, Shanghai Bio-will Co., Ltd.) for 6 h (12:00–18:00 h) during late gestation (GD15–21). Sleep deprivation was ensured by the continuous working of the running belt in the machine (running speed: 0.5 m/min). Mice not subjected to sleep deprivation were also placed in a sleep deprivation machine (BW-NSD404) but with the running belt switched off (0 m/min). Food and water were provided throughout the sleep deprivation period (Wei et al., 2022).

On PND 90, the locomotor activity and anxiety-like behaviors of the offspring were assessed via the OFT. The apparatus consisted of a large black wooden square box (50 cm × 50 cm × 25 cm). The mice were individually placed in the center of the box and allowed to freely explore the environment for 5 min. The experiment was conducted under dim lights (30 lx). The time spent in the central area, the number of entries into the central area, and the total distance moved were recorded and analyzed using ANY-maze software. The box was cleaned with 75% alcohol after each test to eliminate the odor of the previous mouse.

On PND 91, the anxiety-like behaviors of the mice were evaluated using the EPM test. The experimental instrument consisted of a cross-shaped platform with two open arms, two closed arms, and a central area, and was raised 80 cm above the ground. The offspring were placed in the central area of the apparatus with their head facing the open arms and were allowed to freely explore the maze for 6 min. The experiment was conducted under dim lights (30 lx). The time spent in and the number of entries into each arm were recorded and analyzed using the ANY-maze video tracking system. After each recording, the maze was cleaned with 75% alcohol to eliminate the odor of the previous mouse.

On PND 92, behavioral despair in the mice were assessed via the TST. The mice were suspended by the tail with adhesive tape, 50 cm above the bench, for 6 min. The immobility time in the last 4 min of the test was recorded. Immobility is a depression-like behavior. Mice were considered to be immobile when they were suspended completely motionless.

On PND 93, behavioral despair in the mice were investigated using the FST. The mice were placed in a glass cylinder (20 cm × 40 cm) filled with 25 ± 1°C water to a depth of 30 cm for 6 min and the immobility time in the last 4 min was quantified. Mice were considered immobile when they were floating passively in an upright position and made only those movements necessary to keep their nose above the water.

On PND 94, the spatial learning and memory abilities of the offspring were studied using the MWM test. The experiment was carried out for 7 successive days and was performed as previously described (Zhang et al., 2023a). The equipment consisted of a circular black water tank (150 cm in diameter, 30 cm in height), a submerged black escape platform (10 cm in diameter, 24 cm in height), and a camera with a video recording system on the ceiling to analyze the activity of mice in the tank. The tank was divided into four quadrants, each with specific visual cues. The test comprised two parts, namely, a learning phase and a memory phase. In the learning phase, each offspring was separately and randomly placed in a quadrant with their heads facing the wall and was allowed 60 s to find the platform. If a mouse failed to find the platform within the specified time, it was guided to the platform and allowed to stay there for 30 s. Four trials were conducted per day with a 15-min inter-trial interval and the learning phase lasted for 7 days. The escape latency, distance swam, and swimming velocity were recorded and analyzed with the ANY-maze tracking system (Stoelting, USA). In the memory phase, the probe trial was performed 2 h after the final trial of the learning phase. For this trial, the platform was removed, and the mice were placed in the tank in the quadrant opposite the target quadrant and were allowed to freely explore for 60 s. The percentage of time spent and the distance swam in the target quadrant were analyzed using ANY-Maze software (Stoelting, USA).

At PND 101, the mice were deeply anesthetized with 2% sodium pentobarbital, and the hippocampus was harvested on ice, snap-frozen in liquid nitrogen, and stored at −80°C for ELISA, western blot, and RT-PCR analysis.

Hippocampal tissues of offspring were homogenized in PBS buffer and centrifuged at 1,500 × g for 10 min at 4°C. The levels of IL-1β, IL-6, and TNF-α in the resulting supernatants were detected using the respective ELISA kits following the manufacturer’s instructions.

The hippocampus was collected from offspring and lysed in RIPA lysis buffer. After centrifugation at 12,000 rpm for 15 min, the supernatant was harvested, 5 × SDS-PAGE protein loading buffer (1:4) was added to the supernatant, and the proteins were denatured by placing the mixture in a boiling water bath for 15 min. Then, 5–10 μL of the protein sample was added to each of the wells of the SDS-PAGE gel, and electrophoresis was performed at a constant voltage (80 V) for approximately 1 h. The proteins were then transferred to a membrane and, after blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with primary antibodies (rabbit anti-PSD-95, 1:2,000; Abcam, Cambridge, UK; rabbit anti-SYN, 1:1,000; Bioss, Beijing, China). The next day, the membranes were washed three times with PBST (10 min each wash), and then incubated for 1.2 h with secondary antibody [horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG; Zsbio, ZB-2301] at 37 °C, and then washed again three times with PBST, 10 min each wash. The protein bands were imaged using ImageJ software (Media Cybernetics, USA).

Total RNA was extracted from hippocampal tissue using Trizol reagent and reverse transcribed into cDNA using a RT-PCR kit (TaKaRa, RR047A, Japan). The resulting cDNA was used as a template for fluorescence quantification. The qPCR cycling program consisted of one cycle of pre-denaturation for 1 min at 95°C, followed by 40 cycles of 20 s at 95°C and 1 min at 60°C. The relative mRNA levels of the target genes were calculated using the 2^{–Δ Δ Ct} method. The sequences of the primers used for PCR are shown in Table 1.

All data was normal and homogeneous and was presented as means ± standard error of the mean (SEM). Statistical analysis was performed in GraphPad Prism 8.0 and SPSS 23.0. The data for the MWM test outcomes were analyzed using a repeated measures ANOVA with two factors. One-way ANOVA followed by Tukey’s least-significant difference post-hoc test was employed for multiple group comparisons. P-values < 0.05 were considered significant. Correlations were assessed using Pearson’s correlation coefficient.

The one-way ANOVA results showed that the time spent in and the number of entries into the central area differed markedly among the four groups of mice [time spent in the central area: treatment: F_{(3, 28)} = 21.06, P < 0.01; number of entries in the center: treatment: F_{(3, 28)} = 15.12, P < 0.01]. Post hoc analysis indicated that mice in the MLPS, MSD, and MLPS + MSD groups spent significantly less time in and had substantially fewer entries into the central area in comparison with mice in the control group (Ps < 0.05). Furthermore, our data revealed that the time spent in and the number of entries into the central area were both lower among offspring of the MSD + MLPS group than among those of either the MLPS or MSD groups (Ps < 0.05). However, no difference in these parameters was observed between the MLPS and MSD groups (P > 0.05) (Figures 2A–C). Additionally, no difference in the total distance moved in the OFT was recorded among the four groups [treatment: F_{(3, 28)} = 1.76, P > 0.05].

The one-way ANOVA showed significant effects of treatment for time spent in and number of entries into the open arms in the EPM test [time spent in the open arms: F_{(3, 28)} = 25.81, P < 0.01; entries into the open arms: F_{(3, 28)} = 17.21, P < 0.01]. The results indicated that, compared with offspring in the control group, those in the MLPS, MSD, and MLPS + MSD groups spent markedly less time in and had substantially fewer number of entries into the open arms of the EPM (Ps < 0.01). Moreover, post hoc analysis showed that the time spent in and the number of entries into the open arms were reduced in the MSD + MLPS group compared with that seen in the MLPS or MSD groups (Ps < 0.05). However, no difference in either parameter was detected between the MLPS and MSD groups (P > 0.05) (Figures 2D, E).

In the FST, the one-way ANOVA for immobility time demonstrated significant effects of treatment [F_{(3, 28)} = 12.98, P < 0.01]. Our data showed that immobility time was significantly increased in the MLPS, MSD, and MLPS + MSD groups compared with that in the control group (Ps < 0.05). Additionally, the immobility time was significantly longer in the MLPS + MSD group than in the MLPS and MSD groups (Ps < 0.05). However, there was no difference in immobility time between the MLPS and MSD groups (P > 0.05) (Figure 3A).

In the TST, the one-way ANOVA for immobility time showed significant effects of treatment [F_{(3, 28)} = 24.07, P < 0.01]. Post hoc analysis revealed that immobility time differed significantly between the MLPS, MSD, and MLPS + MSD groups when compared with that in the control group (Ps < 0.05). Compared with the MLPS or MSD group, immobility time was significantly increased in the MLPS + MSD group (Ps < 0.01) (Figure 3B). Collectively, these results suggested maternal sleep deprivation exacerbated the anxiety- and depression-like behaviors induced by prenatal LPS exposure in the offspring mice.

We investigated the effect of maternal sleep deprivation and maternal LPS treatment on the levels of the inflammatory factors IL-1β, IL-6, and TNF-α in the hippocampus. The one-way ANOVA showed significant effect of treatment on the levels of all three factors [IL-1β: F_{(3, 28)} = 38.82, P < 0.01; IL-6: F_{(3, 28)} = 24.40, P < 0.01; TNF-α: F_{(3, 28)} = 19.13, P < 0.01]. The hippocampal levels of IL-1β, IL-6, and TNF-α were greatly increased in the MLPS, MSD, and MLPS + MSD groups in comparison with those in the control group (Ps < 0.05). Moreover, the levels of these inflammatory factors were relatively higher in the MLPS + MSD group than in the MLPS or MSD group (Ps < 0.05). However, no differences in the hippocampal levels of IL-1β, IL-6, and TNF-α were detected between the MLPS and MSD groups (Ps > 0.05) (Figures 5A–C). These results indicated that maternal sleep deprivation further increased the elevated proinflammatory cytokines induced by prenatal LPS exposure.

The one-way ANOVA demonstrated that the treatments had a significant effect on the mRNA levels of PSD-95 and SYN in the hippocampus [PSD-95 mRNA: F_{(3, 28)} = 26.22, P < 0.01; SYN mRNA: F_{(3, 28)} = 48.57, P < 0.01]. As shown in Figure 6, the relative mRNA levels of PSD-95 and SYN were significantly reduced in the MLPS, MSD, and MLPS + MSD groups compared with those of the control group (Ps < 0.01). Furthermore, Tukey’s post-hoc tests revealed that the mRNA levels of PSD-95 and SYN in the MLPS + MSD group were significantly lower than those in the MLPS or MSD group (Ps < 0.01). No differences in the mRNA levels of PSD-95 and SYN mRNA were found between the MLPS and MSD groups (Ps > 0.05) (Figures 6A, B).

The one-way ANOVA showed that there was a significant effect of treatments on the protein expression of PSD-95 and SYN in the hippocampus [PSD-95: F_{(3, 20)} = 31.61, P < 0.01; SYN: F_{(3, 20)} = 30.68, P < 0.01]. One-way ANOVA indicated that the protein levels of PSD-95 and SYN were significantly lower in mice of the MLPS, MSD, and MLPS + MSD groups than in those of the control group (Ps < 0.01). Moreover, Tukey’s post-hoc tests revealed that the levels of both proteins were significantly lower in the MLPS + MSD group than in the MLPS or MSD group (Ps < 0.05). However, the hippocampal protein levels of PSD-95 and SYN did not differ between the MLPS and MSD groups (Ps > 0.05) (Figures 7A–C). These results indicated that maternal sleep deprivation further exacerbated the downregulated synaptic proteins induced by prenatal LPS exposure.

Pearson’s correlation analysis revealed that the hippocampal levels of IL-1β, IL-6, and TNF-α were negatively correlated with the time spent in and the number of entries into the central area in the MLPS, MSD, and MLPS + MSD groups in both the OFT (Ps < 0.05) and the EPM test (Ps < 0.05). Additionally, in the MWM test, the hippocampal levels of the three proinflammatory factors were positively correlated with escape latency and the distance swam in the learning phase and negatively correlated with the percentage of time spent and distance swam in the target quadrant in the memory phase in the MLPS, MSD, and MLPS + MSD groups (Ps < 0.05) (Table 2).

Studies have suggested that stress in early life promotes oxidative stress and inflammation and negatively influences the hypothalamic-pituitary-adrenal axis and the neuroendocrine system in offspring, and these effects have long-lasting consequences for emotional and cognitive behaviors (Krugers and Joëls, 2014; Ciafrè et al., 2020; Cattane et al., 2022). LPS administration during pregnancy significantly increased the levels of anxiety- and depression-like behaviors in offspring in both the EPM test and the FST (Enayati et al., 2012). These behaviors have also been observed in the offspring of mother rats subjected to sleep deprivation during the third trimester-equivalent of pregnancy in humans by gentle manual handing (Radhakrishnan et al., 2015). In full agreement with previous findings, our results showed that maternal sleep deprivation or prenatal exposure to inflammation increased the levels of anxiety and depression in offspring, as evidenced by the results of the OFT, TST, FST, and EPM test. Additionally, both of these stressors have been reported to impair cognitive function in both clinical and preclinical studies. Consistent with these observations, we found that maternal sleep deprivation or exposure to inflammation prenatally prolonged the escape latency and increased the distance swam in the learning phase while decreasing the percentage of time spent and distance swam in the target quadrant in the memory phase of the MWM test. This suggested that both environmental stressors impaired learning and memory function in the offspring of affected mothers.

The binding of LPS to Toll-like receptor 4 (TLR4) triggers an inflammatory response (Ren et al., 2023; Tongaonkar et al., 2023). One study showed that exposure to LPS activated the TLR4 signaling pathway and microglia in pregnant mice and led to autism-like behavior in the offspring (Xiao et al., 2021). These findings indicated that LPS-mediated maternal immune activation may be involved in the occurrence of behavioral abnormalities in offspring. Furthermore, sleep deprivation has also been reported to enhance the inflammatory response following exposure to LPS in adult mice (Xu et al., 2020). In the present study, the results showed that maternal sleep deprivation, as a prenatal stress, aggravated the anxiety- and depression-like symptoms and cognitive deficits induced by prenatal exposure to inflammation in the offspring. Previous study showed that chronic sleep deprivation activates the hypothalamic-pituitary-adrenal axis (HPA) and upregulates corticosterone, which enhances vulnerability to LPS challenge (Ito et al., 2020). We suspect that this may have been due to maternal sleep deprivation further exacerbating LPS-activated maternal immunity via modulating the reaction of HPA.

The hippocampus is an important brain region involved in cognitive function, and its function is vulnerable to various stresses (Grigoryan and Segal, 2016). Elevated levels of proinflammatory cytokines in the hippocampus can negatively affect cell proliferation, differentiation, survival, and synaptic function and lead to anxiety, depression, and cognitive dysfunction (Adebayo et al., 2022; Li et al., 2023). Several studies have shown that the intraperitoneal injection of LPS upregulates the expression of proinflammatory cytokines, including IL-1β, IL-6, and TNF-α, leading to anxiety, depression, and cognitive deficits (Huang et al., 2020; Chen et al., 2021). IL-1β- or TNF-α-knockout mice show reduced levels of anxiety, depression, and cognitive decline relative to that seen in their wild-type counterparts (Goshen et al., 2008; Kaster et al., 2012; Murray et al., 2013; Naude et al., 2014). At the high levels detected following prenatal exposure to inflammation, proinflammatory cytokines can cross the placental and blood-brain barriers and induce an inflammatory response in offspring mice, which is associated with abnormal neurobehaviors. Long-term exercise was demonstrated to improve prenatal exposure to inflammation-induced anxiety and depression in offspring by increasing the concentrations of anti-inflammatory cytokines (Rahimi et al., 2020). Meanwhile, maternal sleep deprivation induced a notable inflammatory response and exerted a significant inhibitory effect on neurogenesis, leading to spatial learning and memory impairment in offspring (Zhao et al., 2014). In the current study, we also found that inflammation contributed to anxiety- and depression-like behaviors and cognitive impairment in offspring following maternal sleep deprivation or prenatal exposure to inflammation, as reflected by the high levels of proinflammatory cytokines detected in the MLPS and MSD groups. Importantly, the expression levels of proinflammatory cytokines were higher in the offspring of mice in the MSD + MLPS group than in those of mice in the MSD or MLPS group, suggesting that maternal sleep deprivation may aggravate the inflammatory response induced by prenatal exposure to inflammation, thereby further impairing emotional and cognitive functioning in the offspring.

Substantial evidence supports the existence of a direct link between synaptic plasticity and anxiety, depression, and cognitive function (Wang et al., 2021; Hu et al., 2023). Early life is a critical period for synaptic pruning, synaptic maturation, and neural network establishment, and is susceptible to interference from external environmental factors (Malave et al., 2022; Roszkowska et al., 2022). One study reported that exposure to inflammation in utero increased the levels of synaptic pruning-associated proteins (C3 and CR3A) and decreased dendrite length and dendritic spine density in offspring mice (Xiao et al., 2021). Maternal sleep deprivation, an early-life stress, was reported to impair long-term potentiation and synaptic transmission in the hippocampi of offspring (Peng et al., 2016). The synaptic proteins PSD-95 and SYN play an important role in dendrite development and participate in synaptic plasticity (Martínez-Torres et al., 2021; Li et al., 2022). In the current study, we found that the mRNA and protein levels of PSD-95 and SYN were decreased in the offspring of mice in the MSD and MLPS groups, indicating that a potential impairment in synapse formation and/or plasticity contributed to the abnormal behaviors in the offspring. Furthermore, the combination of prenatal exposure to inflammation and maternal sleep deprivation aggravated the synaptic dysfunction induced by either stressor alone, as evidenced by the downregulated levels of PSD-95 and SYN in the MLPS + MSD group relative to those in the MLPS and MSD groups. This partly explains the mechanisms involved in how maternal sleep deprivation exacerbates the emotional and cognitive dysfunction induced by exposure to inflammation in utero.

Growing evidence indicates that the levels of proinflammatory cytokines and synaptic proteins are closely related to anxiety, depression, and cognitive decline. Elevated levels of proinflammatory cytokines in the blood are associated with high anxiety and depression scores in patients (Zou et al., 2018; Leff Gelman et al., 2019). In our previous study, we showed that the levels of synaptic proteins are strongly correlated with cognitive dysfunction induced by maternal sleep deprivation (Wei et al., 2022). In the present study, the levels of proinflammatory cytokines and synaptic proteins were correlated with indicators of the OFT, EPM test, FST, TST, and MWM test. This indicated that the anxiety, depression, and cognitive impairment induced by prenatal exposure to inflammation coupled with maternal sleep deprivation may be a consequence, at least in part, of the upregulation of the levels of proinflammatory cytokines and the downregulation of the levels of synaptic proteins in the hippocampus.

Our study had some limitations. First, we only evaluated behavioral phenotypes in male offspring mice. Sex differences relating to the effect of maternal sleep deprivation and prenatal exposure to inflammation on the behavior of offspring were not evaluated. Secondly, this study only documented the association between inflammation and synaptic dysfunction and abnormal behaviors in the offspring. The underlying mechanisms were not explored. Thirdly, we examined the alterations in synaptic proteins and did not evaluate the hippocampal synaptic plasticity including long-term potentiation and long-term depression in the offspring mice. Lastly, the levels of markers of inflammation and synaptic function were determined only in the hippocampus and not in other brain regions associated with anxiety, depression, and cognitive impairment (Moore et al., 2023; Wood et al., 2023).