Maternal Immune Activation with High Molecular Weight Poly (I:C) Induces Selective Depressive-Like Phenotype in Adult Offspring

Maternal Immune Activation with High Molecular Weight Poly (I:C) Induces Selective Depressive-Like Phenotype in Adult Offspring | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Maternal Immune Activation with High Molecular Weight Poly (I:C) Induces Selective Depressive-Like Phenotype in Adult Offspring Marco Pistis, Michele Santoni, Andrea Mastio, Luca Concas, Rafaela Mostallino, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7131610/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Translational Psychiatry → Version 1 posted 11 You are reading this latest preprint version Abstract Maternal immune activation (MIA) during pregnancy may increase the risk for neurodevelopmental disorders such as schizophrenia and autism in offspring. Preclinical and human evidence support a potential role for MIA in the development of depressive symptoms in offspring. Among animal models of MIA, prenatal treatment with the synthetic viral mimetic polyinosinic-polycytidylic acid [poly (I:C)] is well established in studying psychotic-like and autism-like phenotypes, while its validity for modeling depressive-like behaviors remains underexplored. In this study, we assessed whether MIA, induced in rats with an injection of high molecular weight (HMW) poly (I:C), at gestational day 15, leads to a depressive-like phenotype in the offspring. In male and female offspring during adolescence and adulthood, we evaluated i) behavioral despair and anhedonia using the forced swim test (FST) and sucrose preference test (SPT); ii) the electrophysiological properties of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in vivo ; iii) serum cytokine profile. We found that MIA offspring exhibited increased immobility and reduced climbing and swimming in the FST, with more pronounced effects in males, while sucrose preference remained unaltered. In vivo recordings revealed a significant increase in 5-HT neuron firing rate in MIA adult males. Peripheral cytokine analysis showed elevated IL-1α in MIA males and decreased GRO/KC levels in MIA females. In conclusion, these findings indicate that prenatal exposure to HMW poly (I:C) selectively affects stress-coping mechanisms without inducing anhedonia, modulates serotonergic signaling in a sex- dependent manner in the absence of widespread inflammatory alterations. Biological sciences/Neuroscience/Molecular neuroscience Health sciences/Diseases/Psychiatric disorders/Depression Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The global impact of the COVID-19 pandemic has underscored the significant burden posed by infectious diseases. Indeed, there is a growing scientific interest in understanding the effects of prenatal COVID-19 infections, and the consequent maternal immune activation (MIA), on the neurodevelopmental trajectories of offspring ( 1 , 2 ). Maternal infections during pregnancy have emerged as a significant risk factor for the onset of neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia ( 3 ). Studying SARS-CoV-2 infection directly in pregnant animal models could offer crucial insights into its impact on offspring neurodevelopment. However, this approach presents significant challenges, as it requires strict adherence to biosafety standards and technical precautions, as pioneering studies have shown using prenatal exposure to live pathogens, such as the influenza virus ( 4 , 5 ). Thus, one of the most characterized MIA models is based on the exposure during pregnancy to the polyinosinic-polycytidylic acid [poly (I:C)], a double-stranded synthetic RNA, which triggers an innate immune response by mimicking a viral infection. MIA models reliably replicate neurodevelopmental disruptions observed in humans, inducing neurodevelopmental disorders in the offspring ( 6 , 7 ). In our laboratory, we previously characterized a poly (I:C)-induced MIA model, demonstrating that inflammation triggered by MIA disrupts endocannabinoid system signaling ( 8 ), ultimately impairing dopaminergic function and contributing to the emergence of a schizophrenia-like phenotype in adult offspring across generations ( 9 – 11 ). Moreover, we demonstrated that activation of peroxisome proliferator-activated receptor-α (PPARα) with the clinically available agonist fenofibrate attenuates the neurodevelopmental disturbances induced by MIA in rat offspring ( 12 ) by reducing the cytokine imbalance during pregnancy ( 13 ). While the MIA model is well-established in studying psychotic-like and autism-like phenotypes ( 14 ), its validity for modeling depressive-like behaviors remains underexplored. While some studies have suggested an association between MIA and depressive-like phenotype in offspring ( 15 – 17 ), the extent to which this relationship reflects a direct causal mechanism remains uncertain. Moreover, among the various challenges in MIA models, experimental conditions represent a crucial factor, as the type of poly (I:C) used (e.g. low molecular weight, LMW vs. high molecular weight, HMW; 18 ), the gestational timing of administration ( 19 ), and laboratory environment coupled with the caging system ( 20 , 21 ) play a key role in shaping developmental outcomes. Notably, a growing body of evidence highlights the importance of the molecular weight of poly (I:C) in modulating maternal and fetal immune responses, as well as pregnancy viability. Studies in mice have shown that HMW poly (I:C) induces stronger cytokine responses but is also associated with a higher rate of fetal loss, leading many laboratories to adopt LMW poly (I:C) as a refinement measure to reduce variability and ameliorate gestational outcomes ( 20 , 21 ). In contrast, our laboratory has consistently utilized HMW poly(I:C) in a rat model of MIA, with well-characterized and reproducible phenotypes ( 8 – 13 , 22 ). Moreover, rats appear less sensitive than mice to the effects of HMW poly(I:C) on abortion rate, allowing for robust and sustained maternal immune activation without compromising pregnancy viability. Thus, we sought to determine whether MIA induced by HMW poly (I:C) (4 mg/kg, i.v.) at gestational day (GD) 15 contributes to the emergence of a depressive-like phenotype in male and female offspring. We investigated longitudinally the neurodevelopmental trajectories in male and female offspring from adolescence (postnatal days, PND 35–45) to adulthood (PND ≥ 80). To this end, we performed a behavioral characterization using the forced swim test and the sucrose preference test, two well-established paradigms for assessing depressive-like behavior, specifically behavioral despair and anhedonia, respectively ( 23 , 24 ). Additionally, we performed electrophysiological recordings of putative serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN), critically involved in mood regulation, to investigate a neural correlate of depressive-like behavior ( 25 ). Due to the growing recognition of inflammation as a critical factor in the pathophysiology of depression, we further evaluated systemic inflammation by analyzing full cytokines/chemokines/growth factors profile in the serum of the male and female offspring. This study aims to elucidate whether prenatal exposure to HMW poly (I:C) at GD 15 in Sprague Dawley rats leads to a depressive-like phenotype in the offspring, providing novel insights into the long-term effects of MIA on mood-related behaviors and underlying neurobiological mechanisms. 2. Methods 2.1 Animals Female Sprague Dawley rats (Envigo, Italy) were mated at the age of 3 months. The first day after the copulation, which was confirmed through the presence of the vaginal plug, was defined as GD 0 ( 26 ). Pregnant dams on GD 15 were randomly assigned to receive either a single injection of poly (I:C) or an equivalent volume of endotoxin-free saline solution in the lateral vein of the tail (vehicle). Offspring were weaned and sexed on PND 21. After weaning, offspring were housed with littermates and maintained undisturbed until experiments. Subsequently, rats were randomly assigned to experimental procedures and care was taken to avoid assigning more than three animals from the same litter to the same experimental group. In fact, for the experiments described here, a total of 25 dams were utilized [13 were treated with vehicle and 12 with poly (I:C)] (Fig. 1). Behavioral tests and in vivo electrophysiological recordings were performed throughout adolescence (PND 35–45) and during adulthood (PND ≥ 80). Blood for the analysis of cytokine levels was collected at two timepoints, PND 35 ( 8 ) and PND 90. For cytokine measurements, rats were deeply anesthetized with isoflurane (5%) (Merial, Toulouse, France), sacrificed by decapitation, and trunk blood was collected into a 8 ml tube and allowed to clot at room temperature. All procedures were performed in accordance with the European legislation EU Directive 2010/63 and were approved by the Animal Ethics Committee of the University of Cagliari and by the Italian Ministry of Health (auth. n. 658/2015-PR; 631/2020-PR). 2.2 Drugs and Treatments Poly (I:C) was purchased from InvivoGen (France). Poly (I:C) was dissolved in endotoxin-free saline solution, and injected at 4.0 mg/kg, i.v in the lateral vein of the tail of pregnant dams. To assess the efficacy of poly (I:C) exposure, all pregnant rats were weighed on the day of the injection and on the first day after the administration of either poly (I:C) or saline to evaluate weight loss as underlined by previous investigations ( 27 ). 2.3. Behavioral Tests During adolescence, behavioral tests began on PND 35, with a two-day recovery interval between each testing condition. In adulthood, behavioral tests began on PND 80, employing the same inter-test recovery period as used during adolescence. 2.3.1. Forced-Swim Test The forced-swim test was conducted as previously described ( 28 ). Briefly, rats were placed in transparent Plexiglas cylinders (45.7 × 30 cm in diameter) filled with water to a depth of 30 cm, maintained at a temperature of 25°C. The test lasted for 10 minutes, with environmental light set at 300 lux. The duration of immobility (defined as the minimal movement required to keep the head above water in the absence of the other two behaviors), climbing (upward movements of the forepaws directed toward the walls of the cylinder), and swimming (horizontal movement throughout the cylinder) was manually recorded. 2.3.2. Sucrose preference test Rats were exposed to a 1.5% or 1% sucrose solution for 48 h in order to acclimate them to the test procedure. On the test day, rats were isolated and provided for 24 h with two bottles, one containing the sucrose solution and the other containing tap water. The positions of the two bottles were changed every 12 h to control for position preference. Fluid (sucrose solution or water) consumption was calculated by weighing each bottle before and after animal exposure. Sucrose preference was calculated as 100% sucrose solution consumption (g)/total fluid consumption (g). 2.4. In vivo electrophysiology Rats were anesthetized with urethane (1.3 g/kg, i.p.) and placed in the stereotaxic apparatus (Kopf, Tujunga, CA, USA) with their body temperature maintained at 37 ± 1°C by a heating pad. For the positioning of a recording electrode, the scalp was retracted and one burr hole was drilled above the DRN according to the Paxinos and Watson stereotaxic rat brain atlas ( 29 ) (DRN coordinates adjusted in adolescence: 6.8 mm posterior from bregma, 0.0-0.1 mm lateral to the midline, 5.0–6.0 mm from the cortical surface; DRN coordinates in adulthood: 7.5 mm posterior from bregma, 0.0-0.1 mm lateral to the midline, 5.0–6.0 mm from the cortical surface). Extracellular single unit activity of putative 5-HT neurons was recorded with glass micropipettes filled with 2% Pontamine sky blue dissolved in 0.5 M sodium acetat with an impedance of 2.5-5 MΩ. Spontaneous population activity of DRN 5-HT cells was determined in 4–6 predetermined tracks separated by 100 µm and the total number of active cells encountered in each brain area was divided by the number of tracks (cells/track). Putative serotoninergic neurons were selected if all identification criteria were met according to previously published criteria ( 30 , 31 ). The action potential is characterized by a duration of 2–5 ms and positive-negative or positive-negative-positive deflections. Spontaneous activity can be either regular or irregular, with a frequency range of 0.1-4.0 Hz ( 31 ). Electrical activity from individual neurons was filtered using a bandpass filter of 0.1–10000 Hz. Individual action potentials were isolated and amplified (DAM80, WPI, Hertfordshire, UK, and Neurolog System, Digitimer, Hertfordshire, UK), displayed on a digital oscilloscope (DL708E, Yokogawa, Japan), and digitally recorded for a period of 2–3 minutes. Experiments were sampled online with Spike2 7.20 software by a computer connected to the CED 1401 interface (Cambridge Electronic Design, Cambridge, UK). At the end of each recording session, direct current (7 µA for 10 minutes) from a constant current isolated stimulator (DS3, Digitimer, Hertfordshire, UK) was applied through the recording micropipette for ejecting PSB to mark the recording site. The position of the electrodes was identified microscopically on 60 µm sections. Only cells from subjects for which correct electrode placement was verified histologically were included in the study. 2.5. Cytokine measurements Blood was collected from MIA adult rats (PND 90). Rats were deeply anesthetized with isoflurane (5%) and sacrificed by decapitation. Trunk blood was collected into 8 ml tube and allowed to clot at room temperature for 45 min. Samples were centrifuged at 1,000g for 15 min at 4°C, and the supernatant was collected and further centrifuged at 10,000g for 10 min at 4°C. The resulting serum was aliquoted and stored at -80°C for subsequent cytokine analysis. 2.5.1. Cytokine assay As previously described ( 8 , 13 ) serum samples were analyzed for cytokines, chemokines and growth factors using a Luminex xMAP-based multiplex bead-based immunoassay, the Bio-Plex ProTM Rat Cytokine Group I Panel 23-Plex (Bio-Rad Laboratories, Inc., USA), which detects cytokines: [Interleukins (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, interferon (IFN)γ, tumor necrosis factor (TNF)α; chemokines: monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)1α, MIP-3α, regulated on activation normal T cells expressed and secreted (RANTES), keratinocyte derived chemokine (GRO/KC or CXCL1), and growth factors: granulocytes macrophage colony-stimulating factor (GM)-CSF, granulocyte (G)-CSF, macrophage (M)-CSF and vascular endothelial growth factor (VEGF)]. Assays were performed in 96-well plates. Each plate included 8 lyophilized cytokine standards in duplicate, two blank wells, and up to 32 serum samples (diluted 1:4 prior to assay). All samples were run in duplicate and control and MIA offspring at PND 90 were analyzed in the same plate. Wash steps were carried out at room temperature using Bio-Plex Pro wash station. A Bio- Plex MagPix Multiplex System by Luminex was used to read the plate and data were analyzed using BioPlex manager 4.1 software. Cytokine concentrations were calculated with a 5-parametric logistic regression (5 PL) curve fitting to determine the standard curve (pg/ml) from 8 reference standards in duplicate for each cytokine. Specifically, unknown sample cytokine concentrations were calculated by Bio-Plex Manager software using a standard curve derived from the known reference cytokine concentrations supplied by the manufacturer. Only standards and samples with coefficients of variance under 5% were included. Data below the assay’s detection limit (i.e., background value) were excluded from analyses. 2.6 Statistical analysis Data are analyzed by two-way ANOVA with treatment and sex as factors. As two-way ANOVA do not detect significant interaction pairwise comparisons were performed by using two-tailed t-test with Bonferroni’s corrected alpha values. All biochemical data, expressed as pg/ml, are mean ± SEM calculated from one experiment performed in duplicate and analyzed using GraphPad Software Prism 10.0. Normal distribution was assessed using the Shapiro–Wilk’s test. 3. Results 3.1 Effects of gestational HMW poly (I:C) on depressive-like behavior in male and female offspring during adolescence and adulthood MIA has been shown to induce depressive-like phenotypes in offspring in a manner dependent on strain, behavioral task, and immunostimulant used ( 30 – 32 ). Therefore, we explored whether gestational exposure to HMW poly (I:C) induces behavioral phenotypes associated with depressive-like states, including anhedonia and coping strategies to stress. Male and female were tested for anhedonia-like behavior using the sucrose preference test during adolescence (PND 35–45) and adulthood (PND ≥ 80). For these experiments we used n = 11 rats, control adolescent and adult male offspring; n = 12 rats, control adolescent and adult female offspring; n = 11 rats, poly (I:C) adolescent and adult male offspring; n = 11 rats, poly (I:C) adolescent and adult female offspring. Rats were given 48h access to two bottles, one containing the sucrose solution (1% or 1.5%) and the other containing tap water. For the dose of sucrose 1% we used n = 10 rats, control and poly (I:C) adolescent male and female offspring, and n = 12 control adult male offspring, n = 11 control adult female offspring and n = 10 poly (I:C) adult male and female offspring. For the dose 1.5% we used n = 11 control and poly (I:C) adolescent and adult male offspring, n = 12 control adolescent and adult female offspring and n = 11 poly (I:C) adolescent and adult female offspring. After 24h, rats were individually housed and re-exposed to both bottles. Two-way ANOVA did not detect any differences between male and female adolescent rats at either the 1% sucrose dose [treatment x sex: F ( 1,36 ) = 1.395, P = 0.245] or the 1.5% sucrose dose [treatment x sex: F (1,41) = 0.137, P = 0.712] (Fig. 2A-2B). Likewise, no significant differences were found in the sucrose preference at both doses of 1% [treatment x sex: F (1,39) = 0.869, P = 0.356] and 1.5% of sucrose [treatment x sex: F (1,41) = 0.072, P = 0.788] during adulthood (Fig. 2C-2D). Active and passive coping behaviors in response to acute stress were assessed using the forced swim test. For this experiment we used n = 11 rats, control and poly (I:C) adolescent male offspring, n = 10 control adolescent female offspring, n = 11 poly (I:C) female offspring. To test the active and passive coping behaviors in response to acute stress during adulthood we used, n = 10 control adult male offspring, n = 11 poly (I:C) adult male offspring, n = 12 control adult female offspring and n = 11 poly (I:C) adult female offspring. Data analysis revealed that gestational exposure to poly (I:C) did not alter the latency to the first immobility episode in adolescent offspring (Fig. 3A) [main effect of treatment: F (1, 39) = 1.548, P = 0.220]. However, poly (I:C) offspring exhibited a significant increase in the immobility time compared to their counterparts [main effect of treatment: F (1, 39) = 9.522, P = 0.003], independent of sex [main effect of sex: F( 1,39 ) = 0.949, P = 0.335] (Fig. 3B). Moreover, the two-way ANOVA revealed a significant reduction in both swimming [main effect of treatment: F( 1, 39 ) = 4.356; P = 0.043] (Fig. 3C) and climbing behavior [main effect of treatment: F (1, 39) = 37.37; P < 0.0001] (Fig. 3D) in both male and female MIA adolescent offspring. Similar to adolescent offspring, no significant differences in the latency to the first immobility episode were observed in adulthood [main effect of treatment: F( 1,40 ) = 0.329, P = 0.569] (Fig. 3E). However, maternal poly (I:C) exposure significantly increased immobility time [main effect of treatment: F( 1,40 ) = 14.91, P = 0.0004] (Fig. 3F). In contrast to the adolescent offspring, ANOVA revealed a main effect of sex in immobility time, with males showing longer immobility durations than females [F (1,40) = 11.69, P = 0.0015] (Fig. 3F). Gestational HMW poly (I:C) exposure also reduced the time spent swimming, with a more pronounced effect in males than in females [main effect of treatment: F (1,40) = 6.209, P = 0.017; main effect of sex: F (1,40) = 19.11, P < 0.0001] (Fig. 3G). In the analyses of the climbing behaviors, a significant interaction between factors treatment x sex was observed (Fig. 3H). Post-hoc test revealed that maternal HMW poly (I:C) exposure decreased climbing time in males but not in females [treatment x sex: F( 1,40 ) = 9.367, P = 0.0039; control males vs. poly (I:C) males: p < 0.0001] (Fig. 3H). 3.2 Effects of gestational HMW poly (I:C) on the electrical activity of putative 5-HT DRN neurons in male and female offspring during adolescence and adulthood Given the role of 5-HT in mood regulation and depressive disorders, we examined the electrical activity of 5-HT neurons in the DRN as a potential neural correlate of depressive-like phenotype ( 25 ). Thus, we performed in vivo single-unit extracellular recordings of putative 5-HT DRN neurons from adolescent (PND 35–45) and adult (PND ≥ 80) offspring under urethane anesthesia. In these experiments, we utilized n = 6 rats (55 cells), control adolescent males; n = 5 rats (42 cells), control adolescent females; n = 5 rats (44 cells), poly (I:C) adolescent males; n = 5 rats (62 cells), poly (I:C) adolescent females; n = 6 rats (70 cells), control adult males; n = 6 rats (53 cells), control adult females; n = 5 rats (52 cells), poly (I:C) adult males; n = 8 rats (66 cells), poly (I:C) adult females. Figure 4A shows the typical broad spike waveform of a putative 5-HT DRN, Fig. 4B depicts representative localization of recording sites from the DRN. Figure 4C depicts demonstrative traces of regularly and irregularly firing putative 5-HT DRN cells. Analysis of the number of cells/track (Fig. 4D), which is an index that represents the spontaneous population activity of a specific brain area, did not reveal any difference during adolescence (Fig. 4D) (Two-way ANOVA, treatment x sex F ( 1, 17 ), P = 0.3294), and neither did during adulthood (Fig. 4F) (treatment x sex F ( 1, 20 ) = 2.730, P = 0.1141). Moreover, no main effect of treatment, sex, nor their interaction was observed on the electrical activity of putative 5-HT DRN neurons in adolescent offspring (Fig. 4E (treatment x sex (F (1, 207) = 1.785), P = 0.183). However, two-way ANOVA revealed an interaction between treatment and sex in the adult offspring (treatment x sex F ( 1, 237 ) = 4.867, P = 0.028. Tukey’s post hoc test revealed a significant increase in the firing frequency of adult poly (I:C) male offspring compared to adult control male offspring (P = 0.023) (Fig. 4G). 3.3 Effect of HMW poly (I:C) on serum cytokine, chemokine, and growth factor levels in male and female offspring during adulthood To evaluate the impact of gestational HMW poly (I:C) on the expression of cytokines, chemokines, and growth factors in adult offspring, we performed a multiplex ELISA at PND 90 on serum collected from control and MIA offspring. The high sensitivity of multiplex ELISA enabled the simultaneous quantification of 23 factors from each sample. All these analytes were detectable in the examined serum samples, apart from IL-18, which was undetectable in the serum of both control and MIA-exposed female offspring. Two-way ANOVA displayed a significant main effect of sex for almost all cytokines, chemokines, and growth factors of poly (I:C) offspring. However, no significant sex X treatment interaction was observed for any of the analytes analyzed. Specifically, the female group showed significantly lower values of all cytokines (except IL-12), chemokines (except VGF), and growth factors compared to their respective male controls and poly (I:C) offspring (see Supplementary Table 2). The main effect of poly (I:C) was significant only for IL-1α [F(1 ,26 ) = 8.2, P = 0.008] and GRO/KC [F(1,26) = 5.4, P = 0.029], revealing higher levels of IL-1αin male serum samples (p < 0.05) and lower levels of GRO/KC in female serum samples (p < 0.05), compared to their respective controls. As shown in Table 1 , the serum of male poly (I:C) offspring exhibited a significant increase in IL-1α (11% vs. Control, p < 0.05). Moreover, female poly (I:C) offspring showed a significant reduction in GRO/KC (20% vs. Control, p < 0.05). No differences in cytokine, chemokine, or growth factor levels were detected in the serum of either male or female offspring (Table 1 ). Table 1 Cytokine, chemokine, and growth factor concentrations in male and female serum Male Female Control Poly (I:C) ↑ or ↓ Control Poly (I:C) ↑ or ↓ Pro- and anti-inflammatory cytokines (pg/ml) IL-1α IL-1β IL-2 IL-4 IL-5 IL-6 IL-7 IL-10 IL-12(p70) IL-13 IL-17 IL-18 IFN-γ TNF-α 240.23 ± 8.72 153.89 ± 3.94 4014.90 ± 161.48 664.24 ± 20.80 908.70 ± 17.63 910.58 ± 45.88 205.17 ± 7.45 535.89 ± 19.62 528.63 ± 33.17 467.95 ± 29.05 143.38 ± 5.38 255.18 ± 21.39 806.12 ± 36.66 933.04 ± 62.43 268.37 ± 7.12 145.13 ± 5.97 3891.06 ± 171.24 626.79 ± 28.48 918.12 ± 13.92 897.85 ± 56.05 212.69 ± 6.50 532.48 ± 26.70 554.81 ± 26.46 450.93 ± 30.82 140.90 ± 3.45 245.15 ± 12.26 792.65 ± 33.33 853.37 ± 59.69 ↑ * 161.13 ± 3.86 93.09 ± 3.24 3517.84 ± 122.54 234.16 ± 8.52 558.82 ± 13.03 522.96 ± 36.73 141.24 ± 7.74 294.88 ± 9.02 590.95 ± 28.21 194.34 ± 21.67 132.71 ± 4.94 N.D. 457.63 ± 25.22 499.31 ± 23.18 170.78 ± 5.06 92.38 ± 2.17 3847.23 ± 202.50 234.30 ± 11.14 561.05 ± 12.02 501.28 ± 22.15 145.39 ± 8.22 283.13 ± 12.70 623.64 ± 25.11 183.04 ± 8.91 133.77 ± 7.06 N.D. 467.11 ± 13.99 493.58 ± 29.52 Chemokines (pg/ml) MCP-1 MIP-1α MIP-3α RANTES GRO/KC 1406.61 ± 49.23 42.75 ± 0.79 41.80 ± 1.92 580.45 ± 58.07 182.82 ± 5.76 1467.61 ± 39.22 44.58 ± 1.14 42.05 ± 1.71 572.44 ± 34.41 178.19 ± 9.54 1158.84 ± 70.95 27.00 ± 1.15 20.25 ± 0.73 228.37 ± 16.21 157.07 ± 10.60 1061.02 ± 78.50 26.92 ± 1.14 18.41 ± 0.80 212.31 ± 24.61 125.95 ± 4.57 ↓ * Growth factors (pg/ml) GM-CSF G-CSF M-CSF VEGF 108.79 ± 4.79 41.59 ± 1.98 33.03 ± 2.31 195.95 ± 17.97 110.64 ± 3.87 40.25 ± 2.83 33.14 ± 3.89 203.79 ± 16.82 88.86 ± 2.73 23.18 ± 1.43 30.80 ± 2.40 260.89 ± 18.76 88.65 ± 3.27 22.55 ± 1.11 28.95 ± 1.79 244.36 ± 18.58 Data, expressed as a pg/ml, are mean ± SEM calculated from one experiment performed in duplicate (n = 8 ctrl males; n = 8 poly (I:C) males; n = 7 ctrl females; n = 8 poly (I:C) females); N.D.; not detectable: cytokine, chemokine or growth factor concentrations under the limit of detection. *p < 0.05 compared to control group, two-tailed t- test with Bonferroni’s corrected alpha values. 4. Discussion Our results suggest that gestational administration of HMW poly (I:C) selectively alters depressive-like in the offspring rather than inducing a comprehensive depressive-like profile. While no differences in anhedonia were observed in the sucrose preference test, which evaluates key behavioral manifestations of negative valence, maternal administration of HMW poly(I:C) induced substantial alterations in stress-coping mechanisms, as assessed by the forced swim test. Specifically, HMW poly(I:C) exposure during pregnancy resulted in increased immobility time in offspring of both sexes and reduced swimming behavior, with the effect being pronounced in males. The significant interaction between sex and poly (I:C) exposure in climbing behavior further suggests sex-dependent differences in adaptive behaviors to acute stress exposures. These findings corroborate previous studies and highlight how MIA may contribute to the onset of depression-related behavioral phenotypes ( 34 ). They also underscore the importance of considering both sex and the timing of exposure when investigating the behavioral consequences in offspring. Given the well-established role of the dopamine system in reward-related behaviors ( 35 ), and several previous works demonstrating MIA-induced alterations in mesolimbic dopamine functions ( 8 , 9 , 27 , 36 – 38 ), we expected anhedonia-like deficits in the sucrose preference test. However, the lack of detectable changes in sucrose preference suggests a more selective impact of MIA on specific domains of the depressive-like phenotype. Previous studies employing poly (I:C) ( 16 , 17 , 39 ) reported concurrent elevations in forced-swim immobility and decreased sucrose preference, indicative of a more encompassing depressive-like phenotype. It should also be emphasized that these studies were performed in different species (C57BL/6N mice vs Sprague Dawley rats), and following different protocols to induce MIA [poly (I:C) 20 mg/kg i.p. at GD 12.5 vs HMW 4 mg/kg i.v at GD 15, and used a different supplier (Sigma-Aldrich vs InvivoGen]. Indeed, several studies highlight the relevance of the poly (I:C) batch, its molecular weight (LMW versus HMW), and the vendor, all of which can critically influence behavioral readouts ( 18 – 20 , 40 ). Furthermore, the behavioral alterations we observed were not paralleled by changes in serotonergic activity in the DRN, suggesting that these outcomes could result from mechanisms beyond classic serotonergic dysfunction. In our in vivo recordings, adult male offspring from maternal exposure to HMW poly (I:C) displayed an increased spontaneous firing rate of DRN 5-HT neurons compared with controls. This result might appear in contrast with earlier work indicating that reduced DRN activity is typically associated with depressive-like behaviors ( 41 ). Furthermore, Csatlosova and colleagues ( 42 ) found a decreased firing rate of 5-HT neurons in the DRN, whereas we detected a stimulatory effect of prenatal HMW poly (I:C) on 5-HT neurons in the DRN. Accordingly, we previously showed that an anhedonic-like status induced by chronic neuropathic pain in rat, alters the electrophysiological activity of DRN 5-HT neurons particularly increasing their average firing frequency among others ( 30 ). It is therefore plausible that functional maladaptations and/or distinct immune challenges [poly (I:C) at GD 15 versus LPS across GD 15–19] engage partially divergent mechanisms, yielding opposite effects on DRN excitability while converging on similar behavioral phenotypes. Mounting evidence underlines the link between immune dysregulation and depression ( 43 , 44 ). Indeed, we next examined the peripheral cytokine and chemokine profile in MIA-exposed offspring. To provide a comprehensive profile of serum cytokines, chemokines, and growth factors in adult MIA offspring, we used a multiplex immunoassay, which maximizes the simultaneous detection of multiple analytes in a single sample. At this developmental stage, the levels of almost all cytokines and chemokines were unaltered in both male and female serum. Of note, IL-1α, which was increased in the maternal serum 24h after poly (I:C) injection (see 13 ), is the only cytokine we found significantly higher in the offspring during adulthood. IL-1α, a member of the interleukin-1 family, is a key mediator of systemic inflammation, brain inflammation and injury ( 45 ). Despite its role in inflammation, most studies have focused almost entirely on IL-1β and not on IL-1α. To our knowledge, only Garay and colleagues ( 46 ) analyzed IL-1α serum levels in MIA male mice offspring, reporting no difference compared to those of respective controls at PND 60. Among the chemokines assessed, only GRO/KC (CXCL1) levels were significantly decreased in the serum of MIA female offspring at adulthood. This chemokine acts as a chemoattractant for several immune cells and other non-hematopoietic cells at the site of injury or infection, playing a crucial role in modulating immune and inflammatory responses. To date, there is a lack of data regarding the role of GRO/KC in the context of MIA. However, it is important to emphasize that the absence of overt peripheral immune activation in adulthood does not rule out the presence of ongoing or latent neuroinflammatory processes. Indeed, neuroinflammation can occur independently of systemic cytokine elevations and may be confined to discrete brain regions involved in mood regulation and depressive-like behaviors ( 47 ). A more detailed characterization of neuroinflammatory markers within regions involved in depression, such as the mesocorticolimbic system, hippocampus, and DRN, would therefore be instrumental in elucidating the central immune mechanisms underlying the observed behavioral alterations. Our previous work demonstrated that MIA male offspring exhibit increased expression of cyclooxygenase-2 (COX-2) and ionized calcium-binding adaptor molecule 1 (IBA-1) in the whole brain ( 8 ). These markers are associated with latent inflammatory states and sustained microglial activation and may reflect long-lasting neuroimmune vulnerability to the emergence of latent depression-related phenotypes. In conclusion, these findings suggest that gestational administration of HMW poly(I:C) selectively alters specific aspects of depressive-like behavior in the offspring rather than inducing a broad depressive phenotype. Notably, these behavioral alterations were not paralleled by changes in serotonergic activity in the DRN, suggesting that mechanisms beyond classic serotonergic dysfunction may underlie these outcomes, and more investigations will be required. Taken together, our results underscore the importance of considering species, immune challenge characteristics, and sex as critical variables producing different output following the MIA model. Future studies should aim to characterize the spatial and temporal dynamics of neuroinflammation across brain regions implicated in mood regulation. Declarations 5. Funding This work was supported by Progetti di Rilevante Interesse Nazionale (Grant Nos. PRIN 2022 P20229CCLB and 2022NSLB3Z [to MP]) from the Italian Ministry of University and Research, by the Assessorato alla Programmazione (Grant No. PNRR-MAD-2022-12375802; Regione Autonoma della Sardegna) [to MP]), and by Hybrid Hub: Modelli cellulari e COMputazionali, micro e nanotEcnologie per la personalizzazione di Terapie innovAtive grant (Grant No. T4-AN-10 [to MP]) from the Italian Ministry of Health. 6. Acknowledgements We thank Marta Tuveri and Dr. Barbara Tuveri for their skillful assistance. References Jaswa EG, Huddleston HG, Lindquist KJ, Wu AHB, Bishop SL, Kim Y. In Utero Exposure to Maternal COVID-19 and Offspring Neurodevelopment Through Age 24 Months. Published online 2024. doi:10.1001/jamanetworkopen.2024.39792 Kim DH, Croen LA, Iosif AM, et al. The association of maternal COVID-19-infection during pregnancy on the neonatal immune profile and associations with later diagnosis of neurodevelopmental disorders. 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Proc Natl Acad Sci U S A . 2007;104(43):17087-17092. doi:10.1073/pnas.0704144104 Csatlosova K, Bogi E, Durisova B, et al. Maternal immune activation in rats attenuates the excitability of monoamine-secreting neurons in adult offspring in a sex-specific way. Eur Neuropsychopharmacol . 2021;43:82-91. doi:10.1016/j.euroneuro.2020.12.002 Pariante CM. Why are depressed patients inflamed? A reflection on 20 years of research on depression, glucocorticoid resistance and inflammation. Eur Neuropsychopharmacol . 2017;27(6):554-559. doi:10.1016/j.euroneuro.2017.04.001 Yin Y, Ju T, Zeng D, et al. “Inflamed” depression: A review of the interactions between depression and inflammation and current anti-inflammatory strategies for depression. Pharmacol Res . 2024;207(May):107322. doi:10.1016/j.phrs.2024.107322 Dinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev . 2018;281(1):8-27. doi:10.1111/imr.12621 Garay PA, Hsiao EY, Patterson PH, McAllister AK. 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Transl Psychiatry . 2023;13(1). doi:10.1038/s41398-022-02297-y Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files 4SupplementaryTable2Santonietal.2025.docx Supplementary table 2 3SupplementarydataSantonietal.2025.pdf Supplementary data Cite Share Download PDF Status: Published Journal Publication published 03 Mar, 2026 Read the published version in Translational Psychiatry → Version 1 posted Editorial decision: revise 22 Oct, 2025 Review # 2 received at journal 26 Aug, 2025 Review # 3 received at journal 06 Aug, 2025 Reviewer # 3 agreed at journal 23 Jul, 2025 Reviewer # 2 agreed at journal 21 Jul, 2025 Reviewer # 1 agreed at journal 21 Jul, 2025 Reviewers invited by journal 18 Jul, 2025 Editor assigned by journal 18 Jul, 2025 Submission checks completed at journal 17 Jul, 2025 First submitted to journal 16 Jul, 2025 Unknown event 16 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7131610","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":487588545,"identity":"8a464692-0caf-4411-aa61-10ff44b1426a","order_by":0,"name":"Marco Pistis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYFACxgYGBgMGOQiDQYJILQcMGIxhWojSw8BwgIEhsQHKJqxFd3Zz8+cPBXXpG243N374wWBRR1CL2Z2DbRIHDA7nbrhzsFmyhxiHmd1IbAP65UDuBhCDh0gtzR8OGNSlGwC1MP4hUksD0GHMCSAtzMTZAvLLGYPDhjOB1knLGEhINhDUcrv98YeKP3XyfDfSH358U1HHT9AWtHgwIKyByBQyCkbBKBgFIxsAADmnPrJ1rmoDAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4622-3205","institution":"University of Cagliari","correspondingAuthor":true,"prefix":"","firstName":"Marco","middleName":"","lastName":"Pistis","suffix":""},{"id":487588546,"identity":"e397834e-d19d-499d-8bb3-2236057d8f96","order_by":1,"name":"Michele Santoni","email":"","orcid":"https://orcid.org/0000-0003-4603-2369","institution":"University of Cagliari","correspondingAuthor":false,"prefix":"","firstName":"Michele","middleName":"","lastName":"Santoni","suffix":""},{"id":487588547,"identity":"a5b0ccd7-e741-4376-9c43-09aacaa7248b","order_by":2,"name":"Andrea Mastio","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Mastio","suffix":""},{"id":487588548,"identity":"8d3c32a2-bff2-4871-8b1a-18019b3175d7","order_by":3,"name":"Luca Concas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Concas","suffix":""},{"id":487588549,"identity":"70349271-fc99-41ff-a2d7-a4a9a45e956e","order_by":4,"name":"Rafaela Mostallino","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rafaela","middleName":"","lastName":"Mostallino","suffix":""},{"id":487588550,"identity":"1f03d4a5-3839-4460-8086-75f944e6977b","order_by":5,"name":"Anna Herres","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Herres","suffix":""},{"id":487588551,"identity":"5f3f001f-cb94-46e1-955f-b78ced526ad4","order_by":6,"name":"Claudia Sagheddu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Sagheddu","suffix":""},{"id":487588552,"identity":"e6e14284-2935-4bb2-a1fd-2bdf98ffce4d","order_by":7,"name":"M. 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Poly (I:C) injection during pregnancy consisted of a single i.v. injection of HMW poly (I:C) (4 mg/kg) or vehicle (sterile pyrogen-free saline) at GD15. Behavioral experiments, \u003cem\u003ein vivo\u003c/em\u003e electrophysiology recordings were performed both during adolescence (PND 35-45) and adulthood (PND ≥ 80). A biochemical assay was performed during adulthood (PND 90). Figure 1 was created with BioRender.\u003c/p\u003e","description":"","filename":"5Figure1Experimentalprotocol.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/2e726dea7907942b2ec26b8b.jpg"},{"id":87407565,"identity":"fa7fab8c-26d9-4c7b-9005-325a3010e64d","added_by":"auto","created_at":"2025-07-23 13:09:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":587032,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MIA induced with gestational HMW poly (I:C) on anhedonic-like phenotype in adolescent (A-B) and adult (C-D) male and female offspring. Rats were exposed to two different concentrations of sucrose: 1% (A, C) and 1.5% (B, D). Gestational HMW poly (I:C) does not induce anhedonia-like phenotype. Sucrose concentration 1% N=10 rats, control and poly (I:C) adolescent male and female offspring, and N=12 control adult male offspring, N=11 control adult female offspring and N =10 poly (I:C) adult male and female offspring. Sucrose concentration 1.5% N=11 control and poly (I:C) adolescent and adult male offspring, N=12 control adolescent and adult female offspring and N=11 poly (I:C) adolescent and adult female offspring. Data are expressed as a percentage of baseline and are the mean ± SEM.\u003c/p\u003e","description":"","filename":"6Figure2SUCROSEDepressivelikephenotype.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/d91ebddfa3619af5a52cb6d8.jpg"},{"id":87406601,"identity":"a52ca2b7-ded9-4883-afd2-1638fd091332","added_by":"auto","created_at":"2025-07-23 13:01:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431862,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MIA induced with gestational HMW poly (I:C) on coping strategies to stress in adolescent and adult male and female offspring. Graphs in (A, E) show the latency to the first episode of immobility (s), and graphs in (B. F) show the immobility time (s). Immobility was described by the minimum amount of movements with the anterior paws to maintain the head above the water surface. Graphs in (C, G) display the time (s) spent swimming. Graphs in (D, H) show the time of (s) spent climbing. Climbing is described by attempts to climb the wall of the cylinder. N=11 rats, control and poly (I:C) adolescent male offspring, N=10 control adolescent female offspring, N=11 poly (I:C) female offspring. N=10 control adult male offspring, N=11 poly (I:C) adult male offspring, N=12 control adult female offspring and N=11 poly (I:C) adult female offspring.Data are expressed as means ± SEM. Main Effect of treatment: ^p \u0026lt; 0.05, ^^ p\u0026lt;0.01, ^^^p\u0026lt; 0.001, ^^^^p\u0026lt;0.0001. Main effect of sex: ## p\u0026lt;0.01, #### p\u0026lt;0.0001. Post hoc analyses: *** p\u0026lt;0.001, ***p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"7Figure3FSTDepressivelikephenotype.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/3b1c4d7c34db9a1640b04f8f.jpg"},{"id":87406603,"identity":"2e15b979-ceb8-4c59-a008-7dfb978bfb89","added_by":"auto","created_at":"2025-07-23 13:01:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727376,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of MIA induced with gestational HMW poly (I:C) on putative DRN 5-HT neuron firing activity in adolescent and adult male and female offspring. (A) Typical spike waveform of a 5-HT neuron. (B) Representative localization of recording sites from the DRN (in red), as verified by histological sections. (C) Example of traces from regular and irregular firing pattern. (D) The scatter plot shows the number of spontaneously active 5-HT neurons during adolescence. (E) The scatter plot depicts individual and average 5-HT neuron firing rates in Ctrl and Poly (I:C), male and female adolescent offspring. (F) The scatter plot shows the number of spontaneously active 5-HT neurons during adulthood. (G) The scatter plot shows individual and average 5-HT neuron firing rates in Ctrl and Poly (I:C), male and female adult offspring. N= 6 rats, (55 cells) control adolescent males; n= 5 rats (42 cells) control adolescent females; n= 5 rats (44 cells) poly (I:C) adolescent males; n= 5 rats (62 cells) poly (I:C) adolescent females; n= 6 control adult males (70 cells); n= 6 control adult females (53 cells); n= 5 poly (I:C) adult males (52 cells); n= 8 poly (I:C) adult females (66 cells). Ctrl: control. Data are expressed as means ± SEM. *P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"8Figure4EFDRNDepressivelikephenotype.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/7defc8952c6b545244c39f11.jpg"},{"id":104545365,"identity":"e37b9e5f-4bec-43f4-aa85-ffd103b23ff7","added_by":"auto","created_at":"2026-03-13 07:12:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2985547,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/d8106865-93c7-461b-b121-4dcefe33c55f.pdf"},{"id":87406597,"identity":"864b133a-8740-40b2-95ea-6b57a49138bb","added_by":"auto","created_at":"2025-07-23 13:01:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20545,"visible":true,"origin":"","legend":"Supplementary table 2","description":"","filename":"4SupplementaryTable2Santonietal.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/303df4b1d94be4cf10102c6c.docx"},{"id":87407566,"identity":"5ac795e0-8d40-4e45-80c9-c85226094cf4","added_by":"auto","created_at":"2025-07-23 13:09:07","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":290336,"visible":true,"origin":"","legend":"Supplementary data","description":"","filename":"3SupplementarydataSantonietal.2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7131610/v1/a67da3b8fb92b0a43a1bbf05.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Maternal Immune Activation with High Molecular Weight Poly (I:C) Induces Selective Depressive-Like Phenotype in Adult Offspring","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global impact of the COVID-19 pandemic has underscored the significant burden posed by infectious diseases. Indeed, there is a growing scientific interest in understanding the effects of prenatal COVID-19 infections, and the consequent maternal immune activation (MIA), on the neurodevelopmental trajectories of offspring (\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). Maternal infections during pregnancy have emerged as a significant risk factor for the onset of neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia (\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e). Studying SARS-CoV-2 infection directly in pregnant animal models could offer crucial insights into its impact on offspring neurodevelopment. However, this approach presents significant challenges, as it requires strict adherence to biosafety standards and technical precautions, as pioneering studies have shown using prenatal exposure to live pathogens, such as the influenza virus (\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e). Thus, one of the most characterized MIA models is based on the exposure during pregnancy to the polyinosinic-polycytidylic acid [poly (I:C)], a double-stranded synthetic RNA, which triggers an innate immune response by mimicking a viral infection. MIA models reliably replicate neurodevelopmental disruptions observed in humans, inducing neurodevelopmental disorders in the offspring (\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e). In our laboratory, we previously characterized a poly (I:C)-induced MIA model, demonstrating that inflammation triggered by MIA disrupts endocannabinoid system signaling (\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e), ultimately impairing dopaminergic function and contributing to the emergence of a schizophrenia-like phenotype in adult offspring across generations (\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e). Moreover, we demonstrated that activation of peroxisome proliferator-activated receptor-α (PPARα) with the clinically available agonist fenofibrate attenuates the neurodevelopmental disturbances induced by MIA in rat offspring (\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e) by reducing the cytokine imbalance during pregnancy (\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e). While the MIA model is well-established in studying psychotic-like and autism-like phenotypes (\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e), its validity for modeling depressive-like behaviors remains underexplored. While some studies have suggested an association between MIA and depressive-like phenotype in offspring (\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e), the extent to which this relationship reflects a direct causal mechanism remains uncertain. Moreover, among the various challenges in MIA models, experimental conditions represent a crucial factor, as the type of poly (I:C) used (e.g. low molecular weight, LMW vs. high molecular weight, HMW; \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e), the gestational timing of administration (\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e), and laboratory environment coupled with the caging system (\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e) play a key role in shaping developmental outcomes. Notably, a growing body of evidence highlights the importance of the molecular weight of poly (I:C) in modulating maternal and fetal immune responses, as well as pregnancy viability. Studies in mice have shown that HMW poly (I:C) induces stronger cytokine responses but is also associated with a higher rate of fetal loss, leading many laboratories to adopt LMW poly (I:C) as a refinement measure to reduce variability and ameliorate gestational outcomes (\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e). In contrast, our laboratory has consistently utilized HMW poly(I:C) in a rat model of MIA, with well-characterized and reproducible phenotypes (\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e). Moreover, rats appear less sensitive than mice to the effects of HMW poly(I:C) on abortion rate, allowing for robust and sustained maternal immune activation without compromising pregnancy viability.\u003c/p\u003e\u003cp\u003eThus, we sought to determine whether MIA induced by HMW poly (I:C) (4 mg/kg, i.v.) at gestational day (GD) 15 contributes to the emergence of a depressive-like phenotype in male and female offspring. We investigated longitudinally the neurodevelopmental trajectories in male and female offspring from adolescence (postnatal days, PND 35\u0026ndash;45) to adulthood (PND\u0026thinsp;\u0026ge;\u0026thinsp;80). To this end, we performed a behavioral characterization using the forced swim test and the sucrose preference test, two well-established paradigms for assessing depressive-like behavior, specifically behavioral despair and anhedonia, respectively (\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e). Additionally, we performed electrophysiological recordings of putative serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN), critically involved in mood regulation, to investigate a neural correlate of depressive-like behavior (\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e). Due to the growing recognition of inflammation as a critical factor in the pathophysiology of depression, we further evaluated systemic inflammation by analyzing full cytokines/chemokines/growth factors profile in the serum of the male and female offspring. This study aims to elucidate whether prenatal exposure to HMW poly (I:C) at GD 15 in Sprague Dawley rats leads to a depressive-like phenotype in the offspring, providing novel insights into the long-term effects of MIA on mood-related behaviors and underlying neurobiological mechanisms.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003eFemale Sprague Dawley rats (Envigo, Italy) were mated at the age of 3 months. The first day after the copulation, which was confirmed through the presence of the vaginal plug, was defined as GD 0 (\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e). Pregnant dams on GD 15 were randomly assigned to receive either a single injection of poly (I:C) or an equivalent volume of endotoxin-free saline solution in the lateral vein of the tail (vehicle). Offspring were weaned and sexed on PND 21. After weaning, offspring were housed with littermates and maintained undisturbed until experiments. Subsequently, rats were randomly assigned to experimental procedures and care was taken to avoid assigning more than three animals from the same litter to the same experimental group. In fact, for the experiments described here, a total of 25 dams were utilized [13 were treated with vehicle and 12 with poly (I:C)] (Fig.\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eBehavioral tests and \u003cem\u003ein vivo\u003c/em\u003e electrophysiological recordings were performed throughout adolescence (PND 35\u0026ndash;45) and during adulthood (PND\u0026thinsp;\u0026ge;\u0026thinsp;80). Blood for the analysis of cytokine levels was collected at two timepoints, PND 35 (\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e) and PND 90. For cytokine measurements, rats were deeply anesthetized with isoflurane (5%) (Merial, Toulouse, France), sacrificed by decapitation, and trunk blood was collected into a 8 ml tube and allowed to clot at room temperature.\u003c/p\u003e\u003cp\u003e All procedures were performed in accordance with the European legislation EU Directive 2010/63 and were approved by the Animal Ethics Committee of the University of Cagliari and by the Italian Ministry of Health (auth. n. 658/2015-PR; 631/2020-PR).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Drugs and Treatments\u003c/h2\u003e\u003cp\u003ePoly (I:C) was purchased from InvivoGen (France). Poly (I:C) was dissolved in endotoxin-free saline solution, and injected at 4.0 mg/kg, i.v in the lateral vein of the tail of pregnant dams. To assess the efficacy of poly (I:C) exposure, all pregnant rats were weighed on the day of the injection and on the first day after the administration of either poly (I:C) or saline to evaluate weight loss as underlined by previous investigations (\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Behavioral Tests\u003c/h2\u003e\u003cp\u003eDuring adolescence, behavioral tests began on PND 35, with a two-day recovery interval between each testing condition. In adulthood, behavioral tests began on PND 80, employing the same inter-test recovery period as used during adolescence.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Forced-Swim Test\u003c/h2\u003e\u003cp\u003eThe forced-swim test was conducted as previously described (\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e). Briefly, rats were placed in transparent Plexiglas cylinders (45.7 \u0026times; 30 cm in diameter) filled with water to a depth of 30 cm, maintained at a temperature of 25\u0026deg;C. The test lasted for 10 minutes, with environmental light set at 300 lux. The duration of immobility (defined as the minimal movement required to keep the head above water in the absence of the other two behaviors), climbing (upward movements of the forepaws directed toward the walls of the cylinder), and swimming (horizontal movement throughout the cylinder) was manually recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Sucrose preference test\u003c/h2\u003e\u003cp\u003eRats were exposed to a 1.5% or 1% sucrose solution for 48 h in order to acclimate them to the test procedure. On the test day, rats were isolated and provided for 24 h with two bottles, one containing the sucrose solution and the other containing tap water. The positions of the two bottles were changed every 12 h to control for position preference. Fluid (sucrose solution or water) consumption was calculated by weighing each bottle before and after animal exposure. Sucrose preference was calculated as 100% sucrose solution consumption (g)/total fluid consumption (g).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. \u003cem\u003eIn vivo\u003c/em\u003e electrophysiology\u003c/h2\u003e\u003cp\u003eRats were anesthetized with urethane (1.3 g/kg, i.p.) and placed in the stereotaxic apparatus (Kopf, Tujunga, CA, USA) with their body temperature maintained at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C by a heating pad.\u003c/p\u003e\u003cp\u003eFor the positioning of a recording electrode, the scalp was retracted and one burr hole was drilled above the DRN according to the Paxinos and Watson stereotaxic rat brain atlas (\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e) (DRN coordinates adjusted in adolescence: 6.8 mm posterior from bregma, 0.0-0.1 mm lateral to the midline, 5.0\u0026ndash;6.0 mm from the cortical surface; DRN coordinates in adulthood: 7.5 mm posterior from bregma, 0.0-0.1 mm lateral to the midline, 5.0\u0026ndash;6.0 mm from the cortical surface). Extracellular single unit activity of putative 5-HT neurons was recorded with glass micropipettes filled with 2% Pontamine sky blue dissolved in 0.5 M sodium acetat with an impedance of 2.5-5 MΩ.\u003c/p\u003e\u003cp\u003eSpontaneous population activity of DRN 5-HT cells was determined in 4\u0026ndash;6 predetermined tracks separated by 100 \u0026micro;m and the total number of active cells encountered in each brain area was divided by the number of tracks (cells/track). Putative serotoninergic neurons were selected if all identification criteria were met according to previously published criteria (\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e,\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e). The action potential is characterized by a duration of 2\u0026ndash;5 ms and positive-negative or positive-negative-positive deflections. Spontaneous activity can be either regular or irregular, with a frequency range of 0.1-4.0 Hz (\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eElectrical activity from individual neurons was filtered using a bandpass filter of 0.1\u0026ndash;10000 Hz. Individual action potentials were isolated and amplified (DAM80, WPI, Hertfordshire, UK, and Neurolog System, Digitimer, Hertfordshire, UK), displayed on a digital oscilloscope (DL708E, Yokogawa, Japan), and digitally recorded for a period of 2\u0026ndash;3 minutes. Experiments were sampled online with Spike2 7.20 software by a computer connected to the CED 1401 interface (Cambridge Electronic Design, Cambridge, UK).\u003c/p\u003e\u003cp\u003eAt the end of each recording session, direct current (7 \u0026micro;A for 10 minutes) from a constant current isolated stimulator (DS3, Digitimer, Hertfordshire, UK) was applied through the recording micropipette for ejecting PSB to mark the recording site. The position of the electrodes was identified microscopically on 60 \u0026micro;m sections. Only cells from subjects for which correct electrode placement was verified histologically were included in the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Cytokine measurements\u003c/h2\u003e\u003cp\u003eBlood was collected from MIA adult rats (PND 90). Rats were deeply anesthetized with isoflurane (5%) and sacrificed by decapitation. Trunk blood was collected into 8 ml tube and allowed to clot at room temperature for 45 min. Samples were centrifuged at 1,000g for 15 min at 4\u0026deg;C, and the supernatant was collected and further centrifuged at 10,000g for 10 min at 4\u0026deg;C. The resulting serum was aliquoted and stored at -80\u0026deg;C for subsequent cytokine analysis.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Cytokine assay\u003c/h2\u003e\u003cp\u003eAs previously described (\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e) serum samples were analyzed for cytokines, chemokines and growth factors using a Luminex xMAP-based multiplex bead-based immunoassay, the Bio-Plex ProTM Rat Cytokine Group I Panel 23-Plex (Bio-Rad Laboratories, Inc., USA), which detects cytokines: [Interleukins (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL- 6, IL-7, IL-10, IL-12 (p70), IL-13, IL-17A, IL-18, interferon (IFN)γ, tumor necrosis factor (TNF)α; chemokines: monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)1α, MIP-3α, regulated on activation normal T cells expressed and secreted (RANTES), keratinocyte derived chemokine (GRO/KC or CXCL1), and growth factors: granulocytes macrophage colony-stimulating factor (GM)-CSF, granulocyte (G)-CSF, macrophage (M)-CSF and vascular endothelial growth factor (VEGF)].\u003c/p\u003e\u003cp\u003eAssays were performed in 96-well plates. Each plate included 8 lyophilized cytokine standards in duplicate, two blank wells, and up to 32 serum samples (diluted 1:4 prior to assay). All samples were run in duplicate and control and MIA offspring at PND 90 were analyzed in the same plate. Wash steps were carried out at room temperature using Bio-Plex Pro wash station. A Bio- Plex MagPix Multiplex System by Luminex was used to read the plate and data were analyzed using BioPlex manager 4.1 software. Cytokine concentrations were calculated with a 5-parametric logistic regression (5 PL) curve fitting to determine the standard curve (pg/ml) from 8 reference standards in duplicate for each cytokine. Specifically, unknown sample cytokine concentrations were calculated by Bio-Plex Manager software using a standard curve derived from the known reference cytokine concentrations supplied by the manufacturer. Only standards and samples with coefficients of variance under 5% were included. Data below the assay\u0026rsquo;s detection limit (i.e., background value) were excluded from analyses.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are analyzed by two-way ANOVA with treatment and sex as factors. As two-way ANOVA do not detect significant interaction pairwise comparisons were performed by using two-tailed t-test with Bonferroni\u0026rsquo;s corrected alpha values. All biochemical data, expressed as pg/ml, are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM calculated from one experiment performed in duplicate and analyzed using GraphPad Software Prism 10.0. Normal distribution was assessed using the Shapiro\u0026ndash;Wilk\u0026rsquo;s test.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cb\u003e3.1 Effects of gestational HMW poly (I:C) on depressive-like behavior in male and female offspring during adolescence and adulthood\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMIA has been shown to induce depressive-like phenotypes in offspring in a manner dependent on strain, behavioral task, and immunostimulant used (\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e). Therefore, we explored whether gestational exposure to HMW poly (I:C) induces behavioral phenotypes associated with depressive-like states, including anhedonia and coping strategies to stress. Male and female were tested for anhedonia-like behavior using the sucrose preference test during adolescence (PND 35\u0026ndash;45) and adulthood (PND\u0026thinsp;\u0026ge;\u0026thinsp;80). For these experiments we used n\u0026thinsp;=\u0026thinsp;11 rats, control adolescent and adult male offspring; n\u0026thinsp;=\u0026thinsp;12 rats, control adolescent and adult female offspring; n\u0026thinsp;=\u0026thinsp;11 rats, poly (I:C) adolescent and adult male offspring; n\u0026thinsp;=\u0026thinsp;11 rats, poly (I:C) adolescent and adult female offspring. Rats were given 48h access to two bottles, one containing the sucrose solution (1% or 1.5%) and the other containing tap water. For the dose of sucrose 1% we used n\u0026thinsp;=\u0026thinsp;10 rats, control and poly (I:C) adolescent male and female offspring, and n\u0026thinsp;=\u0026thinsp;12 control adult male offspring, n\u0026thinsp;=\u0026thinsp;11 control adult female offspring and n\u0026thinsp;=\u0026thinsp;10 poly (I:C) adult male and female offspring. For the dose 1.5% we used n\u0026thinsp;=\u0026thinsp;11 control and poly (I:C) adolescent and adult male offspring, n\u0026thinsp;=\u0026thinsp;12 control adolescent and adult female offspring and n\u0026thinsp;=\u0026thinsp;11 poly (I:C) adolescent and adult female offspring.\u003c/p\u003e\u003cp\u003eAfter 24h, rats were individually housed and re-exposed to both bottles. Two-way ANOVA did not detect any differences between male and female adolescent rats at either the 1% sucrose dose [treatment x sex: F (\u003csub\u003e1,36\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;1.395, P\u0026thinsp;=\u0026thinsp;0.245] or the 1.5% sucrose dose [treatment x sex: F \u003csub\u003e(1,41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.137, P\u0026thinsp;=\u0026thinsp;0.712] (Fig.\u0026nbsp;2A-2B). Likewise, no significant differences were found in the sucrose preference at both doses of 1% [treatment x sex: F \u003csub\u003e(1,39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.869, P\u0026thinsp;=\u0026thinsp;0.356] and 1.5% of sucrose [treatment x sex: F \u003csub\u003e(1,41)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.072, P\u0026thinsp;=\u0026thinsp;0.788] during adulthood (Fig.\u0026nbsp;2C-2D). Active and passive coping behaviors in response to acute stress were assessed using the forced swim test. For this experiment we used n\u0026thinsp;=\u0026thinsp;11 rats, control and poly (I:C) adolescent male offspring, n\u0026thinsp;=\u0026thinsp;10 control adolescent female offspring, n\u0026thinsp;=\u0026thinsp;11 poly (I:C) female offspring. To test the active and passive coping behaviors in response to acute stress during adulthood we used, n\u0026thinsp;=\u0026thinsp;10 control adult male offspring, n\u0026thinsp;=\u0026thinsp;11 poly (I:C) adult male offspring, n\u0026thinsp;=\u0026thinsp;12 control adult female offspring and n\u0026thinsp;=\u0026thinsp;11 poly (I:C) adult female offspring. Data analysis revealed that gestational exposure to poly (I:C) did not alter the latency to the first immobility episode in adolescent offspring (Fig.\u0026nbsp;3A) [main effect of treatment: F\u003csub\u003e(1, 39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.548, P\u0026thinsp;=\u0026thinsp;0.220]. However, poly (I:C) offspring exhibited a significant increase in the immobility time compared to their counterparts [main effect of treatment: F\u003csub\u003e(1, 39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.522, P\u0026thinsp;=\u0026thinsp;0.003], independent of sex [main effect of sex: F(\u003csub\u003e1,39\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;0.949, P\u0026thinsp;=\u0026thinsp;0.335] (Fig.\u0026nbsp;3B). Moreover, the two-way ANOVA revealed a significant reduction in both swimming [main effect of treatment: F(\u003csub\u003e1, 39\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.356; P\u0026thinsp;=\u0026thinsp;0.043] (Fig.\u0026nbsp;3C) and climbing behavior [main effect of treatment: F\u003csub\u003e(1, 39)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;37.37; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;3D) in both male and female MIA adolescent offspring.\u003c/p\u003e\u003cp\u003eSimilar to adolescent offspring, no significant differences in the latency to the first immobility episode were observed in adulthood [main effect of treatment: F(\u003csub\u003e1,40\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;0.329, P\u0026thinsp;=\u0026thinsp;0.569] (Fig.\u0026nbsp;3E). However, maternal poly (I:C) exposure significantly increased immobility time [main effect of treatment: F(\u003csub\u003e1,40\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;14.91, P\u0026thinsp;=\u0026thinsp;0.0004] (Fig.\u0026nbsp;3F). In contrast to the adolescent offspring, ANOVA revealed a main effect of sex in immobility time, with males showing longer immobility durations than females [F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.69, P\u0026thinsp;=\u0026thinsp;0.0015] (Fig.\u0026nbsp;3F). Gestational HMW poly (I:C) exposure also reduced the time spent swimming, with a more pronounced effect in males than in females [main effect of treatment: F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.209, P\u0026thinsp;=\u0026thinsp;0.017; main effect of sex: F\u003csub\u003e(1,40)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;19.11, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;3G). In the analyses of the climbing behaviors, a significant interaction between factors treatment x sex was observed (Fig.\u0026nbsp;3H). Post-hoc test revealed that maternal HMW poly (I:C) exposure decreased climbing time in males but not in females [treatment x sex: F(\u003csub\u003e1,40\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;9.367, P\u0026thinsp;=\u0026thinsp;0.0039; control males vs. poly (I:C) males: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;3H).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.2 Effects of gestational HMW poly (I:C) on the electrical activity of putative 5-HT DRN neurons in male and female offspring during adolescence and adulthood\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the role of 5-HT in mood regulation and depressive disorders, we examined the electrical activity of 5-HT neurons in the DRN as a potential neural correlate of depressive-like phenotype (\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e). Thus, we performed \u003cem\u003ein vivo\u003c/em\u003e single-unit extracellular recordings of putative 5-HT DRN neurons from adolescent (PND 35\u0026ndash;45) and adult (PND\u0026thinsp;\u0026ge;\u0026thinsp;80) offspring under urethane anesthesia. In these experiments, we utilized n\u0026thinsp;=\u0026thinsp;6 rats (55 cells), control adolescent males; n\u0026thinsp;=\u0026thinsp;5 rats (42 cells), control adolescent females; n\u0026thinsp;=\u0026thinsp;5 rats (44 cells), poly (I:C) adolescent males; n\u0026thinsp;=\u0026thinsp;5 rats (62 cells), poly (I:C) adolescent females; n\u0026thinsp;=\u0026thinsp;6 rats (70 cells), control adult males; n\u0026thinsp;=\u0026thinsp;6 rats (53 cells), control adult females; n\u0026thinsp;=\u0026thinsp;5 rats (52 cells), poly (I:C) adult males; n\u0026thinsp;=\u0026thinsp;8 rats (66 cells), poly (I:C) adult females. Figure\u0026nbsp;4A shows the typical broad spike waveform of a putative 5-HT DRN, \u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e depicts representative localization of recording sites from the DRN. Figure\u0026nbsp;4C depicts demonstrative traces of regularly and irregularly firing putative 5-HT DRN cells. Analysis of the number of cells/track (Fig.\u0026nbsp;4D), which is an index that represents the spontaneous population activity of a specific brain area, did not reveal any difference during adolescence (Fig.\u0026nbsp;4D) (Two-way ANOVA, treatment x sex F (\u003csub\u003e1, 17\u003c/sub\u003e), P\u0026thinsp;=\u0026thinsp;0.3294), and neither did during adulthood (Fig.\u0026nbsp;4F) (treatment x sex F (\u003csub\u003e1, 20\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;2.730, P\u0026thinsp;=\u0026thinsp;0.1141). Moreover, no main effect of treatment, sex, nor their interaction was observed on the electrical activity of putative 5-HT DRN neurons in adolescent offspring (Fig.\u0026nbsp;4E (treatment x sex (F \u003csub\u003e(1, 207)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.785), P\u0026thinsp;=\u0026thinsp;0.183). However, two-way ANOVA revealed an interaction between treatment and sex in the adult offspring (treatment x sex F (\u003csub\u003e1, 237\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;4.867, P\u0026thinsp;=\u0026thinsp;0.028. Tukey\u0026rsquo;s post hoc test revealed a significant increase in the firing frequency of adult poly (I:C) male offspring compared to adult control male offspring (P\u0026thinsp;=\u0026thinsp;0.023) (Fig.\u0026nbsp;4G).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Effect of HMW poly (I:C) on serum cytokine, chemokine, and growth factor levels in male and female offspring during adulthood\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the impact of gestational HMW poly (I:C) on the expression of cytokines, chemokines, and growth factors in adult offspring, we performed a multiplex ELISA at PND 90 on serum collected from control and MIA offspring. The high sensitivity of multiplex ELISA enabled the simultaneous quantification of 23 factors from each sample. All these analytes were detectable in the examined serum samples, apart from IL-18, which was undetectable in the serum of both control and MIA-exposed female offspring. Two-way ANOVA displayed a significant main effect of sex for almost all cytokines, chemokines, and growth factors of poly (I:C) offspring. However, no significant sex X treatment interaction was observed for any of the analytes analyzed. Specifically, the female group showed significantly lower values of all cytokines (except IL-12), chemokines (except VGF), and growth factors compared to their respective male controls and poly (I:C) offspring (see Supplementary Table\u0026nbsp;2). The main effect of poly (I:C) was significant only for IL-1α [F(1\u003csub\u003e,26\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;8.2, P\u0026thinsp;=\u0026thinsp;0.008] and GRO/KC [F(1,26)\u0026thinsp;=\u0026thinsp;5.4, P\u0026thinsp;=\u0026thinsp;0.029], revealing higher levels of IL-1αin male serum samples (p \u0026lt; 0.05) and lower levels of GRO/KC in female serum samples (p \u0026lt; 0.05), compared to their respective controls. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the serum of male poly (I:C) offspring exhibited a significant increase in IL-1α (11% vs. Control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, female poly (I:C) offspring showed a significant reduction in GRO/KC (20% vs. Control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No differences in cytokine, chemokine, or growth factor levels were detected in the serum of either male or female offspring (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCytokine, chemokine, and growth factor concentrations in male and female serum\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePoly (I:C)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr; or \u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePoly (I:C)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr; or \u0026darr;\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e\u003cp\u003ePro- and anti-inflammatory cytokines (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIL-1α\u003c/p\u003e\u003cp\u003eIL-1β\u003c/p\u003e\u003cp\u003eIL-2\u003c/p\u003e\u003cp\u003eIL-4\u003c/p\u003e\u003cp\u003eIL-5\u003c/p\u003e\u003cp\u003eIL-6\u003c/p\u003e\u003cp\u003eIL-7\u003c/p\u003e\u003cp\u003eIL-10\u003c/p\u003e\u003cp\u003eIL-12(p70)\u003c/p\u003e\u003cp\u003eIL-13\u003c/p\u003e\u003cp\u003eIL-17\u003c/p\u003e\u003cp\u003eIL-18\u003c/p\u003e\u003cp\u003eIFN-γ\u003c/p\u003e\u003cp\u003eTNF-α\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e240.23\u0026thinsp;\u0026plusmn;\u0026thinsp;8.72\u003c/p\u003e\u003cp\u003e153.89\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94\u003c/p\u003e\u003cp\u003e4014.90\u0026thinsp;\u0026plusmn;\u0026thinsp;161.48\u003c/p\u003e\u003cp\u003e664.24\u0026thinsp;\u0026plusmn;\u0026thinsp;20.80\u003c/p\u003e\u003cp\u003e908.70\u0026thinsp;\u0026plusmn;\u0026thinsp;17.63\u003c/p\u003e\u003cp\u003e910.58\u0026thinsp;\u0026plusmn;\u0026thinsp;45.88\u003c/p\u003e\u003cp\u003e205.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.45\u003c/p\u003e\u003cp\u003e535.89\u0026thinsp;\u0026plusmn;\u0026thinsp;19.62\u003c/p\u003e\u003cp\u003e528.63\u0026thinsp;\u0026plusmn;\u0026thinsp;33.17\u003c/p\u003e\u003cp\u003e467.95\u0026thinsp;\u0026plusmn;\u0026thinsp;29.05\u003c/p\u003e\u003cp\u003e143.38\u0026thinsp;\u0026plusmn;\u0026thinsp;5.38\u003c/p\u003e\u003cp\u003e255.18\u0026thinsp;\u0026plusmn;\u0026thinsp;21.39\u003c/p\u003e\u003cp\u003e806.12\u0026thinsp;\u0026plusmn;\u0026thinsp;36.66\u003c/p\u003e\u003cp\u003e933.04\u0026thinsp;\u0026plusmn;\u0026thinsp;62.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e268.37\u0026thinsp;\u0026plusmn;\u0026thinsp;7.12\u003c/p\u003e\u003cp\u003e145.13\u0026thinsp;\u0026plusmn;\u0026thinsp;5.97\u003c/p\u003e\u003cp\u003e3891.06\u0026thinsp;\u0026plusmn;\u0026thinsp;171.24\u003c/p\u003e\u003cp\u003e626.79\u0026thinsp;\u0026plusmn;\u0026thinsp;28.48\u003c/p\u003e\u003cp\u003e918.12\u0026thinsp;\u0026plusmn;\u0026thinsp;13.92\u003c/p\u003e\u003cp\u003e897.85\u0026thinsp;\u0026plusmn;\u0026thinsp;56.05\u003c/p\u003e\u003cp\u003e212.69\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50\u003c/p\u003e\u003cp\u003e532.48\u0026thinsp;\u0026plusmn;\u0026thinsp;26.70\u003c/p\u003e\u003cp\u003e554.81\u0026thinsp;\u0026plusmn;\u0026thinsp;26.46\u003c/p\u003e\u003cp\u003e450.93\u0026thinsp;\u0026plusmn;\u0026thinsp;30.82\u003c/p\u003e\u003cp\u003e140.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003c/p\u003e\u003cp\u003e245.15\u0026thinsp;\u0026plusmn;\u0026thinsp;12.26\u003c/p\u003e\u003cp\u003e792.65\u0026thinsp;\u0026plusmn;\u0026thinsp;33.33\u003c/p\u003e\u003cp\u003e853.37\u0026thinsp;\u0026plusmn;\u0026thinsp;59.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e\u0026uarr;\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e161.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86\u003c/p\u003e\u003cp\u003e93.09\u0026thinsp;\u0026plusmn;\u0026thinsp;3.24\u003c/p\u003e\u003cp\u003e3517.84\u0026thinsp;\u0026plusmn;\u0026thinsp;122.54\u003c/p\u003e\u003cp\u003e234.16\u0026thinsp;\u0026plusmn;\u0026thinsp;8.52\u003c/p\u003e\u003cp\u003e558.82\u0026thinsp;\u0026plusmn;\u0026thinsp;13.03\u003c/p\u003e\u003cp\u003e522.96\u0026thinsp;\u0026plusmn;\u0026thinsp;36.73\u003c/p\u003e\u003cp\u003e141.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.74\u003c/p\u003e\u003cp\u003e294.88\u0026thinsp;\u0026plusmn;\u0026thinsp;9.02\u003c/p\u003e\u003cp\u003e590.95\u0026thinsp;\u0026plusmn;\u0026thinsp;28.21\u003c/p\u003e\u003cp\u003e194.34\u0026thinsp;\u0026plusmn;\u0026thinsp;21.67\u003c/p\u003e\u003cp\u003e132.71\u0026thinsp;\u0026plusmn;\u0026thinsp;4.94\u003c/p\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003cp\u003e457.63\u0026thinsp;\u0026plusmn;\u0026thinsp;25.22\u003c/p\u003e\u003cp\u003e499.31\u0026thinsp;\u0026plusmn;\u0026thinsp;23.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e170.78\u0026thinsp;\u0026plusmn;\u0026thinsp;5.06\u003c/p\u003e\u003cp\u003e92.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.17\u003c/p\u003e\u003cp\u003e3847.23\u0026thinsp;\u0026plusmn;\u0026thinsp;202.50\u003c/p\u003e\u003cp\u003e234.30\u0026thinsp;\u0026plusmn;\u0026thinsp;11.14\u003c/p\u003e\u003cp\u003e561.05\u0026thinsp;\u0026plusmn;\u0026thinsp;12.02\u003c/p\u003e\u003cp\u003e501.28\u0026thinsp;\u0026plusmn;\u0026thinsp;22.15\u003c/p\u003e\u003cp\u003e145.39\u0026thinsp;\u0026plusmn;\u0026thinsp;8.22\u003c/p\u003e\u003cp\u003e283.13\u0026thinsp;\u0026plusmn;\u0026thinsp;12.70\u003c/p\u003e\u003cp\u003e623.64\u0026thinsp;\u0026plusmn;\u0026thinsp;25.11\u003c/p\u003e\u003cp\u003e183.04\u0026thinsp;\u0026plusmn;\u0026thinsp;8.91\u003c/p\u003e\u003cp\u003e133.77\u0026thinsp;\u0026plusmn;\u0026thinsp;7.06\u003c/p\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003cp\u003e467.11\u0026thinsp;\u0026plusmn;\u0026thinsp;13.99\u003c/p\u003e\u003cp\u003e493.58\u0026thinsp;\u0026plusmn;\u0026thinsp;29.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eChemokines (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCP-1\u003c/p\u003e\u003cp\u003eMIP-1α\u003c/p\u003e\u003cp\u003eMIP-3α\u003c/p\u003e\u003cp\u003eRANTES\u003c/p\u003e\u003cp\u003eGRO/KC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1406.61\u0026thinsp;\u0026plusmn;\u0026thinsp;49.23\u003c/p\u003e\u003cp\u003e42.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003cp\u003e41.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e\u003cp\u003e580.45\u0026thinsp;\u0026plusmn;\u0026thinsp;58.07\u003c/p\u003e\u003cp\u003e182.82\u0026thinsp;\u0026plusmn;\u0026thinsp;5.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1467.61\u0026thinsp;\u0026plusmn;\u0026thinsp;39.22\u003c/p\u003e\u003cp\u003e44.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003cp\u003e42.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.71\u003c/p\u003e\u003cp\u003e572.44\u0026thinsp;\u0026plusmn;\u0026thinsp;34.41\u003c/p\u003e\u003cp\u003e178.19\u0026thinsp;\u0026plusmn;\u0026thinsp;9.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1158.84\u0026thinsp;\u0026plusmn;\u0026thinsp;70.95\u003c/p\u003e\u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003cp\u003e20.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e\u003cp\u003e228.37\u0026thinsp;\u0026plusmn;\u0026thinsp;16.21\u003c/p\u003e\u003cp\u003e157.07\u0026thinsp;\u0026plusmn;\u0026thinsp;10.60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1061.02\u0026thinsp;\u0026plusmn;\u0026thinsp;78.50\u003c/p\u003e\u003cp\u003e26.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e\u003cp\u003e18.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003cp\u003e212.31\u0026thinsp;\u0026plusmn;\u0026thinsp;24.61\u003c/p\u003e\u003cp\u003e125.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e\u0026darr;\u003c/b\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGrowth factors (pg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGM-CSF\u003c/p\u003e\u003cp\u003eG-CSF\u003c/p\u003e\u003cp\u003eM-CSF\u003c/p\u003e\u003cp\u003eVEGF\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e108.79\u0026thinsp;\u0026plusmn;\u0026thinsp;4.79\u003c/p\u003e\u003cp\u003e41.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98\u003c/p\u003e\u003cp\u003e33.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.31\u003c/p\u003e\u003cp\u003e195.95\u0026thinsp;\u0026plusmn;\u0026thinsp;17.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e110.64\u0026thinsp;\u0026plusmn;\u0026thinsp;3.87\u003c/p\u003e\u003cp\u003e40.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e\u003cp\u003e33.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89\u003c/p\u003e\u003cp\u003e203.79\u0026thinsp;\u0026plusmn;\u0026thinsp;16.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e88.86\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e\u003cp\u003e23.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e\u003cp\u003e30.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e\u003cp\u003e260.89\u0026thinsp;\u0026plusmn;\u0026thinsp;18.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e88.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27\u003c/p\u003e\u003cp\u003e22.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e\u003cp\u003e28.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003c/p\u003e\u003cp\u003e244.36\u0026thinsp;\u0026plusmn;\u0026thinsp;18.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eData, expressed as a pg/ml, are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM calculated from one experiment performed in duplicate (n\u0026thinsp;=\u0026thinsp;8 ctrl males; n\u0026thinsp;=\u0026thinsp;8 poly (I:C) males; n\u0026thinsp;=\u0026thinsp;7 ctrl females; n\u0026thinsp;=\u0026thinsp;8 poly (I:C) females); N.D.; not detectable: cytokine, chemokine or growth factor concentrations under the limit of detection. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to control group, two-tailed \u003cem\u003et-\u003c/em\u003etest with Bonferroni\u0026rsquo;s corrected alpha values.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur results suggest that gestational administration of HMW poly (I:C) selectively alters depressive-like in the offspring rather than inducing a comprehensive depressive-like profile. While no differences in anhedonia were observed in the sucrose preference test, which evaluates key behavioral manifestations of negative valence, maternal administration of HMW poly(I:C) induced substantial alterations in stress-coping mechanisms, as assessed by the forced swim test. Specifically, HMW poly(I:C) exposure during pregnancy resulted in increased immobility time in offspring of both sexes and reduced swimming behavior, with the effect being pronounced in males. The significant interaction between sex and poly (I:C) exposure in climbing behavior further suggests sex-dependent differences in adaptive behaviors to acute stress exposures. These findings corroborate previous studies and highlight how MIA may contribute to the onset of depression-related behavioral phenotypes (\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e). They also underscore the importance of considering both sex and the timing of exposure when investigating the behavioral consequences in offspring. Given the well-established role of the dopamine system in reward-related behaviors (\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e), and several previous works demonstrating MIA-induced alterations in mesolimbic dopamine functions (\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e), we expected anhedonia-like deficits in the sucrose preference test. However, the lack of detectable changes in sucrose preference suggests a more selective impact of MIA on specific domains of the depressive-like phenotype. Previous studies employing poly (I:C) (\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e) reported concurrent elevations in forced-swim immobility and decreased sucrose preference, indicative of a more encompassing depressive-like phenotype. It should also be emphasized that these studies were performed in different species (C57BL/6N mice vs Sprague Dawley rats), and following different protocols to induce MIA [poly (I:C) 20 mg/kg i.p. at GD 12.5 vs HMW 4 mg/kg i.v at GD 15, and used a different supplier (Sigma-Aldrich vs InvivoGen]. Indeed, several studies highlight the relevance of the poly (I:C) batch, its molecular weight (LMW versus HMW), and the vendor, all of which can critically influence behavioral readouts (\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e). Furthermore, the behavioral alterations we observed were not paralleled by changes in serotonergic activity in the DRN, suggesting that these outcomes could result from mechanisms beyond classic serotonergic dysfunction. In our \u003cem\u003ein vivo\u003c/em\u003e recordings, adult male offspring from maternal exposure to HMW poly (I:C) displayed an increased spontaneous firing rate of DRN 5-HT neurons compared with controls. This result might appear in contrast with earlier work indicating that reduced DRN activity is typically associated with depressive-like behaviors (\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eFurthermore, Csatlosova and colleagues (\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e) found a decreased firing rate of 5-HT neurons in the DRN, whereas we detected a stimulatory effect of prenatal HMW poly (I:C) on 5-HT neurons in the DRN. Accordingly, we previously showed that an anhedonic-like status induced by chronic neuropathic pain in rat, alters the electrophysiological activity of DRN 5-HT neurons particularly increasing their average firing frequency among others (\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e). It is therefore plausible that functional maladaptations and/or distinct immune challenges [poly (I:C) at GD 15 versus LPS across GD 15\u0026ndash;19] engage partially divergent mechanisms, yielding opposite effects on DRN excitability while converging on similar behavioral phenotypes. Mounting evidence underlines the link between immune dysregulation and depression (\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e). Indeed, we next examined the peripheral cytokine and chemokine profile in MIA-exposed offspring. To provide a comprehensive profile of serum cytokines, chemokines, and growth factors in adult MIA offspring, we used a multiplex immunoassay, which maximizes the simultaneous detection of multiple analytes in a single sample. At this developmental stage, the levels of almost all cytokines and chemokines were unaltered in both male and female serum. Of note, IL-1α, which was increased in the maternal serum 24h after poly (I:C) injection (see \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e), is the only cytokine we found significantly higher in the offspring during adulthood. IL-1α, a member of the interleukin-1 family, is a key mediator of systemic inflammation, brain inflammation and injury (\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e). Despite its role in inflammation, most studies have focused almost entirely on IL-1β and not on IL-1α. To our knowledge, only Garay and colleagues (\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e) analyzed IL-1α serum levels in MIA male mice offspring, reporting no difference compared to those of respective controls at PND 60. Among the chemokines assessed, only GRO/KC (CXCL1) levels were significantly decreased in the serum of MIA female offspring at adulthood. This chemokine acts as a chemoattractant for several immune cells and other non-hematopoietic cells at the site of injury or infection, playing a crucial role in modulating immune and inflammatory responses. To date, there is a lack of data regarding the role of GRO/KC in the context of MIA. However, it is important to emphasize that the absence of overt peripheral immune activation in adulthood does not rule out the presence of ongoing or latent neuroinflammatory processes. Indeed, neuroinflammation can occur independently of systemic cytokine elevations and may be confined to discrete brain regions involved in mood regulation and depressive-like behaviors (\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e). A more detailed characterization of neuroinflammatory markers within regions involved in depression, such as the mesocorticolimbic system, hippocampus, and DRN, would therefore be instrumental in elucidating the central immune mechanisms underlying the observed behavioral alterations. Our previous work demonstrated that MIA male offspring exhibit increased expression of cyclooxygenase-2 (COX-2) and ionized calcium-binding adaptor molecule 1 (IBA-1) in the whole brain (\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e). These markers are associated with latent inflammatory states and sustained microglial activation and may reflect long-lasting neuroimmune vulnerability to the emergence of latent depression-related phenotypes. In conclusion, these findings suggest that gestational administration of HMW poly(I:C) selectively alters specific aspects of depressive-like behavior in the offspring rather than inducing a broad depressive phenotype. Notably, these behavioral alterations were not paralleled by changes in serotonergic activity in the DRN, suggesting that mechanisms beyond classic serotonergic dysfunction may underlie these outcomes, and more investigations will be required. Taken together, our results underscore the importance of considering species, immune challenge characteristics, and sex as critical variables producing different output following the MIA model. Future studies should aim to characterize the spatial and temporal dynamics of neuroinflammation across brain regions implicated in mood regulation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e5. Funding\u003c/h2\u003e\u003cp\u003eThis work was supported by Progetti di Rilevante Interesse Nazionale (Grant Nos. PRIN 2022 P20229CCLB and 2022NSLB3Z [to MP]) from the Italian Ministry of University and Research, by the Assessorato alla Programmazione (Grant No. PNRR-MAD-2022-12375802; Regione Autonoma della Sardegna) [to MP]), and by Hybrid Hub: Modelli cellulari e COMputazionali, micro e nanotEcnologie per la personalizzazione di Terapie innovAtive grant (Grant No. T4-AN-10 [to MP]) from the Italian Ministry of Health.\u003c/p\u003e\u003ch2\u003e6. Acknowledgements\u003c/h2\u003e\u003cp\u003eWe thank Marta Tuveri and Dr. Barbara Tuveri for their skillful assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJaswa EG, Huddleston HG, Lindquist KJ, Wu AHB, Bishop SL, Kim Y. In Utero Exposure to Maternal COVID-19 and Offspring Neurodevelopment Through Age 24 Months. Published online 2024. doi:10.1001/jamanetworkopen.2024.39792\u003c/li\u003e\n\u003cli\u003eKim DH, Croen LA, Iosif AM, et al. The association of maternal COVID-19-infection during pregnancy on the neonatal immune profile and associations with later diagnosis of neurodevelopmental disorders. \u003cem\u003eBrain Behav Immun\u003c/em\u003e. 2025;123(May 2024):1071-1080. doi:10.1016/j.bbi.2024.11.014\u003c/li\u003e\n\u003cli\u003eBrown AS. 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Neuroinflammation mechanisms of neuromodulation therapies for anxiety and depression. \u003cem\u003eTransl Psychiatry\u003c/em\u003e. 2023;13(1). doi:10.1038/s41398-022-02297-y\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7131610/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7131610/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaternal immune activation (MIA) during pregnancy may increase the risk for neurodevelopmental disorders such as schizophrenia and autism in offspring. Preclinical and human evidence support a potential role for MIA in the development of depressive symptoms in offspring. Among animal models of MIA, prenatal treatment with the synthetic viral mimetic polyinosinic-polycytidylic acid [poly (I:C)] is well established in studying psychotic-like and autism-like phenotypes, while its validity for modeling depressive-like behaviors remains underexplored. In this study, we assessed whether MIA, induced in rats with an injection of high molecular weight (HMW) poly (I:C), at gestational day 15, leads to a depressive-like phenotype in the offspring. In male and female offspring during adolescence and adulthood, we evaluated i) behavioral despair and anhedonia using the forced swim test (FST) and sucrose preference test (SPT); ii) the electrophysiological properties of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons \u003cem\u003ein vivo\u003c/em\u003e; iii) serum cytokine profile. We found that MIA offspring exhibited increased immobility and reduced climbing and swimming in the FST, with more pronounced effects in males, while sucrose preference remained unaltered. \u003cem\u003eIn vivo\u003c/em\u003e recordings revealed a significant increase in 5-HT neuron firing rate in MIA adult males. Peripheral cytokine analysis showed elevated IL-1α in MIA males and decreased GRO/KC levels in MIA females. In conclusion, these findings indicate that prenatal exposure to HMW poly (I:C) selectively affects stress-coping mechanisms without inducing anhedonia, modulates serotonergic signaling in a sex- dependent manner in the absence of widespread inflammatory alterations.\u003c/p\u003e","manuscriptTitle":"Maternal Immune Activation with High Molecular Weight Poly (I:C) Induces Selective Depressive-Like Phenotype in Adult Offspring","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 13:01:02","doi":"10.21203/rs.3.rs-7131610/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-10-22T09:31:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-26T17:02:19+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-06T09:18:35+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-23T22:23:43+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-21T15:36:34+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-07-21T09:05:27+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-07-18T17:49:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-18T09:08:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-17T11:52:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2025-07-16T13:18:49+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-07-16T10:32:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f718a418-68f6-4d27-b067-ef1cc7bfa795","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51769744,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":51769745,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2026-03-13T07:12:17+00:00","versionOfRecord":{"articleIdentity":"rs-7131610","link":"https://doi.org/10.1038/s41398-026-03926-6","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2026-03-03 05:00:00","publishedOnDateReadable":"March 3rd, 2026"},"versionCreatedAt":"2025-07-23 13:01:02","video":"","vorDoi":"10.1038/s41398-026-03926-6","vorDoiUrl":"https://doi.org/10.1038/s41398-026-03926-6","workflowStages":[]},"version":"v1","identity":"rs-7131610","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7131610","identity":"rs-7131610","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}