Ventromedial hypothalamus IL-6 signaling mediates the excessive aggressive behavior induced by late-gestational sleep deprivation in male mice

Abstract

Background

Aggressive behavior is an evolutionarily conserved trait that supports resource acquisition and survival; however, excessive or dysregulated aggression is closely associated with neuropsychiatric disorders and social dysfunction. Maternal sleep deprivation (SD) during late gestation is a common yet underexplored environmental stressor that may disrupt offspring neurodevelopment through immune-mediated pathways. The present study investigated whether SD during late pregnancy alters aggressive behavior in offspring during early adulthood, explored the underlying neuroimmune mechanisms, and evaluated the potential of a post-weaning ketogenic diet (KD) as a non-pharmacological intervention.

Results

Behavioral analyses revealed that late-gestational SD significantly increased both defensive and proactive aggressive behaviors in offspring mice during early adulthood. Peripheral immune assessment revealed that serum IL-6 levels were elevated in male offspring, while no significant change was observed in females. Pharmacological experiments demonstrated a causal role of IL-6 in aggression regulation, as peripheral administration of IL-6 increased aggression in control male offspring mice, whereas administration of anti-IL-6 in sleep deprivation-exposed male offspring normalized aggressive behavior. Neurobiological analyses identified enhanced neuronal activation, increased IL-6 and IL-6R expression, and pronounced microglial activation in the ventromedial hypothalamus (VMH). Consistently, VMH administration of IL-6 increased aggressive behavior, while injection of anti-IL-6 antibody attenuated the heightened aggression in SD-exposed male offspring mice. Chemogenetic inhibition of VMH neurons effectively attenuated the heightened aggression, indicating a critical role for this neural circuit. Importantly, post-weaning KD suppressed microglial overactivation and reduced IL-6 signaling in the VMH, restored neuroimmune homeostasis, and reversed abnormal aggressive behaviors.

Conclusions

These findings demonstrate that late-gestational maternal SD promotes excessive aggression in male offspring mice through IL-6-mediated neuroimmune dysregulation within the VMH. Moreover, post-weaning KD intervention effectively rescues both immune imbalance and behavioral abnormalities. This study highlights the long-term behavioral consequences of prenatal sleep loss and identifies dietary modulation of neuroimmune pathways as a promising non-pharmacological strategy for mitigating pathological aggression induced by early-life stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12993-026-00337-x.

Background

Aggressive behavior is one of the most important innate behaviors in most animals, moderate levels of aggression facilitate resource acquisition and species survival [1]. However, excessive aggression can disrupt normal developmental trajectories and pose direct threats to individual safety and social order [2, 3]. Clinically, heightened aggression is closely associated with psychiatric disorders such as schizophrenia, mania and antisocial personality disorder, which often emerge during childhood or adolescence and can persist without timely intervention [4–6]. The increasing prevalence of school violence and extreme social conflicts further underscores the urgency of clarifying the mechanisms underlying aggression dysregulation and developing effective interventions.

Early life represents a highly sensitive window for developmental programming, during which environmental exposures can exert lasting effects on long-term health outcomes [7, 8]. Early-life adversity is increasingly recognized as a major determinant of later vulnerability to psychiatric disorders. Among these, the maternal environment during early life has programming effects on fetal brain development, significantly shaping offspring cognition, emotion and social behavior [9]. Recent studies indicate that maternal experiences of stress during gestation can influence offspring neurodevelopment and behavioral phenotypes via intergenerational transmission mechanisms [10, 11]. In particular, prenatal stress may alter neurodevelopmental trajectories and increase the risk of psychiatric disorders in offspring [12]. Gestational sleep deprivation (SD), a common but often overlooked stressor, can affect fetal development through hormonal alterations and inflammatory responses, and its incidence increases as gestation progresses [13, 14]. Epidemiological studies have reported that poor maternal sleep quality during gestation is associated with offspring behavioral problems, including anxiety, hyperactivity and emotional dysregulation, suggesting that gestational SD represents an important early-life challenge shaping neurodevelopmental and behavioral outcomes [15]. However, the mechanisms by which gestational SD induces changes in offspring aggressive behavior through neuroimmune pathways remain largely unknown.

Gestational SD can influence fetal brain development via immune activation and neuroinflammation [16]. Maternal SD induces peripheral inflammation, which can disrupt offspring immune function and interfere with the integration of developing neural circuits [17]. IL-6 serves as a critical mediator of neuroimmune interactions, and its dysregulated expression in both peripheral and central systems is closely linked to behavioral alterations [18, 19]. Elevated peripheral IL-6 can access the brain via the blood–brain barrier (BBB) or activate microglia, thereby altering neuronal differentiation and circuit integration [20]. Clinical studies also report that increased IL-6 level during gestation is associated with a higher risk of behavioral abnormalities in offspring [21], although the precise mechanisms within discrete brain regions remain unclear.

Within the central nervous system (CNS), the ventromedial hypothalamus (VMH) is a key node regulating aggressive behavior by integrating physiological and environmental information to govern the onset and expression of aggression [22]. Recent evidence suggests that the VMH modulates individual responses to external metabolic and inflammatory challenges [23]. Under systemic inflammation, elevated IL-6 within the CNS disrupts excitatory-inhibitory balance and leads to neuronal hyperexcitability. This localized IL-6 overabundance selectively reduces inhibitory inputs in the rat temporal cortex via trans-signaling, thereby decreasing the inhibition/excitation ratio, leading to neuronal hyperactivation and subsequent escalation of aggressive behavior [24].

Interventions targeting neuroimmune homeostasis may mitigate the long-term impact of early-life adversity. The ketogenic diet (KD), characterized by high-fat, low-carbohydrate and adequate protein, has demonstrated unique benefits in preventing and treating neuropsychiatric disorders [25–27]. Since its introduction in 1921, KD has been shown in multiple animal models to improve metabolism and enhance stress resilience [28]. In the domain of emotional regulation, KD exhibits therapeutic benefits for depression, anxiety and bipolar disorder [29]. KD also exerts strong anti-inflammatory and neuroprotective effects, largely by suppressing microglial activation and reducing neuroinflammation [30]. Microglia, the primary immune cells of the CNS, are central to neuroimmune communication. Under physiological conditions, they maintain homeostasis by clearing cellular debris and participating in synaptic pruning [31]. Under stress, microglia undergo rapid morphological and functional activation, producing inflammatory cytokines and reshaping neural circuits [32]. As microglia are a major source of IL-6 in the CNS [33], KD may modulate IL-6–dependent signaling and restore circuit function, providing a potential strategy to counteract behavioral vulnerability programmed by early-life stress.

In this study, we investigated how late-gestational SD, as a model of early-life adversity, shapes aggressive behavior during early adulthood and explored the underlying neuroimmune mechanisms. We further assessed the therapeutic potential of post-weaning KD. By integrating behavioral analyses with peripheral immune profiling and central neuroinflammatory assessments, our work aims to delineate pathways linking prenatal adversity to offspring aggression and to inform strategies for promoting long-term health outcomes.

Materials and methods

Animals

A total of fifteen ICR mice (10 females and 5 males, aged 6–8 weeks) were obtained from Beijing SPF Biotechnology Co., Ltd. Animals were maintained in the barrier facility of the Experimental Animal Center, Hebei Medical University, under controlled temperature (20–25 °C), humidity (50–55%) and a 12 h light/dark cycle (lights on from 20:00 to 08:00), with ad libitum access to food and water. Mice were housed five per cage and allowed to acclimate for 7 days before breeding. Females were paired with males at a 2:1 ratio, and the presence of a vaginal plug was designated as gestational day 0 (GD0) [34]. All animals were randomly assigned to experimental groups using a random number table. To control for potential litter effects, 3–4 pups from each dam were randomly selected and allocated to the different groups. Each pregnant mouse was used only once. SD procedures were performed during the light phase, while behavioral testing was occurred during the dark phase. All behavioral assays followed standardized protocols to minimize stress-related interference. All animal procedures were performed in accordance with institutional and national regulations, following the NIH Guide for the Care and Use of Laboratory Animals, the ARRIVE 2.0 reporting guidelines and the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). All experimental protocols were reviewed and approved by the Animal Ethics Committee of Hebei Medical University (IACUC Approval No. IACUC-Hebmu-P2024164).

SD protocol

Late-gestational SD was performed using a specialized SD apparatus as previously described [35, 36]. This approach has been widely used in rodents and previously validated to reliably induce sleep loss during the inactive phase [37–40]. SD was conducted during the light phase, corresponding to the natural inactive period of mice, to selectively target sleep without broadly disrupting circadian rhythms. The system comprised a platform and a motor-driven rotating rod beneath it. Slow, continuous rod rotation disrupted the sleep–wake cycle without causing pain or tissue injury. Rotation speed was carefully controlled and gradually increased from 5 to 7 rpm to prevent adaptation while minimizing stress. The platform was lined with corncob bedding, and food and water were provided ad libitum. Pregnant mice in the SD group underwent SD for 6 h daily (22:00–04:00) from gestational day 14 (GD14) to GD20. Pregnant mice in the control group (CON group) were placed in identical apparatuses with the motor off to account for environmental exposure without sleep disruption. After each session, all mice were returned to their home cages. Male and female offspring (from litters with no significant difference in litter size) were subsequently used for experiments.

KD feeding protocol

The compositions of the control diet (CD) and KD were based on previous studies [41]. The CD contained 10.0% fat, 20.0% protein, 63.0% carbohydrates, 5.0% cellulose, 1.0% vitamins, 1.0% minerals and 0.1% choline bitartrate. The KD (XSYT-ED-044; Xiao Shu You Tai Biotechnology Co., Ltd, Beijing, China) consisted of 67.05% fat, 17.54% protein, 0.21% carbohydrates, 8.64% cellulose, 0.17% vitamins, 6.05% minerals and 0.34% choline bitartrate. The CD and KD were formulated to be approximately isocaloric, which ensures that the observed effects of the KD are attributable to differences in macronutrient composition rather than total caloric intake. Offspring were weaned at postnatal day 21 (P21), sex-segregated, housed five per cage and subjected to a 3-week KD intervention. During the first week, mice were gradually adapted to KD (30%, 50%, 70% and 100%), followed by two weeks of full KD feeding. Diet was placed in wire-lid hoppers, with chew sticks supplied throughout and replaced every two days. To examine the effects of KD, two sets of experiments were conducted. In the first set of experiments, we assessed whether postweaning KD could alleviate the increase in aggressive behavior induced by late-pregnancy SD. Following this, a separate set of experiments was performed to examine the effects of KD in CON offspring.

Behavioral tests

Open field test (OFT)

The OFT was conducted as previously described [42, 43]. Male and female mice were placed in the test room for two hours to acclimate and reduce environmental stress (n = 10 per group). Mice were individually placed in an open-field chamber (40 × 40 × 40 cm, Plexiglas), divided into a central zone (20 × 20 cm) and a peripheral zone. Each mouse was placed in the center and allowed to explore freely for 5 min. Total distance traveled and time spent in the central zone were recorded and analyzed using SMART 3.0 software (Panlab, Barcelona, Spain).

Three-chamber social interaction test (SIT)

The SIT was performed according to established methods [42, 44]. The apparatus (50 × 25 cm, Plexiglas) comprised three interconnected chambers with 5 cm openings. Male and female mice were placed in the test room for two hours to acclimate and reduce environmental stress (n = 10 per group). After a 5 min adaptation phase with empty wire cages in the side chambers, an unfamiliar same-sex and same-age mouse (stranger) was placed in one cage while the other remained empty. Time spent sniffing each cage during a 5 min test phase was recorded.

Defensive aggression test (DAT)

Defensive aggression was assessed as previously described [45]. Male and female mice were placed in the test room for two hours to acclimate and reduce environmental stress (n = 10 per group). A realistic rubber snake model with a metal alligator clip was used to simulate a predator attack. The clip was gently attached to the mouse’s tail, inducing defensive aggression without causing tissue injury. Mice were tested individually in a closed arena (21 × 21 × 14 cm) for 1 min. The latency, frequency and time of aggressive attacks were recorded.

Resident-intruder test (RIT)

The RIT was conducted following established protocols [46, 47]. In this paradigm, the resident mouse typically exhibits proactive aggression in response to territorial intrusion, whereas the intruder displays defensive aggression. Prior to testing, each resident mouse was housed individually for 7 days to establish territoriality. Male and female mice were placed in the test room for two hours to acclimate and reduce environmental stress (n = 10 per group). During the test, a slightly smaller, unfamiliar same-sex intruder of the same strain was introduced into the resident’s cage for 10 min. Behavioral parameters of the resident mice, including the latency, frequency and time of wrestling (grappling, body-to-body holding and vigorous tumbling), freezing and chasing, were analyzed using VisuTrack 3.0 software (XinRuan, Shanghai, China).

For each test, experimenters were blinded to group assignments and mice were selected at random. The apparatus was cleaned with 75% ethanol after each trial. All tests were performed double-blind.

Cytokine measurement by Luminex assay

Peripheral blood was collected one day after the completion of behavioral testing, and serum was isolated for analysis. Cytokine concentrations were determined using a multiplex magnetic bead-based Luminex assay (Mouse Cytokine/Chemokine Magnetic Bead Panel, MCYTOMAG-70 K; Millipore, MO, USA) according to the manufacturer’s instructions (n = 6 per group).

Immunofluorescence staining

For c-Fos immunofluorescence analysis, brain tissues were collected 1.5 h after the defensive aggression test (DAT). For IL-6, IL-6R, and IBA1 staining, brain tissues were collected one day after completion of the behavioral tests. Mice were anesthetized with intraperitoneal chloral hydrate (20 mg/mL) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA for 12 h and cryoprotected in 30% sucrose, then embedded in OCT compound for sectioning. Coronal Sects. (30 μm) were prepared using a cryostat (Leica, Wetzlar, Germany). Sections were incubated with primary antibodies against c-Fos (1:1000, 2250S, CST, MA, USA), IL-6 (1:200, 12,912, CST, MA, USA), IL-6R (1:100, 23,457-1-AP, Proteintech, Wuhan, China) and Iba1 (1:200, MABN92, Millipore, CA, USA) (n = 3 mice per group, 3 representative sections per mouse) and then secondary antibody (1:500, 4414S, CST, Massachusetts, USA; 1:500, ab150173, Abcam, MA, USA). Images were acquired under identical exposure parameters with a 3DHISTECH pathology scanner (Hungary), and anatomically defined regions were delineated according to a standard mouse brain atlas. For c-Fos quantification, c-Fos-positive cells were quantified by averaging counts from three representative sections per mouse. For each experimental condition, n = 3 mice were analyzed with 3 sections evaluated per mouse. The number of positive cells within each region was counted using Slideviewer software and normalized to the corresponding area (cells/mm²).

Peripheral IL-6 intervention protocol

The design of this IL-6 intervention experiment was based on previously published studies using peripheral IL-6 administration in mice [48, 49].

Intraperitoneal injection of IL-6

Six-week-old male offspring were randomly assigned to receive daily intraperitoneal injections of recombinant mouse IL-6 (CG39, Novoprotein; 3 ng/g, dissolved in sterile PBS, 0.2 ml) or vehicle (sterile PBS, 0.2 ml) for 7 consecutive days (n = 10 per group). The dosage and preparation followed previous reports [50, 51]. DAT was performed 24 h after the final injection to assess defensive aggressive behavior.

Intraperitoneal injection of anti-IL-6

Six-week-old male offspring of pregnant mice were exposed to daily intraperitoneal injections of anti-IL-6 mAb (MP5-20F3, Bio X Cell, NH, USA; 5 ng/g, dissolved in sterile PBS, 0.2 ml) or vehicle (sterile PBS, 0.2 ml) for 7 days (n = 10 per group) randomly. The dosage and preparation followed previous reports [52, 53]. DAT was performed 24 h after the final injection to assess defensive aggressive behavior.

Stereotaxic surgery and chemogenetic viral injection

Five-week-old male mice were anesthetized with pentobarbital sodium (1%, 50 mg/kg, i.p.) and secured in a stereotaxic apparatus (RWD, Shenzhen, China). Eyes were protected with erythromycin ointment, the scalp shaved and disinfected and a 1 cm midline incision made to expose the skull. According to the mouse brain atlas (2nd edition), the injection site was targeted to the VMH (AP − 1.34 mm, ML ± 0.391 mm, DV − 5.88 mm). A 0.5 mm burr hole was drilled, and AAV2/9-hSyn-hM3D(Gq)-ER2-P2A-EGFP-WPRE-pA (1.25 × 10¹³ GC/mL) or AAV2/9-hSyn-hM4D(Gi)-ER2-P2A-EGFP-WPRE-pA (1.27 × 10¹³ GC/mL) (Taitool Bioscience, Shanghai, China) was bilaterally injected (200 nL per side) at 0.1 μL/min using a 10 μL microsyringe (Gaoge, Shanghai, China). The needle remained in place for 10 min before slow withdrawal. Incisions were sutured and disinfected, and mice recovered on a 37 ℃ heating pad with daily monitoring. After 3–4 weeks, viral transduction in the VMH was confirmed via EGFP immunofluorescence. For chemogenetic activation, mice received an intraperitoneal injection of deschloroclozapine (DCZ) (1 mg/kg) or PBS 30 min prior to DAT.

Cannula implantation and IL-6 microinjection

Five-week-old male mice were anesthetized with pentobarbital sodium (1%, 50 mg/kg, i.p.) and secured in a stereotaxic apparatus (RWD, Shenzhen, China). Eyes were protected with erythromycin ointment, the scalp shaved and disinfected, and a 1 cm midline incision made. According to the mouse brain atlas (2nd edition), a guide cannula (c = 5.5 mm, g1 = 0.5 mm, g2 = 0 mm, C.C = 0.8) was implanted in the VMH (AP − 1.34 mm, ML ± 0.391 mm, DV − 5.38 mm). A small burr hole was drilled at the target site and three additional sites for anchor screws. The cannula and screws were fixed with dental cement, and mice recovered on a 37 ℃ heating pad. Microinjections and behavioral testing were performed one week after surgery. For microinjection, an inner injector was connected to a microsyringe via PU tubing and mounted on an injection pump. CON and SD groups received PBS; CON + IL-6 group received IL-6 (50,136-MNAE, Sino Biological, Beijing, China); SD + anti-IL-6 group received anti-IL-6 mAb (MP5-20F3, Bio X Cell, NH, USA). Injections (0.2 μL) were delivered at 0.2 μL/min, with the injector left in place for 2 min to prevent reflux. DAT was performed 30 min post-injection, and mice were perfused 1.5 h after behavioral assessment.

Statistical analysis

All data are expressed as mean ± standard error of the mean (SEM). Statistical analyses and graphical representations were generated using GraphPad Prism version 8.0.1. Prior to hypothesis testing, data were assessed for normality using the Shapiro–Wilk test. For comparisons between two groups, normally distributed data were analyzed using Student’s t-test, while non-normally distributed data were analyzed using the Mann–Whitney U test. For comparisons among multiple groups, homogeneity of variance was tested using Levene’s test. Normally distributed data with equal variances were analyzed by one-way analysis of variance (ANOVA) followed by Fisher’s LSD post hoc test; if assumptions were not met, the Kruskal–Wallis test followed by Dunn’s post hoc test was applied. All statistical tests were two-tailed, and a P value less than 0.05 was considered statistically significant.

Results

Late-gestational SD increases aggressive behavior in offspring during early adulthood

The experimental procedure is illustrated in Fig. 1A. Late-pregnancy SD had no significant effects on body weight, food and water intake (Figure S1), locomotor activity, or social interaction behavior (Figure S2) in either male or female mice. In the DAT, SD male offspring exhibited significantly shorter bite latency (t₁₈ = 5.450, P < 0.0001) (Fig. 1B), as well as markedly increased bite frequency (t₁₈ = 4.940, P < 0.0001) (Fig. 1C) and bite time (U = 5.450, P < 0.0001) (Fig. 1D) compared with CON offspring. In the RIT, SD male offspring displayed reduced latency to wrestling (t₁₈ = 3.015, P = 0.0074) (Fig. 1E) and freezing (U = 18, P = 0.0147) (Fig. 1H), along with increased wrestling frequency (t₁₈ = 5.563, P < 0.0001) (Fig. 1F), wrestling time (t₁₈ = 3.054, P = 0.0068) (Fig. 1G), freezing frequency (t₁₈ = 3.488, P = 0.0026) (Fig. 1I) and freezing time (t₁₈ = 2.984, P = 0.0080) (Fig. 1J). No significant differences were observed in chasing latency (U = 44.00, P = 0.0842) (Fig. 1K), chasing frequency (t₁₈ = 1.217, P = 0.2395) (Fig. 1L), or chasing time (U = 28.50, P = 0.1090) (Fig. 1M).

Fig. 1.

Late-gestational SD enhances both defensive and proactive aggressive behavior in male offspring. A Experimental procedure. B Latency, C frequency and D time of biting attack of defensive aggressive behavior in offspring. E Latency, F frequency and G time of wrestling of proactive aggressive behavior in offspring. H Latency, I frequency and J time of freezing of proactive aggressive behavior in offspring. K Latency, L frequency and M time of chasing of proactive aggressive behavior in offspring. Data are presented as mean ± SEM. Comparisons between two groups were performed using Student’s t-test or the Mann–Whitney U test, as appropriate. n = 10 per group. ^*P < 0.05

In female offspring, late-gestational SD increased defensive aggressive behavior, as indicated by decreased attack latency and increased attack frequency and duration. In contrast, the effect on proactive aggression was relatively limited, with reductions in the latency to wrestling and chasing and an increase in freezing time (Figure S3).

These behavioral results suggest that late-gestational SD significantly increases proactive and defensive aggressive behaviors in both male and female mice during early adulthood, with a more significant effect observed in male mice.

Late-gestational SD disrupts peripheral immune status in offspring during early adulthood

Multiplex cytokine analysis revealed elevated serum IL-6 in SD males (t₉ = 2.427, P = 0.0382) (Fig. 2F) without changes in IL-1β, IL-2, IL-4, IL-10, IL-17, or TNF-α (Fig. 2G–L). In contrast, none of these cytokines were significantly altered in female offspring (Figure S4E-K).

Fig. 2.

Late-gestational SD links peripheral immune disruption and elevated IL-6 to defensive aggression in male offspring. A–G Serum levels of IL-6, IL-1β, IL-2, IL-4, IL-10, IL-17 and TNF-α (pg/ml). Data are presented as mean ± SEM. n = 5–6 per group. Comparisons between two groups were performed using Student’s t-test or the Mann–Whitney U test, as appropriate. H Experimental procedure of peripheral IL-6/anti-IL-6 injection. I Latency, J frequency and K time of biting attack of defensive aggressive behavior in the DAT after peripheral IL-6 or anti-IL-6 injection. Data are presented as mean ± SEM. n = 10 per group. Multiple-group comparisons were conducted using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. ^*P < 0.05

To further verify the role of peripheral IL-6 in late-gestational SD induced-aggressive behavior, male mice received intraperitoneal IL-6 or anti-IL-6 mAb (Fig. 2M). Results showed that intraperitoneal injection of IL-6 did not alter bite latency (H = 17.94, P = 0.0005; CON + vehicle vs. CON + IL-6: P = 0.6007; Fig. 2N), but increased bite frequency (H = 20.26, P = 0.0002; CON + vehicle vs. CON + IL-6: P = 0.0376; Fig. 2O) and time among groups (H = 18.34, P = 0.0004; CON + vehicle vs. CON + IL-6: P = 0.0287; Fig. 2P). Conversely, IL-6 blockade prolonged latency (H = 17.94, P = 0.0005; SD + vehicle vs. SD + anti-IL-6: P < 0.0001; Fig. 2N) and reduced both frequency (H = 20.26, P = 0.0002; SD + vehicle vs. SD + anti-IL-6: P < 0.0001; Fig. 2O) and time of biting among groups (H = 18.34, P = 0.0004; SD + vehicle vs. SD + anti-IL-6: P = 0.0003; Fig. 2P). In total, exogenous IL-6 enhanced defensive aggression in control male mice, whereas anti-IL-6 treatment reduced excessive aggression in SD male offspring, indicating that peripheral IL-6 is a key mediator of SD-induced aggression.

Chemogenetic inhibition of VMH reverses the heightened aggression in male offspring exposed to late-gestational SD

Whole-brain c-Fos immunostaining revealed that SD male offspring exhibited significantly enhanced neuronal activation in aggression-related regions, including the anterior hypothalamic nucleus (AHN) (t₁₆ = 11.34, P < 0.0001), VMH (t₁₆ = 5.510, P < 0.0001), periaqueductal gray (PAG) (t₁₆ = 5.928, P < 0.0001), basolateral amygdala (BLA) (t₁₆ = 10.56, P < 0.0001), posterior hypothalamus (PH) (t₁₆ = 2.305, P = 0.0349), posteromedial amygdala (MePD) (t₁₆ = 7.805, P < 0.0001) and posteroventral amygdala (MePV) (U = 5, P = 0.0008) (Fig. 3A, B).

Fig. 3.

VMH neuronal activity correlates with defensive aggression in male offspring after late-gestational SD. A Quantification of c-Fos + neurons in aggression-related regions. B Representative c-Fos immunofluorescence staining image of offspring in CON and SD groups. Data are presented as mean ± SEM. n = 3 mice × 3 slides per group. Comparisons between two groups were performed using Student’s t-test or the Mann–Whitney U test, as appropriate. C Experimental schematic. D Experimental timeline of chemogenetic manipulation. E Verification of hM3Dq viral injection point using fluorescence staining. F Verification of hM4Di viral injection point using fluorescence staining. G Latency, H frequency and I time of biting attack of defensive aggressive behavior in the DAT after VMH activation with hM3Dq and hM4Di. Data are presented as mean ± SEM. n = 10 per group. Multiple-group comparisons were conducted using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. ^*P < 0.05

Considering the key role of VMH region in regulation of aggressive behavior, and to assess the potential role of VMH region in enhanced aggression of male offspring mice exposed to late-gestational SD, chemogenetic manipulation was performed (Fig. 3C, D). Viral targeting was confirmed via fluorescence imaging (Fig. 3, F). Activation of VMH neurons via AAV2/9-hSyn-hM3D(Gq) + DCZ reduced bite latency (H = 26.78, P < 0.0001; CON + hM3D(Gq) + vehicle vs. CON + hM3D(Gq) + DCZ: P = 0.0007; Fig. 3G) and increased bite frequency (H = 28.63, P < 0.0001; CON + hM3D(Gq) + vehicle vs. CON + hM3D(Gq) + DCZ: P = 0.0134; Fig. 3H) and time (H = 28.57, P < 0.0001; CON + hM3D(Gq) + vehicle vs. CON + hM3D(Gq) + DCZ: P = 0.0007; Fig. 3I) compared with CON + hM3D(Gq) + vehicle. Conversely, VMH inhibition in SD offspring via AAV2/9-hSyn-hM4D(Gi) + DCZ increased bite latency (H = 26.78, P < 0.0001; SD + hM4D(Gi) + vehicle vs. SD + hM4D(Gi) + DCZ: P < 0.0001; Fig. 3G) and reduced bite frequency (H = 28.63, P < 0.0001; SD + hM4D(Gi) + vehicle vs. SD + hM4D(Gi) + DCZ: P = 0.0002; Fig. 3H) and time (H = 28.57, P < 0.0001; SD + hM4D(Gi) + vehicle vs. SD + hM4D(Gi) + DCZ: P < 0.0001; Fig. 3I) relative to SD + hM3D(Gi) + vehicle.

IL-6/IL-6R signaling in the VMH may contribute to the increased aggression in male early adult offspring mice exposed to late-gestational SD

Immunofluorescence analysis revealed that SD offspring displayed significantly elevated VMH IL-6 (t₁₆ = 3.542, P = 0.0027) (Fig. 4A) and IL-6R (t₁₆ = 9.614, P < 0.0001) (Fig. 4B) compared with CON offspring. To validate the role of IL-6 in the VMH, IL-6 or anti-IL-6 was microinjected (Fig. 4C, D). IL-6 microinjection in CON male offspring decreased bite latency (H = 25.62, P < 0.0001; CON + vehicle vs. CON + IL-6: P = 0.0035; Fig. 4E) and increased bite frequency (H = 27.69, P < 0.0001; CON + vehicle vs. CON + IL-6: P = 0.0009; Fig. 4F) and time (H = 28.91, P < 0.0001; CON + vehicle vs. CON + IL-6: P = 0.0003; Fig. 4G). Conversely, anti-IL-6 microinjection in SD male offspring prolonged bite latency (H = 25.62, P < 0.0001; SD + vehicle vs. SD + anti-IL-6: P = 0.0002; Fig. 4E) and reduced bite frequency (H = 27.69, P < 0.0001; SD + vehicle vs. SD + anti-IL-6: P < 0.0001; Fig. 4F) and time (H = 28.91, P < 0.0001; SD + vehicle vs. SD + anti-IL-6: P = 0.0002; Fig. 4G).

Fig. 4.

VMH IL-6/IL-6R signaling correlates with defensive aggression in male offspring after late-gestational SD. A IL-6 expression in the VMH of CON group and SD group offspring. B IL-6R expression in the VMH of CON group and SD group offspring. Data are presented as mean ± SEM. n = 3 mice per condition with 3 sections analyzed per mouse. Comparisons between two groups were performed using Student’s t-test or the Mann–Whitney U test, as appropriate. C Experimental schematic of VMH cannula injection. D Experimental timeline of microinjection. E Latency, F frequency and G time of biting attack of defensive aggressive behavior in the DAT after VMH IL-6 and anti-IL-6 injection. Data are presented as mean ± SEM. n = 10 per group. Multiple-group comparisons were conducted using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. ^*P < 0.05

Inhibitory effects of KD exposure on aggression in in male early adult offspring mice exposed to late gestational SD

To explore a potential intervention, SD offspring received a 3-week KD. In the DAT, KD significantly prolonged bite latency (H = 13.96, P = 0.0009; SD vs. SD + KD: P = 0.0004; Fig. 5A) and reduced bite frequency (H = 11.86, P = 0.0027; SD vs. SD + KD: P = 0.0011; Fig. 5B) and bite time (H = 10.43, P = 0.0054; SD vs. SD + KD: P = 0.0022; Fig. 5C) compared with SD alone. In the RIT, KD prolonged latency to wrestling (H = 14.29, P = 0.0008; SD vs. SD + KD: P = 0.0021; Fig. 5D) and freezing (H = 11.40, P = 0.0033; SD vs. SD + KD: P = 0.0238; Fig. 5G), while decreasing wrestling frequency (H = 10.65, P = 0.0049; SD vs. SD + KD: P = 0.0013; Fig. 5E) and time (H = 14.68, P = 0.0006; SD vs. SD + KD: P = 0.0002; Fig. 5F), decreased freezing frequency (H = 11.25, P = 0.0036; SD vs. SD + KD: P = 0.0005; Fig. 5H) and time (H = 17.16, P = 0.0002; SD vs. SD + KD: P = 0.0289; Fig. 5I), as well as chasing time (H = 10.83, P = 0.0044; SD vs. SD + KD: P = 0.0017; Fig. 5L) without affecting chasing latency (Fig. 5J) or frequency (Fig. 5K).

Fig. 5.

KD reverses prenatal SD-induced excessive aggressive behavior in male offspring. A Latency, B frequency and C time of biting attack of defensive aggressive behavior in the DAT. D Latency, E frequency and F time of wrestling of proactive aggressive behavior in the RIT. G Latency, H frequency and I time of freezing of proactive aggressive behavior in the RIT. J Latency, K frequency and L time of chasing of proactive aggressive behavior in the RIT. Data are presented as mean ± SEM. n = 10 per group. Multiple-group comparisons were conducted using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. ^*P < 0.05

After demonstrating that postweaning KD alleviates the increase in aggressive behavior induced by late-pregnancy SD, we further examined whether KD could influence aggression in CON offspring. To this end, CON offspring without SD were maintained on a KD after weaning and assessed aggression (Figure S5A-C). In male mice, KD significantly reduced defensive aggression in the DAT, as indicated by prolonged attack latency and decreased attack frequency and time compared with CON mice (latency: t₁₈ = 5.702, P < 0.0001; frequency: t₁₈ = 3.456, P = 0.0028; time: t₁₈ = 3.078, P = 0.0065; Figure S5D-F). Similarly, in the RIT, KD increased the latency of wrestling(U = 23.5, P = 0.0452), freezing (t₁₈ = 3.045, P = 0.0070),and chasing (t₁₈ = 2.169, P = 0.0437), while reducing the frequency (freezing: t₁₈ = 3.034, P = 0.0071; chasing: U = 22, P = 0.0332) and time (wrestling: U = 22.5, P = 0.0374; freezing: t₁₈ = 2.590, P = 0.0185; chasing: U = 18.5, P = 0.0155) of these aggressive behaviors. Only the frequency of wrestling did not differ between groups. (Figure S5G-O). In female mice, KD also reduced defensive aggression in the DAT (latency: t₁₈ = 2.287, P = 0.0345; frequency: t₁₈ = 2.310, P = 0.0329; time: t₁₈ = 2.163, P = 0.0442; Figure S6A-C). In the RIT, KD reduced the time of wrestling (t₁₈ = 2.607, P = 0.0178) and freezing (t₁₈ = 2.426, P = 0.0260) and increased chasing latency (t₁₈ = 2.905, P = 0.0095), whereas other proactive aggression indicators were not significantly affected (Figure S6D-L), indicating a relatively modest effect compared with males.

KD exposure reverses late SD-induced neuroimmune abnormalities in late-gestational SD-exposed male offspring during early adulthood

Morphological analysis of VMH microglia revealed that SD decreased fractal dimension (F_2,9 = 16.29, P = 0.0010; CON vs. SD: P = 0.0011), increased circularity (F_2,9 = 118.4, P < 0.0001; CON vs. SD: P < 0.0001) and reduced branch endpoints (F_2,9 = 35.56, P < 0.0001; CON vs. SD: P < 0.0001). KD partially restored microglial fractal dimension (F_2,9 = 16.29, P = 0.0010; SD vs. SD + KD: P = 0.0006), reduced circularity (F_2,9 = 118.4, P < 0.0001; SD vs. SD + KD: P < 0.0001), and increased branch endpoints (F_2,9 = 35.56, P < 0.0001; SD vs. SD + KD: P = 0.0015) (Fig. 6A). Immunofluorescence demonstrated that SD elevated total Iba1⁺ cells, Iba1⁺/IL-6⁺ co-labeled, IL-6⁺ and IL-6R⁺ cells in the VMH, all of which were significantly reduced by KD (total Iba1⁺: H = 17.43, P = 0.0002; CON vs. SD: P = 0.0005; SD vs. SD + KD: P = 0.0002; Iba1⁺/IL-6⁺: H = 13.56, P = 0.0011; CON vs. SD: P = 0.0003; SD vs. SD + KD: P = 0.0259; IL-6⁺: H = 10.12, P = 0.0063; CON vs. SD: P = 0.0022; SD vs. SD + KD: P = 0.0222; IL-6R⁺: H = 17.51, P = 0.0002; CON vs. SD: P = 0.0006; SD vs. SD + KD: P = 0.0001) (Fig. 6B).

Fig. 6.

KD reverses prenatal SD-induced VMH immune dysregulation in male offspring. A Morphological analysis of VMH microglia: fractal dimension, circularity, maximum branch length and end-point voxels. n = 4 per group. B Number of Iba1⁺ cells, Iba1⁺IL-6⁺ cells, IL-6⁺ and IL-6R⁺ cells in the VMH. Images/data for IL-6 and IL-6R in Panel B are reproduced from Fig. 4 for comparison purposes; an additional group (SD + KD) is included in this figure. Data are presented as mean ± SEM. n = 3 mice × 3 slides per group. Multiple-group comparisons were conducted using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test. ^*P < 0.05

Discussion

This study provides a systematic investigation of how late-gestational SD, a clinically relevant form of early-life adversity, programs vulnerability to heightened aggression in offspring during early adulthood. Our findings suggest that late-gestational SD results in heightened aggressive tendencies in offspring, consistent with clinical evidence linking maternal stress during pregnancy to increased psychiatric vulnerability in progeny [54], emphasizing the essential role of sleep during pregnancy in shaping offspring neurobehavioral development. We found that SD increased peripheral IL-6 levels and induced hyperactivation of the VMH, accompanied by elevated IL-6/IL-6R expression and aberrant microglial activation. These neuroimmune alterations underlie the increased defensive and proactive aggressive behaviors observed in offspring, highlighting a mechanistic link between prenatal adversity, developmental programming of neural circuits, and long-term behavioral outcomes. Importantly, post-weaning KD intervention partially normalized peripheral and central IL-6 signaling, restored microglial homeostasis and attenuated aggressive behavior. These findings indicate that late-gestational SD promotes offspring aggression via neuroimmune dysregulation, and KD may provide a viable non-pharmacological strategy to improve long-term health outcomes in individuals exposed to early-life adversity.

Prenatal stress is a well-established risk factor for psychiatric disorders, contributing to heightened impulsivity and aggression in offspring [55]. For instance, reduced maternal care enhances offspring aggression [56], and prenatal stress disrupted hippocampal synaptic plasticity, impairing social behavior and indirectly promoting defensive aggression [57]. Our findings indicate that late-gestational SD, as a prenatal chronic stressor, may alter aggression-regulating neural networks via neuroimmune mechanisms. Epidemiological studies reveal that males are more susceptible to neurodevelopmental disorders such as autism, ADHD and schizophrenia [58, 59]. Although depression prevalence is lower in men, suicide rates, which correlate with depressive severity, are markedly higher [60], implying greater male vulnerability to early-life environmental stressors.

Recent studies indicate that maternal stressors such as hypoxia or SD can profoundly impact offspring neurodevelopment and behavior through immune activation. Clinically, poor sleep quality correlates with elevated serum IL-6 levels [61]. Additionally, genetic polymorphisms of IL-10 and TNF-α have been positively correlated with aggression [62], and IL-1 and IL-2 may modulate aggression through central neuroinflammatory mechanisms [63]. Increased IL-17 and decreased C3 levels could serve as potential biomarkers of aggression in schizophrenia [64]. In the initial exploration of underlying mechanisms, peripheral IL-6 levels were measured in both male and female offspring. Notably, significant differences in response to late-gestational SD were observed only in males, which also exhibited a stronger aggressive behavioral phenotype. Accordingly, subsequent mechanistic experiments focused on male offspring, and aggression was assessed using the DAT, which exhibited a stable and reproducible behavioral phenotype in our experiment without requiring prolonged single housing. Based on these results, all subsequent mechanistic experiments were conducted in male offspring to directly probe the causal pathways relevant to the aggression phenotype. The elevated serum IL-6 observed in males may originate from multiple immune or non-immune cell populations, such as monocytes/macrophages or other cytokine-producing cells, and the precise cellular source could be further explored in future studies.

Evidence suggests that maternal late-gestational SD can induce systemic inflammatory responses, including elevated levels of IL-6 and other cytokines, which may influence fetal development through maternal-placental-fetal signaling pathways [65]. Previous preclinical studies have identified IL-6 as a key mediator linking adverse prenatal environments to long-term neurobehavioral alterations. For instance, maternal immune activation models have demonstrated that increased maternal IL-6 during gestation can alter fetal brain development and lead to persistent behavioral abnormalities in offspring, while blockade of IL-6 signaling attenuates these effects [21]. In addition, placental IL-6 signaling has been shown to regulate communication between maternal inflammation and the developing fetal brain, thereby shaping neural circuit development and later behavioral phenotypes [66]. Other studies have further reported that prenatal stress-related inflammatory responses can influence offspring neuroimmune regulation and increase vulnerability to neuropsychiatric-like behaviors in later life [67, 68].

Although the present study did not directly measure maternal cytokine levels, our findings showing increased IL-6 levels in offspring and behavioral alterations are broadly consistent with these previous observations. Compared with classical maternal immune activation models that rely on immune stimulants such as poly(I:C) or lipopolysaccharide, our model uses late-gestational SD as an environmental stressor, which may represent a more physiologically relevant condition. Furthermore, while many prior studies have primarily focused on social deficits or anxiety-like behaviors, the present study emphasizes aggression-related behavioral outcomes and their potential association with neuroimmune alterations. Together, these similarities and differences suggest that SD-induced maternal stress may converge on inflammatory signaling pathways, including IL-6-related mechanisms, to influence offspring neurodevelopment and long-term behavioral phenotypes.

Peripheral IL-6 may contribute to inflammatory signaling associated with prenatal stress, whereas behavioral regulation ultimately relies on central neural circuit activity. We therefore performed c-Fos immunofluorescence to assess brain-wide neuronal activity following late-gestational SD and observed increased c-Fos expression in several regions. Among these regions, the VMH is particularly notable, as it is closely associated with both aggressive behavior and inflammatory responses, suggesting hyperactivation of aggression-related circuits. Abundant evidence identifies the VMH as a central hub for aggression regulation. Early electrical stimulation studies demonstrated that VMH activation elicited attack responses [69], while recent optogenetic studies show that transient activation of VMH glutamatergic neurons triggers aggression, and their inhibition suppresses it [70, 71]. The VMH integrates emotional, endocrine and environmental cues from upstream regions such as the BLA and AHN to coordinate behavioral responses to threats or social conflicts [72], and projects to the PAG and brainstem to drive downstream execution and defensive circuits [73]. Thus, VMH hyperexcitability likely represents a causal driver rather than a downstream of aggression, the other activated regions likely contribute to the phenotype through their connectivity and modulatory influence on VMH circuits. As part of the hypothalamus, the VMH resides in a region with a relatively permeable BBB, rendering it particularly sensitive to peripheral cytokine fluctuations [74]. Proinflammatory cytokines such as IL-6 can modulate hypothalamic neuronal excitability and metabolism, influencing energy balance and social behaviors [75]. Given the observed hyperactivation of the VMH, a core region closely linked to aggressive behavior, we employed chemogenetic approaches to selectively modulate VMH neuron activity. Our results indicate that VMH excitability plays a critical role in mediating defensive aggression and suggest that late-gestational SD shapes offspring aggressive behavior through alterations in VMH neural activity.

Accumulating evidence suggests that circulating cytokines can access the hypothalamus through permeable BBB regions or active transport mechanisms [76, 77]. Elevated hippocampal IL-6 has been shown to inhibit neurogenesis and induces emotional and cognitive deficits [78]. Zhang et al. [17] reported that gestational SD impairs offspring cognition through neuroinflammation and synaptic dysfunction, and another study confirmed that prenatal SD elevates brain IL-6, impairing neurogenesis and inducing behavioral abnormalities [79]. These findings suggest that IL-6 may cross the BBB to amplify inflammatory signaling and disrupt neuroimmune homeostasis in offspring. Our results suggest that SD-induced behavioral alterations may be associated with upregulation of IL-6/IL-6R signaling. Increased IL-6R expression could enhance local neuronal sensitivity to IL-6, thereby amplifying aggression-related neural signaling. To further validate this, stereotaxic microinjections of IL-6 or anti-IL-6 were performed in the VMH. The results demonstrated that IL-6 not only mediates central neural function via peripheral inflammatory signaling but also acts directly on VMH neurons to modulate excitability and drive heightened aggression. Although some studies have suggested that IL-6 administration primarily induces fever without clear behavioral effects, whereas IL-6 knockout mice display enhanced aggression, these seemingly contradictory findings likely reflect the context-dependent nature of IL-6 signaling, as differences in experimental paradigms and developmental compensation associated with lifelong genetic deletion may lead to distinct neuroimmune and behavioral outcomes.

Both peripheral and central inflammatory mechanisms may contribute to the behavioral alterations observed in the present study. Elevated IL-6 levels in the periphery can act as upstream signals that influence central nervous system function through multiple pathways, including cytokine transport across the BBB, activation of microglial cells, and neural-immune communication pathways [80, 81]. Previous studies have shown that peripheral inflammatory cytokines are capable of modulating brain activity and altering behavioral responses [82]. Importantly, behavioral regulation is ultimately governed by central neural circuits. Cytokine signaling within the brain can influence neurotransmission, synaptic plasticity, and neuroendocrine responses, thereby shaping behavioral outputs. For example, IL-6 and other inflammatory cytokines can modulate serotonergic and dopaminergic signaling and influence neural circuits involved in motivation and emotional regulation [83]. In this context, peripheral IL-6 may serve as an upstream inflammatory trigger, while central neural circuits translate these signals into behavioral responses. Therefore, the increased aggression observed in our model may reflect the combined effects of peripheral inflammatory signaling and central neural circuit regulation. This integrated perspective highlights the potential interaction between systemic immune signals and brain circuits involved in aggression.

Currently, dietary interventions such as KD offer a safe and noninvasive approach to mitigating aggression, complementing existing clinical strategies that are often limited by invasiveness or compliance. Microglia serve as a key interface linking environmental stressors to neural circuit function, and their overactivation is closely associated with neuropsychiatric disorders [84, 85]. In aggression and emotion regulation, microglia are considered an important bridge linking environmental stressors to functional alterations in neural circuits [86, 87]. Furthermore, KD has been reported to suppress microglial activation, reduce inflammation and ameliorate neural function [88]. Consistent with previously reported morphological characteristics of activated microglia [89], SD offspring in the present study exhibited microglial morphological changes accompanied by increased IL-6 expression in the VMH, indicating a neuroinflammatory state associated with heightened aggression. These findings suggest that late-pregnancy SD may promote microglial activation and establish a proinflammatory environment in the offspring brain. Importantly, postweaning KD partially reversed these microglial morphological abnormalities and normalized IL-6/IL-6R expression in the VMH, suggesting that its anti-aggressive effects may be mediated through modulation of the neuroimmune environment. Supporting this interpretation, no significant differences in body weight were observed among groups, which likely reflects the controlled dietary protocol and the timing of the post-weaning intervention rather than a lack of physiological impact. Together, these findings indicate that KD mitigates SD-induced aggressive behavior, at least in part, by restoring neuroimmune balance.

Notably, the effects of KD on inflammation appear to be context-dependent. While several studies report anti-inflammatory properties of KD, others have described neutral or variable outcomes, particularly in peripheral inflammatory responses. Therefore, the beneficial effects observed in the present study are likely not attributable to a single pathway but rather to an integrated modulation of metabolic, neurotransmitter, and neuroimmune mechanisms. These observations expand the potential relevance of KD as a preventive strategy for stress-related neuropsychiatric disorders, while highlighting the need for further studies to clarify its context-specific effects on inflammatory signaling.

To determine whether KD specifically targets SD-induced aggression or also exerts broader behavioral effects, we applied KD to CON offspring and found that it influenced aggression-related behaviors even in the absence of prenatal stress, suggesting a potential intrinsic anti-aggressive effect. These findings help distinguish the restorative and preventive roles of KD, providing a more comprehensive basis for its potential clinical application. Notably, the effect appeared more pronounced in males, which may reflect sex-related differences in hormonal regulation or neuroimmune responsiveness and warrants further investigation.

Despite revealing the neuroimmune mechanisms underlying gestational SD-induced offspring aggression, several limitations should be acknowledged. First, given the stability of behavioral phenotypes and pronounced differences in peripheral IL-6 levels in male offspring, immunofluorescence observations and subsequent intervention experiments were conducted only in males. Considering the regulatory effects of sex hormones on neuroimmune function [90] and previously reported sex differences in neuropsychiatric disorders [91], whether these findings can be generalized to female offspring remains to be determined. Sex differences may involve estrogen-mediated modulation of microglial activation and IL-6 signaling, which warrants further investigation in future studies. Notably, the estrous stage was not monitored in the present study because mechanistic investigations were conducted exclusively in male mice. Given that fluctuations in ovarian hormones across the estrous cycle may influence behavioral variability and neuroimmune responses, this factor should be considered in future studies. Furthermore, while our findings highlight a potential involvement of IL-6 signaling, the broader or long-term systemic effects of IL-6 modulation were not extensively examined in the present study and therefore merit further investigation. Additional experiments such as IL-6 administration in SD-exposed mice and IL-6 blockade in control mice could also be considered in future studies to strengthen causal evidence. The inclusion of matched rat IgG isotype antibodies as controls, as well as electrophysiological recordings before and after DCZ application to verify the effectiveness of the manipulation, could further improve the experimental rigor of future studies. Additionally, the VMH was examined as a whole region rather than separating its anterior and posterior parts. These subdivisions may contribute differently to defensive and offensive aggression, so analyzing them together could reduce the ability to detect potential subregion-specific changes. Future work that differentiates these VMH subregions will help refine the anatomical understanding of aggression circuits. Finally, this study primarily focused on the VMH and IL-6 signaling, whereas aggressive behavior may also involve broader neural circuits such as the prefrontal-amygdala pathways and various inflammatory factors. Future studies integrating multi-omics and neural circuit manipulation techniques are needed to construct a comprehensive immune-neural-behavioral regulatory map.

Conclusion

Late-gestational SD mediates enhanced aggression in offspring through peripheral immune dysregulation and hyperactivation of IL-6/IL-6R signaling within the VMH. KD intervention effectively reversed these neuroimmune abnormalities and excessive aggression, indicating that IL-6 signaling in the VMH plays a critical regulatory role in stress-related behavioral disturbances during pregnancy and providing mechanistic evidence for non-pharmacological strategies targeting neuroimmune pathways to improve health outcomes.

Abbreviations

VMH

Ventromedial hypothalamus

IL-6

Interleukin-6

IL-6R

Interleukin-6 receptor

SD

Sleep deprivation

ANOVA

Analysis of variance

AVMA

American Veterinary Medical Association

BBB

Blood-brain barrier

CD

Control diet

KD

Ketogenic diet

CNS

Central nervous system

GD

Gestational day

OFT

Open field test

SIT

Three-chamber social interaction test

DAT

Defensive aggression test

RIT

Resident-intruder test

P21

Postnatal day 21

PBS

Phosphate-buffered saline

PFA

Paraformaldehyde

RBC

Red blood cell

LC/MS

Liquid chromatography–mass spectrometry

AP

Anterior-posterior

DV

Dorsal-ventral

ML

Medial-lateral

mAb

Monoclonal antibody

DCZ

Deschloroclozapine

EGFP

Enhanced green fluorescent protein

NIH

National Institutes of Health

SEM

Standard error of the mean

Author contributions

Shi HS designed the study. Zhou F wrote the manuscript. Zhou F, Yu X, Gong M, Liu XY, Li XC, Li YL, Song L and Wang SJ performed the experiments and analyzed the data. Guo QJ and Shi HS revised the manuscript. The manuscript was reviewed and approved by all authors.

Funding

This study was partly financed by the Key Project Foundation of Hebei Education Department (JCZX2026027), the National Natural Science Foundation of China (82171536), the Hebei Province Central Leading Local Science and Technology Development Fund Project (246Z7746G) and the Key laboratory of Neural and Vascular Biology, Ministry of Education of China (NV20250010). Thanks to the Hebei Key Laboratory of Forensic Medicine for providing technical guidance.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All experimental protocols were reviewed and approved by the Animal Ethics Committee of Hebei Medical University (IACUC Approval No. IACUC-Hebmu-P2024164).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.