Stage-Dependent Disruptions in Neurogenesis and Neurotrophins’ Production Following Prenatal and Postnatal Valproic Acid Exposure: Implications for Autism Spectrum Disorders

Stage-Dependent Disruptions in Neurogenesis and Neurotrophins’ Production Following Prenatal and Postnatal Valproic Acid Exposure: Implications for Autism Spectrum Disorders | 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 Research Article Stage-Dependent Disruptions in Neurogenesis and Neurotrophins’ Production Following Prenatal and Postnatal Valproic Acid Exposure: Implications for Autism Spectrum Disorders Katarine Fereshetyan, Margarita Danielyan, Konstantin Yenkoyan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5920890/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Cellular and Molecular Neurobiology → Version 1 posted 8 You are reading this latest preprint version Abstract Autism spectrum disorders (ASD) are neurodevelopmental conditions involving impaired neuronal processes such as connectivity, synaptogenesis, and migration. Prenatal exposure to valproic acid (VPA), an anticonvulsant and mood stabilizer, is linked to increased ASD risk, with timing as a key factor. However, the molecular mechanisms of VPA-induced neurodevelopmental disruptions remain unclear. Building on our previous study, which characterized VPA-induced prenatal and postnatal ASD models with impaired social behavior, repetitive patterns, and altered brain connectivity, this study examines molecular changes in neurogenic brain regions. We analyzed the prefrontal cortex, hippocampus, and subventricular zone at key developmental time points (postnatal days 14 and 21), assessing neurotrophins (BDNF, Nt-3, IGF-β, GDNF) and markers of cell migration (DCX), differentiation (NeuN, GFAP), and synaptogenesis (synaptophysin). Our findings show that both prenatal and postnatal VPA exposure disrupt neurogenesis, with prenatal effects being more severe and persistent. Prenatal VPA significantly reduced BDNF in the SVZ and DCX in the olfactory bulb, indicating impaired migration, while morphological analysis revealed increased ependymal proliferation and disrupted SVZ organization. Postnatal exposure led to transient neurotrophin changes, including delayed IGF-β production and an abnormal rise of BDNF levels. Elevated GFAP and reduced synaptophysin in the PFC, alongside increased neuronal markers in the hippocampus, suggest region-specific neuro-glial imbalances. These findings highlight the stage-dependent vulnerability of the developing brain to VPA exposure, revealing distinct mechanisms of disruption in prenatal and postnatal administration. They underscore the need to minimize exposure risks during late gestation and early postnatal periods, which are crucial for neurodevelopment. autism spectrum disorders valproic acid pre-and postnatal models neurotrophins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Autism spectrum disorders (ASD) are neurodevelopmental disorders that are characterized by altered verbal and non-verbal communication, and repetitive and stereotyped behavior (Home APA DSM-5; Lai et al. 2014 ). Heterogeneity of disorder is one of the main challenges that complicates the identification of the pathological mechanisms and treatment approaches. Among the most prominent modern theories of ASD etiology and pathogenesis are the impairment of neural connectivity and neuronal migration, imbalance in excitatory-inhibitory neural activity, damaged synaptogenesis (Rubenstein and Merzenich 2003 ; Belmonte et al. 2004 ; Nelson and Valakh 2015 ; Yenkoyan et al. 2017 ). Moreover, multiple studies provided strong evidence that chaos in the autistic brain is caused by dysregulated stages of neurodevelopment starting from the gestational period up to early postnatal development (Watts 2008 ; Amaral et al. 2008 ; Levy et al. 2009 ). Neural tube formation occurs in the first trimester of pregnancy, synaptic connections, cell communication in the second trimester, and arborization and myelinization in the third trimester(Tau and Peterson 2010 ). Many clinical studies demonstrate that the anticonvulsant and mood-stabilizing therapy using valproic acid (VPA) during pregnancy results in higher incidences of ASD. However, the relative sensitivity of different trimesters to ASD development in children exposed to VPA in utero is still not clear. Studies on the risk of ASD development depending on the trimester of exposure produced rather controversial data (Christensen et al. 2013 ; Coste et al. 2020 ). Therefore, exploring the significance of exposure timing on ASD-related changes is considered to be of a high priority. Assuming that ASD may affect different stages of neurogenesis, such as the proliferation of stem and progenitor cells, migration, differentiation, synaptic network formation, pruning and elimination of synapsis we hypothesized that the extent of valproic acid-induced damage may similarly vary throughout the prenatal and early postnatal neurodevelopment. Therefore, we carried out a comparison of two VPA-induced models of ASD: prenatal and postnatal. As the VPA autism model was developed in rats we had to consider the differences in brain development between humans and rats. Postnatal days (PND) 1–14 in rats are predicted to correspond to the third trimester of human gestation in terms of brain development stages (Clancy et al. 2007 ). Thus, in rats, brain cell death, synaptic pruning, and myelination activity peak after birth during the postnatal 2–3 weeks (Zeiss 2021 )(Bandeira et al. 2009 ). In our previous study, we evaluated and compared VPA-induced prenatal and postnatal ASD models in terms of long-term behavioral, electrophysiological, and morphological changes (Fereshetyan et al. 2021 ). We validated both models on postnatal development day 30 with rats manifesting impaired social activity, playing behavior, and patterns of repetitive behavior. Detected changes were only partially saved in postnatally treated animals on postnatal development day 60. Reversibility of behavioral alterations showed the possible recovery of brain damage inflicted in the postnatal period. A similar pattern of recovery was observed in the morphological changes of cells over the course of two months of development. On postnatal development day 70 electrophysiological studies showed impaired connectivity between the prefrontal cortex and hippocampus, and cerebellum, and amygdala. These target brain regions were chosen due to their involvement in the regulation of ASD-related behavior. Based on these data in the current study we aimed to assess the molecular alterations occurring in the neurogenic brain regions at early stages following pre- and postnatal VPA administration. Many studies showed the association of neurotrophins with the development of the different regions of the brain. For instance, postnatal day 14 is the peak period for the expression of neurotrophins in the hippocampus and prefrontal cortex (Das et al. 2001 ; Bandeira et al. 2009 ). This peak is typically replaced by a gradual decline in the following week, eventually leveling off after the 21st day of postnatal development. Moreover, recent clinical studies indicate changes in the level of neurotrophins in autistic children's blood or cerebrospinal fluid, which makes their involvement in autism development more evident(Nelson et al. 2001 ; Nickl-Jockschat and Michel 2010 ; Galvez-Contreras et al. 2017 ). However, whether VPA affects the neurotrophins during postnatal development remains unclear. Therefore, we compared the effects of prenatal and postnatal administration of VPA on the levels of neurotrophins in the main neurogenic regions of the brain at critical periods (PND 14 and 21). A number of studies showed increased growth of autistic children’s brains associated with an increased number of neurons and general disorganization of brain cytoarchitecture (Kemper and Bauman 1993 ; Casanova et al. 2006 ; Courchesne et al. 2011 ). About 20% of children with diagnosed autism had macrocephaly (McCaffery and Deutsch 2005 ; Sacco et al. 2015 ). One of the hypotheses explaining the cause of macrocephaly is an imbalance between the processes of neuronal proliferation and elimination during neurogenesis (Courchesne et al. 2001 ). Synaptic dysfunction can be considered as another pathological pattern underlying ASD. Many studies indicate abnormalities in synaptic elimination, synaptic transmission, and plasticity caused by ASD-associated gene mutations or environmental factors leading to synaptic dysfunction (Guang et al. 2018 ). The progenitor cell migration is another pathological mechanism contributing to ASD. Therefore, we chose several protein targets in neurogenic regions of brain to assess the possible impairment of the above-mentioned processes. For instance, doublecortin modulates and stabilizes microtubules, aiding cell migration in neurogenesis (Francis et al. 1999 ; Merz and Lie 2013 ). NeuN is a stable marker for post-mitotic neurons (Duan et al. 2016 ). Synaptophysin, a key synaptic protein, is consistently distributed in neural synapses (Kwon and Chapman 2011 ). Glial fibrillary acidic protein (GFAP) is an astrocytic structural marker protein and is present in the majority of CNS astrocytes (Jurga et al. 2021 ). Therefore, we chose to detect the regional expression of DCX, GFAP, NeuN, and SYP to assess cell migration, differentiation, and synaptic networks during critical developmental periods. Methods Animals Experiments were performed on adult 4–6 months old female Sprague–Dawley rats, as well as pups and young rats during the early postnatal life. Animals were purchased from the YSMU vivarium and kept under conditions of 12-h light/dark cycle, with controlled temperature (22 ± 2°C) and free access to food and water. All animals were randomly divided into groups of 3–4 animals per cage. All cages were labeled, so the animals' group identity was non-blinded. The experimental protocol adhered to the European Communities Council Directive (86/609/EEC) and was conducted in compliance with the ARRIVE guidelines for animal research (Percie du Sert et al. 2020 ). Study design The whole study was performed in parallel on prenatal and postnatal VPA-treated groups with respective controls (Fig. 1 ). Prenatal model Female rats were paired with males overnight. The next morning vaginal smears were collected, sperm positive cases were registered as day 0 of gestation. On the 12th day of gestation VPA (Sigma-Aldrich, P4543) was injected intraperitoneally in a dose of 500 mg/kg (150 mg/ml), and the control group received the same volume of saline. Postnatal model Pups born to untreated rats received VPA intraperitoneally in a dose of 200 mg/kg on PND 5–12, and control groups received saline injection (Chomiak et al. 2010 ; Reynolds et al. 2012 ). A dosage of 400 mg/kg was also evaluated but subsequently deemed unsuitable due to the observation of hyperactive and agitated behavior in the pups, as well as excessive body weight gain following the injections. Sample preparation On PND 14 and 21 pups were euthanized with inhaled isoflurane followed by an overdose of nembutal (Sigma-Aldrich, Y0002194) i.p. (100 mg/kg) injection and were transcardially perfused with isotonic saline. Prefrontal cortex, hippocampus, olfactory bulbs, and subventricular zone were separated from the isolated brains. For the detection of specific neurotrophins and protein markers by ELISA and Western blot tissues were mechanically lysed (FastPrep-24, Lysing Matrix D, SKU:1169130-CF) in ice-cold lysis buffer containing 50 mM Tris/HCl pH 7.5 (Sigma-Aldrich, #1185-53-1), 150 mM NaCl (Sigma-Aldrich, #7647-14-5), 1% triton X-100 (Sigma-Aldrich, #T8787), protease inhibitors (Roche, #04693132001), and then incubated for 20 min at + 4°C. After centrifugation at 14000 rpm for 20 min, supernatants were collected and stored at − 80°C. Total protein concentration was detected by Bradford assay. Western blot Fifty micrograms of supernatants were resolved through 12% acrylamide SDS-PAGE gel and then transferred onto PVDF membranes (Sigma-Aldrich, GE10600023) with a trans-blot semi-dry transfer cell (Bio-Rad). Membranes were incubated for 1 h at room temperature in a blocking solution containing 5% non-fat dry milk (Cell signaling, #9999S) in TBS-T (tris-buffered saline, 0.1% tween 20). Membranes were incubated overnight at + 4°C with the primary antibodies against GFAP (Dako, Z0334, 1:1000), DCX (sc-271390, 1;500), NeuN (MAB377, 1:500), Synaptophysin (SAB4200544, 1:1000). After incubation membranes were rinsed in 0.05% TBS-T and incubated with secondary HRP-conjugated antibody for 1.5 h at room temperature (Goat Anti-Rabbit IgG H&L, ab6721, 1:1000, Goat Anti-Mouse IgG H&L, ab97023, 1:1000). Immunoblots were detected by ECL kit (Thermo Scientific, 32106) and visualized using FusionFX (Vilber). Values were normalized to those of β-actin (Cell signaling, #4967). All proteins were assessed on the same membrane ( see WBs membrane images in Supplementary file 2_WBs ). ELISA BDNF (Rat BDNF ELISA Kit PicoKine, EK0308), IGF-1 (Rat IGF-1 ELISA Kit PicoKine, E0377), Neurot-3 (Rat NT-3 ELISA Kit PicoKine, E0474) and GDNF (Rat GDNF ELISA Kit PicoKine, EK0363) were detected by corresponding kit assays as to the manufacturer’s instructions. Morphological study Cell structural assessment was done by detecting the activity of Ca 2+ -dependent acidic phosphatase. The method allows for identification of both large and small-size cells and is based on the modifications of Nissl staining and Golgi silver impregnation (Yenkoyan et al. 2011 ). Brains were isolated and fixed on PND14 and 21 in the 5% buffered neutral formalin (containing 0.1 M phosphate buffer pH 7.4, 0.3% CaCl 2 , 15% sucrose) for 24–48 h at 4 0 C. The frontal free-flow frozen slices (40–50 µm thick) of the hippocampus and SVZ were isolated. Slices were washed in distilled water and incubated in the following solution: 0.4% lead acetate, 1 M acetate buffer (pH 5.6), and 2% sodium glycerophosphate, for 2–3 h at 37 0 C. Thereafter the slices were washed in distilled water, transferred to a 3% sodium sulfide solution, rewashed in distilled water, and embedded into the Canada balsam (Sigma-Aldrich, #8007-47-4). The sections were analyzed using an Opton light microscope (West Germany) at magnifications of ×400 and ×1000. Neurons were counted on every 5th section obtained from the studied areas of the rat brain. The distance between the analyzed sections was 200 µm. The number of neurons in digital micrographs was counted manually in the ImageJ program (U.S. National Institutes of Health, Bethesda, MD, USA) using the Mult-point tool per 100 µm of cell layer length in different areas of the brain: CA3 field of the hippocampus, PFC, olfactory bulb, and subventricular zone. Statistical comparisons of quantitative analyses were performed using Student's t-test. Statistical analyses were conducted using GraphPad Prism, version 7 (GraphPad Software Inc., San Diego, CA, USA). Statistical analysis GraphPad Prism Software 8.0.1 (San Diego, CA, USA) was used to perform the statistical analyses and to generate the graphs. A priori sample size estimation was conducted based on expected effect sizes from previous studies and pilot experiments. A significance level of 0.05, power of 0.8 and equal group allocation were assumed. For the Western blotting method, 6 rats were used per group, and for the ELISA method, 4 to 6 rats were used per group. To assess the normality and homogeneity of variance, the Shapiro-Wilk test and F-test were used, respectively. The analysis results were provided in the Supplementary Information (Supplementary file 1). The results of two groups (Control 1 vs PreVPA, Control 2 vs PostVPA) were statistically analyzed using unpaired Student’s t -test. Mann Whitney U test was used for analysis of the non-parametric data with two groups. P values less than 0.05 were considered statistically significant. Data represent the mean values ± SD. Results Changes in the level of neurotrophins, neuroglial, and synaptic markers in brain target zones of VPA-treated groups in the early postnatal period To assess the possible shifts in early neurogenesis, the main modulators of this process – neurotrophins, were analyzed in the neurogenic niches of the brain. It is known that in the SVZ neuroblasts migrate long distances to integrate into the olfactory bulb (OB), however, in the dentate gyrus, they integrate into the local hippocampal network (Alvarez-Buylla and García-Verdugo 2002 ; Ming and Song 2011 ; Lim and Alvarez-Buylla 2016 ). Thus, on PND 14 and 21 the olfactory bulb, prefrontal cortex, SVZ, and hippocampus were isolated and tested to detect the levels of BDNF, NT-3, IGF-1, and GDNF. In addition, we have tested also specific markers of differentiation and maturation stage of the cells during the neurodevelopment (DCX, NeuN, GFAP, Syp). In the prefrontal cortex, the main changes were detected in the levels of GFAP, NeuN, and synaptophysin which were found dysregulated in opposite directions. Particularly, the level of GFAP was increased in prenatally and postnatally VPA-treated groups on both PNDs, whereas the level of synaptophysin was decreased (Fig. 2 A, C, E, G). A significant decrease of NeuN was detected only in prenatally treated rats (Fig. 2 A, E). In the hippocampus significant increase of GFAP was detected in the postnatally VPA-treated group on PND 14 (Fig. 3 C). Significantly decreased GFAP and doublecortin, and increased synaptophysin were detected in VPA-treated both groups on PND 21 (Fig. 3 E, G). A significantly increased level of NeuN was detected in the postnatally VPA-treated group on PND 14 (Fig. 3 C) and the prenatally VPA-treated group on PND 21 (Fig. 3 E). Similar changes were also registered in the olfactory bulb. Specifically, the GFAP was found increased in the both VPA-treated groups on PND 14 (Fig. 4 A, C), and in postnatally VPA-treated group on PND21 (Fig. 4 G). Significantly decreased expression of doublecortin was found in both VPA-treated groups on PND 21 (Fig. 4 E, G). Interestingly, changes in BDNF expression in the prenatally and postnatally VPA-treated groups were similar on both PNDs. Particularly, a significant increase in BDNF level was observed in the olfactory bulb of VPA-treated rats in comparison to control on PND 14 (Fig. 4 B, D) and 21 (Fig. 4 F, H). In SVZ the level of BDNF was significantly decreased on PND 14 in both the VPA-treated groups (Fig. 5 A, B), whereas on PND 21 BDNF was significantly increased in the prenatally VPA-treated group (Fig. 5 C). Postnatally VPA-treated group did not show a significant difference in comparison to the control group on PND 21, however, the tendency of changes was the same. Significantly increased level of BDNF was detected in the hippocampus in both of the VPA-treated groups on PND14 (Fig. 3 B) and PND21 (Fig. 3 F, H). In the postnatally VPA-treated group on PND 14 no significant changes were detected in BDNF level as compared to the control group (Fig. 3 D). In PFC no significant changes were detected in all of the tested groups (Fig. 2 B, D, F, H). A consistent pattern of changes in Nt-3 levels was identified in both the prenatally and postnatally VPA-treated groups on PND 14. A mild decrease was detected in the PFC of the prenatally VPA-treated group (Fig. 2 B), whereas a more significant one was shown in the postnatally VPA-treated group (Fig. 2 D). In SVZ the level of Nt-3 was significantly decreased in both of the groups, though in the postnatally VPA-treated group, it was more pronounced (Fig. 5 A, B). In the hippocampus, a significantly increased level of Nt-3 was identified in both groups (Fig. 3 B, D). There were no changes in the olfactory bulb (Fig. 4 B, D). However, this pattern was completely different in the prenatally VPA-treated group on PND 21. In PFC the level of Nt-3 was elevated in the prenatally VPA-treated group (Fig. 2 F), whereas in the hippocampus it was decreased compared to the control group (Fig. 3 F). The decreased level of Nt-3 was detected in the olfactory bulb of the prenatally VPA-treated group (Fig. 4 F). In the postnatally VPA-treated group significantly decreased level of Nt-3 was identified only in the hippocampus on PND 21 (Fig. 3 H). The alterations in IGF-1 levels across all examined structures were consistent between the two VPA-treated groups. A general reduction in IGF-1 was observed on PND 14 and 21 in both groups (Fig. 2 D, F, Fig. 3 B, D, F, Fig. 4 B, D, F,), except for the postnatally VPA-treated group on PND 21 (Fig. 2 H, Fig. 3 H, Fig. 4 H), where IGF-1 levels in all target structures were notably increased compared to the control group. The GDNF level was increased in the hippocampus of postnatally VPA-treated rats on PND 14 (Fig. 3 D) and decreased in prenatally VPA-treated group on PND 21 (Fig. 3 F). The significant decrease of GDNF levels was identified in the olfactory bulb on PND 21 of prenatally VPA-treated rats (Fig. 4 F). The expression of GDNF in the prefrontal cortex (Fig. 2 B, D, F, H) and subventricular zone (Fig. 5 B, D, F, H) were below the sensitivity range of the assay kit. A comparison of the two models suggests that the pattern of dysregulation of neurotrophins (BDNF, Nt-3, IGF) was similar primarily on PND 14, with pronounced differences emerging by PND 21. Additionally, time-dependent (PND14 vs PND21) intragroup changes in BDNF and Nt-3 levels were observed in the prenatally treated group. Morphological changes in the prefrontal cortex, hippocampus, olfactory bulb, and subventricular zone of VPA-treated groups in the early postnatal period Morphological changes in target structures were assessed on PND 14 and PND 21. Swollen pyramidal cells with central chromatolysis and non-clear contours were detected in the prefrontal cortex of prenatally VPA-treated rats on PND 14 in comparison to the control group (Fig. 6 I). Damaged cells showed morphology characterized by short and more colorless stained dendrites. Similar changes with less pronounced intensity were observed on PND 21 (Fig. 7 I). Damaged cellular structures were observed in the dentate gyrus of the hippocampus in prenatally VPA-treated rats on PND 14 (Fig. 6 III) as well as neurons of abnormal shape and size. Granular cells were less stained but their morphological characteristics were close to the controls. The cellular barrier was visualized, and centrally localized nuclei were detected. The pyramidal cells of CA3 and CA4 were hypertrophic with no clear cellular barrier with a mostly elongated shape. All the described morphological changes were normalized on PND 21 (Fig. 7 III). Similar changes were observed in the CA2 and CA1 regions of the hippocampus on PND 14. Interestingly, there were significant numbers of degenerated cells with chromatolysis in CA2 and CA1 on PND 21. In the olfactory bulb of prenatally VPA-treated rats round or oval-shaped cells with central chromatolysis were seen on PND 14 (Fig. 6 II) and PND 21 (Fig. 7 II). Neurons in certain areas preserved their shape and size. In the subventricular zone of prenatally VPA-treated rats multilayer proliferation of round-shaped ependymal cells was observed on both PNDs (Fig. 6 , 7 IV). Meanwhile, in the control group, the walls of the third ventricle were in close contact, and nuclei were mostly localized in the base of the cylinder-shaped cells. In the hippocampus of postnatally VPA-treated group cells remained intact on PND 14 (Fig. 6 VII) and PND 21 (Fig. 7 VII). Cells had clearly defined cellular barriers, basically localized nuclei. In the subventricular zone of postnatally VPA-treated rats intensive proliferation of ependymal cells was registered on PND 14 and PND 21 (Fig. 6 , 7 VIII). Desquamation of ependymal cells was detected in some places, but the close contact between cells remained mostly intact. In the prefrontal cortex (Fig. 6 , 7 V) and olfactory bulbs (Fig. 6 , 7 VI) of postnatally VPA-treated rats cells mostly preserve the normal structure and shape. Only several cells with swollen nuclei were detected. The morphological comparisons showed more severe cellular damage in the prenatal model, affecting all tested structures, while the postnatal model exhibited changes only in the SVZ. On PND 21, damage persisted in the olfactory bulb and SVZ but was normalized in the PFC and hippocampus in prenatally VPA-treated animals. Discussion This study aimed to evaluate and compare the effects of prenatal and postnatal valproic acid (VPA) exposure on neurotrophin levels, cellular differentiation, and brain organization in primary neurogenic regions during critical early postnatal periods. Building on our previous findings on behavioral and electrophysiological alterations in VPA-induced ASD models (Fereshetyan et al. 2021 ), we investigated the early molecular mechanisms underlying these changes. Given the established role of neurotrophins in neurodevelopmental processes such as cell migration, differentiation, and synaptic network formation, we sought to clarify the molecular and cellular alterations contributing to ASD-like pathophysiology. Neurogenesis markers (DCX, NeuN, SYP, GFAP) were chosen to assess VPA-induced brain alterations, contextualized within ASD pathogenesis. Key findings highlight distinct impacts of prenatal and postnatal VPA exposure on cell migration and neurogenesis. Reduced BDNF levels in the SVZ on PND 14 and decreased DCX in the olfactory bulb by PND 21 suggest impaired migration of progenitor cells, consistent with literature emphasizing BDNF's role in guiding premature cell migration (Henry et al. 2007 ). The comprehensive reduction of neurotrophins in the SVZ underscores VPA’s suppressive effect. Dysregulated migration of the cells during the neurogenesis leads to abnormal distribution and disorganized cortical lamination, which is one of the common features of the autistic brain (Wegiel et al. 2010 ). Brain heterotopias are highly associated with doublecortin defects, which is involved in the formation of microtubules required for cell migration. Moreover, decreased DCX levels in the hippocampus of prenatally VPA-treated rats (PND 21) align with previous studies on ASD models (Aranarochana et al. 2021 ). Ependymal barrier disruptions observed on PND 14 in both VPA groups, including increased cell numbers and desquamations, may attenuate NSC migration by disrupting CSF-mediated signaling (Kaneko and Sawamoto 2018 ). Another major finding is the altered neuro-glial ratio in the PFC, characterized by elevated GFAP and reduced synaptophysin on PND 14 and PND 21, indicative of reactive gliosis or impaired neuronal differentiation. This is supported by various studies indicating the involvement of astrocyte-mediated neuroinflammation in the pathogenesis of autism (Gzielo and Nikiforuk 2021 ). Similar patterns have been observed in ASD models and postmortem studies (Edmonson et al. 2014 ). The study on non-human primates showed a decreased level of doublecortin and NeuN in PFC and increasing in GFAP level after VPA treatment in utero (Zhao et al. 2019 ). Currently, limited literature is available addressing the alterations in neuroglial markers following postnatal VPA injections. DiCicco-Bloom et al showed increased GFAP levels in the frontal cortex after PND 2–4 VPA exposure on PND 21 (Mony et al. 2016 ). Interestingly, the pattern of hippocampal neuroglial dynamics did not match our data, with increased NeuN and synaptophysin levels reflecting enhanced neuronal differentiation, potentially driven by elevated BDNF on PND 21. Several studies indicate that the reduction of BDNF expression in the hippocampus does affect the number of neurons, but suppresses the genes encoding proteins of vesicular trafficking and synaptic communication (Gao et al. 2009 ; Rauskolb et al. 2010 ; Wang et al. 2015 ). However, reduced NT-3 suggests a dominant role for BDNF in hippocampal neurogenesis during this period. The other piece of data worth discussing is the changes in neurotrophins’ expression during the development. Although under normal conditions BDNF expression gradually increases to the maximum level during postnatal development (Karege et al. 2002 ), the VPA-induced rapid increase of BDNF in the brain appears certainly abnormal. Our results indicate such a transition in both of the models. However, it should be noted that in the postnatally VPA-treated group, the BDNF changes at PND 14 and 21 were particularly dramatic. VPA-induced BDNF elevation may cause alterations in synapto- and gliogenesis, neurite outgrowth, dendritic arborization, and spine formation (Vilar and Mira 2016 ; Kowiański et al. 2017 ). The data on IGF-β expression (increase during the first two weeks of postnatal life in control groups) is consistent with previous studies(Bondy et al. 1992 ). IGF levels peaked at PND 14 and gradually declined by PND 21, reflecting the typical developmental pattern. In contrast, the postnatally VPA-treated group showed continued IGF increase until PND 21, exceeding control levels at this stage. Interestingly, the IGF levels at PND 21 matched the control group’s PND 14 peak, suggesting that postnatal VPA exposure shifts the IGF production peak by one week. Further studies on subsequent postnatal days are needed to determine whether IGF levels decline thereafter. In the prenatally VPA-treated group, IGF levels were consistently lower across all structures at both time points, mirroring the pathophysiology of ASD. However, the developmental trend paralleled the control group. These findings indicate distinct differences in neurotrophin dynamics within neurogenic brain regions between the two models. In conclusion, VPA exposure induces significant neurogenesis disruptions that may underlie autism-like pathogenesis in rodents. Both prenatal and postnatal VPA exposure result in alterations in neurogenesis, such as dysregulated neurotrophin levels and impaired cell migration, reflecting some shared mechanisms of disruption. However, prenatal exposure exerts stronger and more persistent effects compared to postnatal exposure, as evidenced by our prior findings on the reversibility of ASD-associated behaviors in postnatally VPA-treated rats (Fereshetyan et al. 2021 ). These findings underscore the critical vulnerability of brain development to VPA, particularly during the third trimester of human pregnancy, reinforcing the need for vigilant risk assessment and preventive strategies. Limitation A notable limitation of this study is the absence of sex-specific analysis in the investigation of neurotrophins and neurogenic markers in the valproic acid induced rat model of autism. Autism spectrum disorder is known to be more prevalent in males, with an estimated male-to-female ratio of 4:1 (Baio et al. 2018 ). However, this sex-based disparity has not been conclusively established in VPA-exposed children with autism (Honybun et al. 2021 ). While numerous studies have reported sex-associated differences in behavioral patterns in VPA-exposed animal models (Chaliha et al. 2020 ), data regarding sex-specific molecular alterations remain limited. Given these findings, the present study focused on identifying overall differences between the treated and control groups without stratifying by sex, which can be considered a reasonable approach in this exploratory phase of research. Nonetheless, future studies should incorporate sex as a biological variable to better elucidate potential sex-specific differences, particularly in the context of developmental trajectories and molecular responses. Declarations Acknowledgments The authors thank Dr Souren Mkrtchian (Karolinska Institute, Stockholm) for the fruitful discussions and critical reading of the manuscript, and Hasmik Harutyunyan (COBRAIN Center, Yerevan State Medical University) for help with graphical illustrations. Author contributions Katarine Fereshetyan: conceptualization, methodology, investigation, data curation, writing original draft. Margarita Danielyan: methodology, investigation. Konstantin Yenkoyan: conceptualization, methodology, project administration, funding acquisition, resources, supervision, writing - review & editing. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by the Higher Education and Science Committee, Ministry of Education, Science, Culture and Sports of RA (23LCG-3A020 and 25YSMU-CON-I-3A), and YSMU. Data availability Data can be made available by the corresponding author upon reasonable request. Conflict of interests The authors declare no competing interests. Ethical Approval The experimental protocol adhered to the conditions set forth by the European Communities Council Directive (86/609/EEC) and was approved by the Ethics Committee of Yerevan State Medical University after Mkhitar Heratsi (Approval Nos: N1, 20.09.2018; N8-3/24, 14.03.2024; N3, 14.11.2024). The study was conducted in compliance with the ARRIVE guidelines for animal research (Percie du Sert et al., 2020). 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Acta Neuropathol 119:755–770. https://doi.org/10.1007/s00401-010-0655-4 Yenkoyan K, Grigoryan A, Fereshetyan K, Yepremyan D (2017) Advances in understanding the pathophysiology of autism spectrum disorders. Behav Brain Res 331:92–101. https://doi.org/10.1016/j.bbr.2017.04.038 Yenkoyan K, Safaryan K, Chavushyan V et al (2011) Neuroprotective action of proline-rich polypeptide-1 in ??-amyloid induced neurodegeneration in rats. Brain Res Bull 86:262–271. https://doi.org/10.1016/j.brainresbull.2011.08.003 Zeiss CJ (2021) Comparative Milestones in Rodent and Human Postnatal Central Nervous System Development. Toxicol Pathol 49:1368–1373. https://doi.org/10.1177/01926233211046933 Zhao H, Wang Q, Yan T et al (2019) Maternal valproic acid exposure leads to neurogenesis defects and autism-like behaviors in non-human primates. Transl Psychiatry 9:267. https://doi.org/10.1038/s41398-019-0608-1 Home | APA DSM-5 http://www.dsm5.org/Pages/Default.aspx . Accessed 14 Nov 2015 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile1.pdf Supplementaryfile2WBs.pdf Graphicalabstract.png Graphical abstract Dynamic and comparative assessment of neurotrophins, neuroglial and synaptic markers, morphological changes both in prenatal and postnatal models of ASD. The graphical abstract illustrates the main significant changes observed in prenatal (left section) and postnatal (write section) models in comparison to the control (central section) group. In the middle (PND 14) and top rows (PND 21), the molecular and morphological changes schematically are illustrated in the relevant structures of the hemisphere (olfactory bulb, prefrontal cortex, subventricular zone, and hippocampus, respectively from the left to the write). Morphological changes of neuronal cells are expressed by different colors: red neurons with abnormal morphology, and morphologically healthy transparent pink neurons. Slow migration of neuroblasts on the rostral way in prenatal (left section) and postnatal models (right section) is expressed by a red background in comparison with the green color in the control group (central section). The changes in protein levels are expressed by different numbers of cells. Decreased doublecortin level in the olfactory bulb on PND 21 is expressed by illustration of one cell instead of three as it is shown on PND 14. Changes in GFAP and NeuN, SYP markers indicating disbalance in the neuroglial ratio in the prefrontal cortex and hippocampus are expressed by comparatively different numbers of the cells (green astrocytes, red neurons). Inside the magnified synaptic clefts vesicles with the different numbers of SYP on the surface are illustrated. The level of neurotrophins in SVZ is expressed as three-color rectangular, which is compressed in prenatal (left middle section) and postnatal (right middle section) on PND 14 in comparison to the control group (central middle row). Cite Share Download PDF Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Cellular and Molecular Neurobiology → Version 1 posted Editorial decision: Revision requested 22 May, 2025 Reviews received at journal 21 May, 2025 Reviews received at journal 03 May, 2025 Reviewers agreed at journal 10 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers invited by journal 05 Apr, 2025 Submission checks completed at journal 02 Apr, 2025 First submitted to journal 29 Mar, 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-5920890","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":439648655,"identity":"87107d43-972a-4705-808f-c3b4dfa393a8","order_by":0,"name":"Katarine Fereshetyan","email":"","orcid":"","institution":"Yerevan State Medical University after M. Heratsi","correspondingAuthor":false,"prefix":"","firstName":"Katarine","middleName":"","lastName":"Fereshetyan","suffix":""},{"id":439648656,"identity":"0cb6dedb-ffa1-47ed-940f-82a6c34eedc8","order_by":1,"name":"Margarita Danielyan","email":"","orcid":"","institution":"L. A. Orbeli Institute of Physiology NAS","correspondingAuthor":false,"prefix":"","firstName":"Margarita","middleName":"","lastName":"Danielyan","suffix":""},{"id":439648657,"identity":"cc066c66-59cb-4331-8444-bdaa14e33da0","order_by":2,"name":"Konstantin Yenkoyan","email":"data:image/png;base64,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","orcid":"","institution":"Yerevan State Medical University after M. Heratsi","correspondingAuthor":true,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Yenkoyan","suffix":""}],"badges":[],"createdAt":"2025-01-28 21:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5920890/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5920890/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10571-025-01623-4","type":"published","date":"2025-11-05T15:57:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80345662,"identity":"2aa46a24-5993-4349-b61d-836a62d1077b","added_by":"auto","created_at":"2025-04-10 19:47:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":649110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design. \u003c/strong\u003eThe whole study was performed in parallel on prenatal and postnatal VPA-treated groups with respective controls. For the prenatal model pregnant rats were injected intraperitoneally VPA in a dose of 500 mg/kg (150 mg/ml) on the 12\u003csup\u003eth\u003c/sup\u003e day of gestation (GD12), and the control group received saline. For the postnatal model pups born to untreated rats were injected intraperitoneally VPA in a dose of 200 mg/kg on PND 5-12, and control groups received a saline injection. Molecular and morphological assessments were conducted on PND14 and PND 21. Neurotrophins (BDNF, Nt-3, IGF-1, GDNF) and cellular markers (GFAP, DCX, NeuN, SYP) were detected in the prefrontal cortex, olfactory bulbs, hippocampus, and subventricular zone by ELISA and western blot methods respectively. Morphological changes were assessed by detection of Ca\u003csup\u003e2+\u003c/sup\u003e-dependent acidic phosphatase activity.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/202aa5d53e9040345411bed1.png"},{"id":80345664,"identity":"1441e0a7-abf4-4d72-a4f2-f9dc8ba6df7a","added_by":"auto","created_at":"2025-04-10 19:47:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3143074,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of biochemical changes in the prefrontal cortex of VPA-treated rats on PND 14 and PND 21. \u003c/strong\u003eThe expression of the GFAP, DCX, NeuN, and SYP proteins in the control, prenatally and postnatally VPA-exposed rats was estimated by the densitometric analysis (A, C, PND 14 and E, G, PND 21) of western blot images (shown are representative immunoblots). Results were normalized to actin. Unpaired \u003cem\u003et-test\u003c/em\u003e, Control vs PreVPA, PND14, t(df)=10.28(4), p=0.0005 (GFAP, A); t(df)=0.3585(4), p=0.7381 (DCX, A); t(df)=6.188(4), p=0.0035 (NeuN, A); t(df)=7.425(4), p=0.0018 (SYP, A). Control vs PostVPA, PND14, t(df)=7.326(4), p=0.002 (GFAP, C); t(df)=2.775(4), p=0.05 (DCX, C); t(df)=2.079(4), p=0.1 (NeuN, C); t(df)=3.641(4), p=0.02 (SYP, C). Control vs PreVPA, PND21, t(df)=4.567(4), p=0.01 (GFAP, E); t(df)=1.473(4), p=0.21 (DCX, E); Mann Whitney test U=0, p=0.1 (NeuN, E); t(df)=6.575(4), p=0.003 (SYP, E). Control vs PostVPA, PND21, t(df)=49.881(4), p=0.0001 (GFAP, G); t(df)=0.5367(4), p=0.62 (DCX, G); t(df)=0.342(4), p=0.75 (NeuN, G); t(df)=3.641(4), p=0.02 (SYP, G). The estimation of the BDNF, NT-3, IGF, and GDNF levels in the control, prenatally and postnatally VPA-exposed rats was done by ELISA analysis on PND 14 (B, D) and PND 21 (F, H). Unpaired \u003cem\u003et-test\u003c/em\u003e, Control vs PreVPA, PND14, t(df)= 0.4178(10), p=0.68(BDNF, B); t(df)= 2.676(9), p=0.025 (NT-3, B); t(df)=2.183(10), p=0.05 (IGF, B); Control vs PostVPA, PND14, t(df)=0.08198(9), p=0.94 (BDNF, D); t(df)= 5.522(7), p=0.0009 (NT-3, D); t(df)=4.388(10), p=0.0014 (IGF, D). Control vs PreVPA, PND21, t(df)= 0.6484(8), p=0.53 (BDNF, F); t(df)=1.925(9), p=0.086 (NT-3, F); t(df)= 7.239(8), p=0.0001 (IGF, F); Control vs PostVPA, PND21, t(df)=1.476(9), p=0.17 (BDNF, H); t(df)=0.1689(9), p=0.87 (NT-3, H); t(df)=3.725(9), p=0.0047 (IGF, H). Data represent the mean values ± SD from n = (4-6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/dafce2e110ddab5dbb3e8c83.png"},{"id":80345668,"identity":"45705f6c-57e0-45d5-aebf-cdb6dfc49b22","added_by":"auto","created_at":"2025-04-10 19:47:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3066709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of biochemical changes in the hippocampus of VPA-treated rats on PND 14 and PND 21. \u003c/strong\u003eThe expression of the GFAP, DCX, NeuN, and SYP proteins in the control, prenatally and postnatally VPA-exposed rats was estimated by the densitometric analysis (A, C, PND 14 and E, G, PND 21) of western blot images (shown are representative immunoblots). Results were normalized to actin. Control vs PreVPA, PND14, Mann Whitney test U=3, p=0.7 (GFAP, A); Unpaired \u003cem\u003et-test\u003c/em\u003e, t(df)=1.972(4), p=0.12 (DCX, A); t(df)=0.4268(4), p=0.69 (NeuN, A); Mann Whitney test U=3, p=0.7 (SYP, A). Control vs PostVPA, PND14, t(df)=3.196(4), p=0.03 (GFAP, C); t(df)=0.5367(4), p=0.62 (DCX, C); t(df)=0.342(4), p=0.75 (NeuN, C); t(df)=6.268(4), p=0.003 (SYP, C). Control vs PreVPA, PND21, t(df)=4.142(4), p=0.014 (GFAP, E); t(df)=3.217(4), p=0.03 (DCX, E); t(df)=0.5585(4), p=0.6 (NeuN, E); t(df)=4.794(4), p=0.009 (SYP, E). Control vs PostVPA, PND21, t(df)=5.474(4), p=0.005 (GFAP, G); t(df)=3.303(4), p=0.03 (DCX, G); t(df)=1.929(4), p=0.13 (NeuN, G); t(df)=6.268(4), p=0.003 (SYP, G). The estimation of the BDNF, NT-3, IGF, and GDNF levels in the control, prenatally and postnatally VPA-exposed rats was done by ELISA analysis on PND 14 (B, D) and PND 21 (F, H). Unpaired \u003cem\u003et-test\u003c/em\u003e, Control vs PreVPA, PND14, t(df)=4.276(10), p=0.0016 (BDNF, B); t(df)=5.769(7), p=0.0007 (NT-3, B); t(df)=7.133(9), p=0.0001 (IGF, B), t(df)=1.715(8), p=0.12 (GDNF, B); Control vs PostVPA, PND14, t(df)=1.445(9), p=0.18 (BDNF, D); t(df)=4.090(8), p=0.0035 (NT-3, D); Mann Whitney test U=0, p=0.004 (IGF, D), t(df)=7.286(8), p=0.0001 (GDNF, D). Control vs PreVPA, PND21, t(df)=3.956(8), p=0.0042 (BDNF, F); t(df)=3.470(8), p=0.0084 (NT-3, F); t(df)=3.261(8), p=0.0115 (IGF, F), t(df)=3.198(8), p=0.01 (GDNF, F); Control vs PostVPA, PND21, t(df)=3.506(10), p=0.0057 (BDNF, H); t(df)= 12.94(8), p=0.0001 (NT-3, H); Mann Whitney test U=0, p=0.004 (IGF, H), t(df)=4.796(10), p=0.0007 (GDNF, H). Data represent the mean values ± SD from n = (4-6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/d213296a573d8cf8f6339855.png"},{"id":80345981,"identity":"d8c6919c-58f8-4250-8254-b76fa86060af","added_by":"auto","created_at":"2025-04-10 19:55:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2891194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of biochemical changes in olfactory bulbs of VPA-treated rats on PND 14 and PND 21. \u003c/strong\u003eThe expression of the GFAP, DCX, NeuN, and SYP proteins in the control, prenatally and postnatally VPA-exposed rats was estimated by the densitometric analysis (A, C, PND 14 and E, G, PND 21) of western blot images (shown are representative immunoblots). Results were normalized to actin. Unpaired \u003cem\u003et-test\u003c/em\u003e, Control vs PreVPA, PND14, t(df)=15.55(4), p= 0.0001 (GFAP, A); t(df)=1.972(4), p=0.12 (DCX, A); t(df)=1.149(4), p=0.31 (NeuN, A). Control vs PostVPA, PND14, t(df)=7.015(4), p=0.0022 (GFAP, C); t(df)=0.568(4), p=0.6 (DCX, C); t(df)=2.703(4), p=0.054 (NeuN, C). Control vs PreVPA, PND21, t(df)=1.512(4), p=0.2 (GFAP, E); t(df)=3.961(4), p=0.02 (DCX, E); t(df)=2.183(4), p=0.09 (NeuN, E). Control vs PostVPA, PND21, t(df)=7.926(4), p=0.001 (GFAP, G); t(df)=3.312(4), p=0.03 (DCX, G); t(df)=0.6569(4), p=0.55 (NeuN, G). The estimation of the BDNF, NT-3, IGF, and GDNF levels in the control, prenatally and postnatally VPA-exposed rats was done by ELISA analysis on PND 14 (B, D) and PND 21 (F, H). Unpaired \u003cem\u003et-test\u003c/em\u003e, Control vs PreVPA, PND14, t(df)=6.345(8), p=0.0002 (BDNF, B); t(df)=0.3692(10), p=0.72 (NT-3, B); t(df)= 2.255(10), p=0.05 (IGF, B); t(df)=1.938(8), p=0.09 (GDNF, B); Control vs PostVPA, PND14, Mann Whitney test U=0, p=0.008 (BDNF, D); t(df)=1.522(10), p=0.16 (NT-3, D); t(df)=4.214(10), p=0.002 (IGF, D), t(df)=0.03552(8), p=0.97 (GDNF, D). Control vs PreVPA, PND21, Mann Whitney test U=1, p=0.02 (BDNF, F); t(df)=13.97(8), p=0.0001 (NT-3, F); t(df)=2.044(8), p=0.0752 (IGF, F), Mann Whitney test U=0, p=0.008 (GDNF, F); Control vs PostVPA, PND21, t(df)=2.501(10), p=0.0314 (BDNF, H); t(df)=1.207(10), p=0.26 (NT-3, H); t(df)=3.695(10), p=0.0041 (IGF, H), t(df)=0.2424(10), p=0.81 (GDNF, H). Data represent the mean values ± SD from n = (5-6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/391abe9a093e64bb28ef7852.png"},{"id":80345979,"identity":"bd3813fa-8b93-40d6-8fbc-1edb70fca3c0","added_by":"auto","created_at":"2025-04-10 19:55:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2201330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of biochemical changes in the subventricular zone of VPA-treated rats on PND 14 and PND 21. \u003c/strong\u003eThe estimation of the BDNF, NT-3, IGF, and GDNF levels in the control, prenatally and postnatally VPA-exposed rats was done by ELISA analysis on PND 14 (A, B) and PND 21 (C, D). Control vs PreVPA, PND14, Mann Whitney test U=0, p=0.008 (BDNF, A); Unpaired \u003cem\u003et-test\u003c/em\u003e, t(df)=2.723(8), p=0.026 (NT-3, A); Mann Whitney test U=0, p=0.004 (IGF, A); Control vs PostVPA, PND14, t(df)=3.882(8), p=0.005 (BDNF, B); t(df)=4.373(6), p=0.005 (NT-3, B); Mann Whitney test U=0, p=0.004 (IGF, B). Control vs PreVPA, PND21, t(df)=2.258(8), p=0.54 (BDNF, C); t(df)=0.8564(8), p=0.42 (NT-3, C); t(df)=11.31(8), p=0.0001 (IGF, C); Control vs PostVPA, PND21, t(df)=1.513(8), p=0.17 (BDNF, D); t(df)=0.02193(9), p=0.98 (NT-3, D); Mann Whitney test U=2, p=0.02 (IGF, D). Data represent the mean values ± SD from n = (4-6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 vs. control.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/7aeb4efc825b0ae1fab288df.png"},{"id":80345985,"identity":"2d07e6b6-b0df-4e5b-b7e5-bdf435c3410e","added_by":"auto","created_at":"2025-04-10 19:55:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5449619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of morphological changes in the different brain regions of VPA-treated rats at P14. \u003c/strong\u003eMorphological changes in the prefrontal cortex, olfactory bulb, hippocampus and subventricular zone of prenatally (I, II, III, IV) and postnatally (V, VI, VII, VIII) VPA-treated rats compared to control were assessed by detection of Ca\u003csup\u003e2+\u003c/sup\u003e dependent acidic phosphatase activity. The arrows show damaged cells (PFC, olfactory bulb, hippocampus) and proliferated ependymal cells (SVZ). Magnification x400 (A, C), x1000 (B, D). The number of damaged and normal neurons per field of view in the prefrontal cortex, olfactory bulb, and hippocampal regions (IX, X, XI), as well as the number of cells in the subventricular zone (XII), are presented. Data represent the mean values ± SD, ***p \u0026lt; 0.001 number of damaged neurons in VPA-treated groups vs. control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/82470588623be9c9fb666ab8.png"},{"id":80345986,"identity":"e2b23f10-4928-432a-8f5a-4f88c20075b1","added_by":"auto","created_at":"2025-04-10 19:55:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5509997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of morphological changes in the different brain regions of VPA-treated rats at P21. \u003c/strong\u003eMorphological changes in the prefrontal cortex, olfactory bulb, hippocampus and subventricular zone of prenatally (I, II, III, IV) and postnatally (V, VI, VII, VIII) VPA-treated rats compared to control were assessed by detection of Ca\u003csup\u003e2+\u003c/sup\u003e dependent acidic phosphatase activity. The arrows show damaged cells (PFC, olfactory bulb, hippocampus) and proliferated ependymal cells (SVZ). Magnification x400 (A, C), x1000 (B, D). The number of damaged and normal neurons per field of view in the prefrontal cortex, olfactory bulb, and hippocampal regions (IX, X, XI), as well as the number of cells in the subventricular zone (XII), are presented. Data represent the mean values ± SD, ***p \u0026lt; 0.001 number of damaged neurons in VPA-treated groups vs. control.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/08eca43d54a855f5f7a9e341.png"},{"id":95564063,"identity":"83471cbf-d542-4d77-954e-0689caf225c0","added_by":"auto","created_at":"2025-11-10 16:07:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23838734,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/36f50adc-15f7-4c90-a59c-bd07447693bc.pdf"},{"id":80346206,"identity":"9002b29a-05e4-41f5-bdac-c45d0d9ed48a","added_by":"auto","created_at":"2025-04-10 20:03:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1283030,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/4d0391ca970a54e0c7dd5884.pdf"},{"id":80346616,"identity":"a610bc44-b40b-4feb-aea0-399528220174","added_by":"auto","created_at":"2025-04-10 20:11:47","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":460822,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2WBs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/c3a6cc741e2bf0a026e9f096.pdf"},{"id":80345676,"identity":"0989e2ac-5c9f-4366-8ee3-a91cffbb5644","added_by":"auto","created_at":"2025-04-10 19:47:48","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5992293,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical abstract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamic and comparative assessment of neurotrophins, neuroglial and synaptic markers, morphological changes both in prenatal and postnatal models of ASD. \u003c/strong\u003eThe graphical abstract illustrates the main significant changes observed in prenatal (left section) and postnatal (write section) models in comparison to the control (central section) group. In the middle (PND 14) and top rows (PND 21), the molecular and morphological changes schematically are illustrated in the relevant structures of the hemisphere (olfactory bulb, prefrontal cortex, subventricular zone, and hippocampus, respectively from the left to the write). Morphological changes of neuronal cells are expressed by different colors: red neurons with abnormal morphology, and morphologically healthy transparent pink neurons. Slow migration of neuroblasts on the rostral way in prenatal (left section) and postnatal models (right section) is expressed by a red background in comparison with the green color in the control group (central section). The changes in protein levels are expressed by different numbers of cells. Decreased doublecortin level in the olfactory bulb on PND 21 is expressed by illustration of one cell instead of three as it is shown on PND 14. Changes in GFAP and NeuN, SYP markers indicating disbalance in the neuroglial ratio in the prefrontal cortex and hippocampus are expressed by comparatively different numbers of the cells (green astrocytes, red neurons). Inside the magnified synaptic clefts vesicles with the different numbers of SYP on the surface are illustrated. The level of neurotrophins in SVZ is expressed as three-color rectangular, which is compressed in prenatal (left middle section) and postnatal (right middle section) on PND 14 in comparison to the control group (central middle row).\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-5920890/v1/6144db01f9c45b7d740e0ff9.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stage-Dependent Disruptions in Neurogenesis and Neurotrophins’ Production Following Prenatal and Postnatal Valproic Acid Exposure: Implications for Autism Spectrum Disorders","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutism spectrum disorders (ASD) are neurodevelopmental disorders that are characterized by altered verbal and non-verbal communication, and repetitive and stereotyped behavior (Home APA DSM-5; Lai et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Heterogeneity of disorder is one of the main challenges that complicates the identification of the pathological mechanisms and treatment approaches. Among the most prominent modern theories of ASD etiology and pathogenesis are the impairment of neural connectivity and neuronal migration, imbalance in excitatory-inhibitory neural activity, damaged synaptogenesis (Rubenstein and Merzenich \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Belmonte et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Nelson and Valakh \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yenkoyan et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, multiple studies provided strong evidence that chaos in the autistic brain is caused by dysregulated stages of neurodevelopment starting from the gestational period up to early postnatal development (Watts \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Amaral et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Levy et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Neural tube formation occurs in the first trimester of pregnancy, synaptic connections, cell communication in the second trimester, and arborization and myelinization in the third trimester(Tau and Peterson \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMany clinical studies demonstrate that the anticonvulsant and mood-stabilizing therapy using valproic acid (VPA) during pregnancy results in higher incidences of ASD. However, the relative sensitivity of different trimesters to ASD development in children exposed to VPA in utero is still not clear. Studies on the risk of ASD development depending on the trimester of exposure produced rather controversial data (Christensen et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Coste et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, exploring the significance of exposure timing on ASD-related changes is considered to be of a high priority.\u003c/p\u003e \u003cp\u003eAssuming that ASD may affect different stages of neurogenesis, such as the proliferation of stem and progenitor cells, migration, differentiation, synaptic network formation, pruning and elimination of synapsis we hypothesized that the extent of valproic acid-induced damage may similarly vary throughout the prenatal and early postnatal neurodevelopment. Therefore, we carried out a comparison of two VPA-induced models of ASD: prenatal and postnatal. As the VPA autism model was developed in rats we had to consider the differences in brain development between humans and rats. Postnatal days (PND) 1\u0026ndash;14 in rats are predicted to correspond to the third trimester of human gestation in terms of brain development stages (Clancy et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Thus, in rats, brain cell death, synaptic pruning, and myelination activity peak after birth during the postnatal 2\u0026ndash;3 weeks (Zeiss \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)(Bandeira et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our previous study, we evaluated and compared VPA-induced prenatal and postnatal ASD models in terms of long-term behavioral, electrophysiological, and morphological changes (Fereshetyan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We validated both models on postnatal development day 30 with rats manifesting impaired social activity, playing behavior, and patterns of repetitive behavior. Detected changes were only partially saved in postnatally treated animals on postnatal development day 60. Reversibility of behavioral alterations showed the possible recovery of brain damage inflicted in the postnatal period. A similar pattern of recovery was observed in the morphological changes of cells over the course of two months of development. On postnatal development day 70 electrophysiological studies showed impaired connectivity between the prefrontal cortex and hippocampus, and cerebellum, and amygdala. These target brain regions were chosen due to their involvement in the regulation of ASD-related behavior. Based on these data in the current study we aimed to assess the molecular alterations occurring in the neurogenic brain regions at early stages following pre- and postnatal VPA administration.\u003c/p\u003e \u003cp\u003eMany studies showed the association of neurotrophins with the development of the different regions of the brain. For instance, postnatal day 14 is the peak period for the expression of neurotrophins in the hippocampus and prefrontal cortex (Das et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Bandeira et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). This peak is typically replaced by a gradual decline in the following week, eventually leveling off after the 21st day of postnatal development. Moreover, recent clinical studies indicate changes in the level of neurotrophins in autistic children's blood or cerebrospinal fluid, which makes their involvement in autism development more evident(Nelson et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Nickl-Jockschat and Michel \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Galvez-Contreras et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, whether VPA affects the neurotrophins during postnatal development remains unclear. Therefore, we compared the effects of prenatal and postnatal administration of VPA on the levels of neurotrophins in the main neurogenic regions of the brain at critical periods (PND 14 and 21).\u003c/p\u003e \u003cp\u003eA number of studies showed increased growth of autistic children\u0026rsquo;s brains associated with an increased number of neurons and general disorganization of brain cytoarchitecture (Kemper and Bauman \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Casanova et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Courchesne et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). About 20% of children with diagnosed autism had macrocephaly (McCaffery and Deutsch \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sacco et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). One of the hypotheses explaining the cause of macrocephaly is an imbalance between the processes of neuronal proliferation and elimination during neurogenesis (Courchesne et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Synaptic dysfunction can be considered as another pathological pattern underlying ASD. Many studies indicate abnormalities in synaptic elimination, synaptic transmission, and plasticity caused by ASD-associated gene mutations or environmental factors leading to synaptic dysfunction (Guang et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The progenitor cell migration is another pathological mechanism contributing to ASD. Therefore, we chose several protein targets in neurogenic regions of brain to assess the possible impairment of the above-mentioned processes. For instance, doublecortin modulates and stabilizes microtubules, aiding cell migration in neurogenesis (Francis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Merz and Lie \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). NeuN is a stable marker for post-mitotic neurons (Duan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Synaptophysin, a key synaptic protein, is consistently distributed in neural synapses (Kwon and Chapman \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Glial fibrillary acidic protein (GFAP) is an astrocytic structural marker protein and is present in the majority of CNS astrocytes (Jurga et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, we chose to detect the regional expression of DCX, GFAP, NeuN, and SYP to assess cell migration, differentiation, and synaptic networks during critical developmental periods.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eExperiments were performed on adult 4\u0026ndash;6 months old female Sprague\u0026ndash;Dawley rats, as well as pups and young rats during the early postnatal life. Animals were purchased from the YSMU vivarium and kept under conditions of 12-h light/dark cycle, with controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and free access to food and water. All animals were randomly divided into groups of 3\u0026ndash;4 animals per cage. All cages were labeled, so the animals' group identity was non-blinded. The experimental protocol adhered to the European Communities Council Directive (86/609/EEC) and was conducted in compliance with the ARRIVE guidelines for animal research (Percie du Sert et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eThe whole study was performed in parallel on prenatal and postnatal VPA-treated groups with respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePrenatal model\u003c/h3\u003e\n\u003cp\u003eFemale rats were paired with males overnight. The next morning vaginal smears were collected, sperm positive cases were registered as day 0 of gestation. On the 12th day of gestation VPA (Sigma-Aldrich, P4543) was injected intraperitoneally in a dose of 500 mg/kg (150 mg/ml), and the control group received the same volume of saline.\u003c/p\u003e\n\u003ch3\u003ePostnatal model\u003c/h3\u003e\n\u003cp\u003ePups born to untreated rats received VPA intraperitoneally in a dose of 200 mg/kg on PND 5\u0026ndash;12, and control groups received saline injection (Chomiak et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Reynolds et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A dosage of 400 mg/kg was also evaluated but subsequently deemed unsuitable due to the observation of hyperactive and agitated behavior in the pups, as well as excessive body weight gain following the injections.\u003c/p\u003e\n\u003ch3\u003eSample preparation\u003c/h3\u003e\n\u003cp\u003eOn PND 14 and 21 pups were euthanized with inhaled isoflurane followed by an overdose of nembutal (Sigma-Aldrich, Y0002194) i.p. (100 mg/kg) injection and were transcardially perfused with isotonic saline. Prefrontal cortex, hippocampus, olfactory bulbs, and subventricular zone were separated from the isolated brains. For the detection of specific neurotrophins and protein markers by ELISA and Western blot tissues were mechanically lysed (FastPrep-24, Lysing Matrix D, SKU:1169130-CF) in ice-cold lysis buffer containing 50 mM Tris/HCl pH 7.5 (Sigma-Aldrich, #1185-53-1), 150 mM NaCl (Sigma-Aldrich, #7647-14-5), 1% triton X-100 (Sigma-Aldrich, #T8787), protease inhibitors (Roche, #04693132001), and then incubated for 20 min at +\u0026thinsp;4\u0026deg;C. After centrifugation at 14000 rpm for 20 min, supernatants were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Total protein concentration was detected by Bradford assay.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eFifty micrograms of supernatants were resolved through 12% acrylamide SDS-PAGE gel and then transferred onto PVDF membranes (Sigma-Aldrich, GE10600023) with a trans-blot semi-dry transfer cell (Bio-Rad). Membranes were incubated for 1 h at room temperature in a blocking solution containing 5% non-fat dry milk (Cell signaling, #9999S) in TBS-T (tris-buffered saline, 0.1% tween 20). Membranes were incubated overnight at +\u0026thinsp;4\u0026deg;C with the primary antibodies against GFAP (Dako, Z0334, 1:1000), DCX (sc-271390, 1;500), NeuN (MAB377, 1:500), Synaptophysin (SAB4200544, 1:1000). After incubation membranes were rinsed in 0.05% TBS-T and incubated with secondary HRP-conjugated antibody for 1.5 h at room temperature (Goat Anti-Rabbit IgG H\u0026amp;L, ab6721, 1:1000, Goat Anti-Mouse IgG H\u0026amp;L, ab97023, 1:1000). Immunoblots were detected by ECL kit (Thermo Scientific, 32106) and visualized using FusionFX (Vilber). Values were normalized to those of β-actin (Cell signaling, #4967). All proteins were assessed on the same membrane (\u003cem\u003esee WBs membrane images in Supplementary file 2_WBs\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eBDNF (Rat BDNF ELISA Kit PicoKine, EK0308), IGF-1 (Rat IGF-1 ELISA Kit PicoKine, E0377), Neurot-3 (Rat NT-3 ELISA Kit PicoKine, E0474) and GDNF (Rat GDNF ELISA Kit PicoKine, EK0363) were detected by corresponding kit assays as to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003ch3\u003eMorphological study\u003c/h3\u003e\n\u003cp\u003eCell structural assessment was done by detecting the activity of Ca\u003csup\u003e2+\u003c/sup\u003e-dependent acidic phosphatase. The method allows for identification of both large and small-size cells and is based on the modifications of Nissl staining and Golgi silver impregnation (Yenkoyan et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Brains were isolated and fixed on PND14 and 21 in the 5% buffered neutral formalin (containing 0.1 M phosphate buffer pH 7.4, 0.3% CaCl\u003csub\u003e2\u003c/sub\u003e, 15% sucrose) for 24\u0026ndash;48 h at 4 \u003csup\u003e0\u003c/sup\u003eC. The frontal free-flow frozen slices (40\u0026ndash;50 \u0026micro;m thick) of the hippocampus and SVZ were isolated. Slices were washed in distilled water and incubated in the following solution: 0.4% lead acetate, 1 M acetate buffer (pH 5.6), and 2% sodium glycerophosphate, for 2\u0026ndash;3 h at 37 \u003csup\u003e0\u003c/sup\u003eC. Thereafter the slices were washed in distilled water, transferred to a 3% sodium sulfide solution, rewashed in distilled water, and embedded into the Canada balsam (Sigma-Aldrich, #8007-47-4). The sections were analyzed using an Opton light microscope (West Germany) at magnifications of \u0026times;400 and \u0026times;1000. Neurons were counted on every 5th section obtained from the studied areas of the rat brain. The distance between the analyzed sections was 200 \u0026micro;m. The number of neurons in digital micrographs was counted manually in the ImageJ program (U.S. National Institutes of Health, Bethesda, MD, USA) using the Mult-point tool per 100 \u0026micro;m of cell layer length in different areas of the brain: CA3 field of the hippocampus, PFC, olfactory bulb, and subventricular zone. Statistical comparisons of quantitative analyses were performed using Student's t-test. Statistical analyses were conducted using GraphPad Prism, version 7 (GraphPad Software Inc., San Diego, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism Software 8.0.1 (San Diego, CA, USA) was used to perform the statistical analyses and to generate the graphs. A priori sample size estimation was conducted based on expected effect sizes from previous studies and pilot experiments. A significance level of 0.05, power of 0.8 and equal group allocation were assumed. For the Western blotting method, 6 rats were used per group, and for the ELISA method, 4 to 6 rats were used per group. To assess the normality and homogeneity of variance, the Shapiro-Wilk test and F-test were used, respectively. The analysis results were provided in the Supplementary Information (Supplementary file 1). The results of two groups (Control 1 vs PreVPA, Control 2 vs PostVPA) were statistically analyzed using unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Mann Whitney U test was used for analysis of the non-parametric data with two groups. P values less than 0.05 were considered statistically significant. Data represent the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eChanges in the level of neurotrophins, neuroglial, and synaptic markers in brain target zones of VPA-treated groups in the early postnatal period\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the possible shifts in early neurogenesis, the main modulators of this process \u0026ndash; neurotrophins, were analyzed in the neurogenic niches of the brain. It is known that in the SVZ neuroblasts migrate long distances to integrate into the olfactory bulb (OB), however, in the dentate gyrus, they integrate into the local hippocampal network (Alvarez-Buylla and Garc\u0026iacute;a-Verdugo \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ming and Song \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lim and Alvarez-Buylla \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, on PND 14 and 21 the olfactory bulb, prefrontal cortex, SVZ, and hippocampus were isolated and tested to detect the levels of BDNF, NT-3, IGF-1, and GDNF. In addition, we have tested also specific markers of differentiation and maturation stage of the cells during the neurodevelopment (DCX, NeuN, GFAP, Syp).\u003c/p\u003e \u003cp\u003eIn the prefrontal cortex, the main changes were detected in the levels of GFAP, NeuN, and synaptophysin which were found dysregulated in opposite directions. Particularly, the level of GFAP was increased in prenatally and postnatally VPA-treated groups on both PNDs, whereas the level of synaptophysin was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C, E, G). A significant decrease of NeuN was detected only in prenatally treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, E). In the hippocampus significant increase of GFAP was detected in the postnatally VPA-treated group on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Significantly decreased GFAP and doublecortin, and increased synaptophysin were detected in VPA-treated both groups on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, G). A significantly increased level of NeuN was detected in the postnatally VPA-treated group on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) and the prenatally VPA-treated group on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Similar changes were also registered in the olfactory bulb. Specifically, the GFAP was found increased in the both VPA-treated groups on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C), and in postnatally VPA-treated group on PND21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Significantly decreased expression of doublecortin was found in both VPA-treated groups on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, changes in BDNF expression in the prenatally and postnatally VPA-treated groups were similar on both PNDs. Particularly, a significant increase in BDNF level was observed in the olfactory bulb of VPA-treated rats in comparison to control on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D) and 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, H). In SVZ the level of BDNF was significantly decreased on PND 14 in both the VPA-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), whereas on PND 21 BDNF was significantly increased in the prenatally VPA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Postnatally VPA-treated group did not show a significant difference in comparison to the control group on PND 21, however, the tendency of changes was the same. Significantly increased level of BDNF was detected in the hippocampus in both of the VPA-treated groups on PND14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and PND21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, H). In the postnatally VPA-treated group on PND 14 no significant changes were detected in BDNF level as compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In PFC no significant changes were detected in all of the tested groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D, F, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA consistent pattern of changes in Nt-3 levels was identified in both the prenatally and postnatally VPA-treated groups on PND 14. A mild decrease was detected in the PFC of the prenatally VPA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), whereas a more significant one was shown in the postnatally VPA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In SVZ the level of Nt-3 was significantly decreased in both of the groups, though in the postnatally VPA-treated group, it was more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). In the hippocampus, a significantly increased level of Nt-3 was identified in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D). There were no changes in the olfactory bulb (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D). However, this pattern was completely different in the prenatally VPA-treated group on PND 21. In PFC the level of Nt-3 was elevated in the prenatally VPA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), whereas in the hippocampus it was decreased compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The decreased level of Nt-3 was detected in the olfactory bulb of the prenatally VPA-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In the postnatally VPA-treated group significantly decreased level of Nt-3 was identified only in the hippocampus on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eThe alterations in IGF-1 levels across all examined structures were consistent between the two VPA-treated groups. A general reduction in IGF-1 was observed on PND 14 and 21 in both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, F, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, D, F, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D, F,), except for the postnatally VPA-treated group on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), where IGF-1 levels in all target structures were notably increased compared to the control group.\u003c/p\u003e \u003cp\u003eThe GDNF level was increased in the hippocampus of postnatally VPA-treated rats on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and decreased in prenatally VPA-treated group on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The significant decrease of GDNF levels was identified in the olfactory bulb on PND 21 of prenatally VPA-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The expression of GDNF in the prefrontal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D, F, H) and subventricular zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, D, F, H) were below the sensitivity range of the assay kit.\u003c/p\u003e \u003cp\u003eA comparison of the two models suggests that the pattern of dysregulation of neurotrophins (BDNF, Nt-3, IGF) was similar primarily on PND 14, with pronounced differences emerging by PND 21. Additionally, time-dependent (PND14 vs PND21) intragroup changes in BDNF and Nt-3 levels were observed in the prenatally treated group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMorphological changes in the prefrontal cortex, hippocampus, olfactory bulb, and subventricular zone of VPA-treated groups in the early postnatal period\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMorphological changes in target structures were assessed on PND 14 and PND 21.\u003c/p\u003e \u003cp\u003eSwollen pyramidal cells with central chromatolysis and non-clear contours were detected in the prefrontal cortex of prenatally VPA-treated rats on PND 14 in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Damaged cells showed morphology characterized by short and more colorless stained dendrites. Similar changes with less pronounced intensity were observed on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDamaged cellular structures were observed in the dentate gyrus of the hippocampus in prenatally VPA-treated rats on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e III) as well as neurons of abnormal shape and size. Granular cells were less stained but their morphological characteristics were close to the controls. The cellular barrier was visualized, and centrally localized nuclei were detected. The pyramidal cells of CA3 and CA4 were hypertrophic with no clear cellular barrier with a mostly elongated shape. All the described morphological changes were normalized on PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e III). Similar changes were observed in the CA2 and CA1 regions of the hippocampus on PND 14. Interestingly, there were significant numbers of degenerated cells with chromatolysis in CA2 and CA1 on PND 21.\u003c/p\u003e \u003cp\u003eIn the olfactory bulb of prenatally VPA-treated rats round or oval-shaped cells with central chromatolysis were seen on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e II) and PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e II). Neurons in certain areas preserved their shape and size.\u003c/p\u003e \u003cp\u003eIn the subventricular zone of prenatally VPA-treated rats multilayer proliferation of round-shaped ependymal cells was observed on both PNDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e IV). Meanwhile, in the control group, the walls of the third ventricle were in close contact, and nuclei were mostly localized in the base of the cylinder-shaped cells.\u003c/p\u003e \u003cp\u003eIn the hippocampus of postnatally VPA-treated group cells remained intact on PND 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e VII) and PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e VII). Cells had clearly defined cellular barriers, basically localized nuclei. In the subventricular zone of postnatally VPA-treated rats intensive proliferation of ependymal cells was registered on PND 14 and PND 21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eVIII). Desquamation of ependymal cells was detected in some places, but the close contact between cells remained mostly intact. In the prefrontal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eV) and olfactory bulbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eVI) of postnatally VPA-treated rats cells mostly preserve the normal structure and shape. Only several cells with swollen nuclei were detected.\u003c/p\u003e \u003cp\u003eThe morphological comparisons showed more severe cellular damage in the prenatal model, affecting all tested structures, while the postnatal model exhibited changes only in the SVZ. On PND 21, damage persisted in the olfactory bulb and SVZ but was normalized in the PFC and hippocampus in prenatally VPA-treated animals.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study aimed to evaluate and compare the effects of prenatal and postnatal valproic acid (VPA) exposure on neurotrophin levels, cellular differentiation, and brain organization in primary neurogenic regions during critical early postnatal periods. Building on our previous findings on behavioral and electrophysiological alterations in VPA-induced ASD models (Fereshetyan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), we investigated the early molecular mechanisms underlying these changes. Given the established role of neurotrophins in neurodevelopmental processes such as cell migration, differentiation, and synaptic network formation, we sought to clarify the molecular and cellular alterations contributing to ASD-like pathophysiology. Neurogenesis markers (DCX, NeuN, SYP, GFAP) were chosen to assess VPA-induced brain alterations, contextualized within ASD pathogenesis.\u003c/p\u003e \u003cp\u003eKey findings highlight distinct impacts of prenatal and postnatal VPA exposure on cell migration and neurogenesis. Reduced BDNF levels in the SVZ on PND 14 and decreased DCX in the olfactory bulb by PND 21 suggest impaired migration of progenitor cells, consistent with literature emphasizing BDNF's role in guiding premature cell migration (Henry et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The comprehensive reduction of neurotrophins in the SVZ underscores VPA\u0026rsquo;s suppressive effect. Dysregulated migration of the cells during the neurogenesis leads to abnormal distribution and disorganized cortical lamination, which is one of the common features of the autistic brain (Wegiel et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Brain heterotopias are highly associated with doublecortin defects, which is involved in the formation of microtubules required for cell migration. Moreover, decreased DCX levels in the hippocampus of prenatally VPA-treated rats (PND 21) align with previous studies on ASD models (Aranarochana et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ependymal barrier disruptions observed on PND 14 in both VPA groups, including increased cell numbers and desquamations, may attenuate NSC migration by disrupting CSF-mediated signaling (Kaneko and Sawamoto \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother major finding is the altered neuro-glial ratio in the PFC, characterized by elevated GFAP and reduced synaptophysin on PND 14 and PND 21, indicative of reactive gliosis or impaired neuronal differentiation. This is supported by various studies indicating the involvement of astrocyte-mediated neuroinflammation in the pathogenesis of autism (Gzielo and Nikiforuk \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar patterns have been observed in ASD models and postmortem studies (Edmonson et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The study on non-human primates showed a decreased level of doublecortin and NeuN in PFC and increasing in GFAP level after VPA treatment in utero (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Currently, limited literature is available addressing the alterations in neuroglial markers following postnatal VPA injections. DiCicco-Bloom et al showed increased GFAP levels in the frontal cortex after PND 2\u0026ndash;4 VPA exposure on PND 21 (Mony et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Interestingly, the pattern of hippocampal neuroglial dynamics did not match our data, with increased NeuN and synaptophysin levels reflecting enhanced neuronal differentiation, potentially driven by elevated BDNF on PND 21. Several studies indicate that the reduction of BDNF expression in the hippocampus does affect the number of neurons, but suppresses the genes encoding proteins of vesicular trafficking and synaptic communication (Gao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rauskolb et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, reduced NT-3 suggests a dominant role for BDNF in hippocampal neurogenesis during this period.\u003c/p\u003e \u003cp\u003eThe other piece of data worth discussing is the changes in neurotrophins\u0026rsquo; expression during the development. Although under normal conditions BDNF expression gradually increases to the maximum level during postnatal development (Karege et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), the VPA-induced rapid increase of BDNF in the brain appears certainly abnormal. Our results indicate such a transition in both of the models. However, it should be noted that in the postnatally VPA-treated group, the BDNF changes at PND 14 and 21 were particularly dramatic. VPA-induced BDNF elevation may cause alterations in synapto- and gliogenesis, neurite outgrowth, dendritic arborization, and spine formation (Vilar and Mira \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Kowiański et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The data on IGF-β expression (increase during the first two weeks of postnatal life in control groups) is consistent with previous studies(Bondy et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). IGF levels peaked at PND 14 and gradually declined by PND 21, reflecting the typical developmental pattern. In contrast, the postnatally VPA-treated group showed continued IGF increase until PND 21, exceeding control levels at this stage. Interestingly, the IGF levels at PND 21 matched the control group\u0026rsquo;s PND 14 peak, suggesting that postnatal VPA exposure shifts the IGF production peak by one week. Further studies on subsequent postnatal days are needed to determine whether IGF levels decline thereafter.\u003c/p\u003e \u003cp\u003eIn the prenatally VPA-treated group, IGF levels were consistently lower across all structures at both time points, mirroring the pathophysiology of ASD. However, the developmental trend paralleled the control group. These findings indicate distinct differences in neurotrophin dynamics within neurogenic brain regions between the two models.\u003c/p\u003e \u003cp\u003eIn conclusion, VPA exposure induces significant neurogenesis disruptions that may underlie autism-like pathogenesis in rodents. Both prenatal and postnatal VPA exposure result in alterations in neurogenesis, such as dysregulated neurotrophin levels and impaired cell migration, reflecting some shared mechanisms of disruption. However, prenatal exposure exerts stronger and more persistent effects compared to postnatal exposure, as evidenced by our prior findings on the reversibility of ASD-associated behaviors in postnatally VPA-treated rats (Fereshetyan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings underscore the critical vulnerability of brain development to VPA, particularly during the third trimester of human pregnancy, reinforcing the need for vigilant risk assessment and preventive strategies.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLimitation\u003c/h2\u003e \u003cp\u003eA notable limitation of this study is the absence of sex-specific analysis in the investigation of neurotrophins and neurogenic markers in the valproic acid induced rat model of autism. Autism spectrum disorder is known to be more prevalent in males, with an estimated male-to-female ratio of 4:1 (Baio et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, this sex-based disparity has not been conclusively established in VPA-exposed children with autism (Honybun et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While numerous studies have reported sex-associated differences in behavioral patterns in VPA-exposed animal models (Chaliha et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), data regarding sex-specific molecular alterations remain limited. Given these findings, the present study focused on identifying overall differences between the treated and control groups without stratifying by sex, which can be considered a reasonable approach in this exploratory phase of research. Nonetheless, future studies should incorporate sex as a biological variable to better elucidate potential sex-specific differences, particularly in the context of developmental trajectories and molecular responses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Dr Souren Mkrtchian (Karolinska Institute, Stockholm) for the fruitful discussions and critical reading of the manuscript, and Hasmik Harutyunyan (COBRAIN Center, Yerevan State Medical University) for help with graphical illustrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKatarine Fereshetyan: conceptualization, methodology, investigation, data curation, writing original draft. Margarita Danielyan: methodology, investigation. Konstantin Yenkoyan: conceptualization, methodology, project administration, funding acquisition, resources, supervision, writing - review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Higher Education and Science Committee, Ministry of Education, Science, Culture and Sports of RA (23LCG-3A020\u0026nbsp;and 25YSMU-CON-I-3A), and YSMU.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be made available by the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol adhered to the conditions set forth by the European Communities Council Directive (86/609/EEC) and was approved by the Ethics Committee of Yerevan State Medical University after Mkhitar Heratsi (Approval Nos: N1, 20.09.2018; N8-3/24, 14.03.2024; N3, 14.11.2024). The study was conducted in compliance with the ARRIVE guidelines for animal research (Percie du Sert et al., 2020).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlvarez-Buylla A, Garc\u0026iacute;a-Verdugo JM (2002) Neurogenesis in adult subventricular zone. J Neurosci 22:629\u0026ndash;634\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmaral DG, Schumann CM, Nordahl CW (2008) Neuroanatomy of autism. Trends Neurosci 31:137\u0026ndash;145\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAranarochana A, Sirichoat A, Pannangrong W et al (2021) Melatonin Ameliorates Valproic Acid-Induced Neurogenesis Impairment: The Role of Oxidative Stress in Adult Rats. 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Accessed 14 Nov 2015\u003c/span\u003e\u003c/li\u003e\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":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"autism spectrum disorders, valproic acid, pre-and postnatal models, neurotrophins","lastPublishedDoi":"10.21203/rs.3.rs-5920890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5920890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutism spectrum disorders (ASD) are neurodevelopmental conditions involving impaired neuronal processes such as connectivity, synaptogenesis, and migration. Prenatal exposure to valproic acid (VPA), an anticonvulsant and mood stabilizer, is linked to increased ASD risk, with timing as a key factor. However, the molecular mechanisms of VPA-induced neurodevelopmental disruptions remain unclear.\u003c/p\u003e \u003cp\u003eBuilding on our previous study, which characterized VPA-induced prenatal and postnatal ASD models with impaired social behavior, repetitive patterns, and altered brain connectivity, this study examines molecular changes in neurogenic brain regions. We analyzed the prefrontal cortex, hippocampus, and subventricular zone at key developmental time points (postnatal days 14 and 21), assessing neurotrophins (BDNF, Nt-3, IGF-β, GDNF) and markers of cell migration (DCX), differentiation (NeuN, GFAP), and synaptogenesis (synaptophysin).\u003c/p\u003e \u003cp\u003eOur findings show that both prenatal and postnatal VPA exposure disrupt neurogenesis, with prenatal effects being more severe and persistent. Prenatal VPA significantly reduced BDNF in the SVZ and DCX in the olfactory bulb, indicating impaired migration, while morphological analysis revealed increased ependymal proliferation and disrupted SVZ organization. Postnatal exposure led to transient neurotrophin changes, including delayed IGF-β production and an abnormal rise of BDNF levels. Elevated GFAP and reduced synaptophysin in the PFC, alongside increased neuronal markers in the hippocampus, suggest region-specific neuro-glial imbalances.\u003c/p\u003e \u003cp\u003eThese findings highlight the stage-dependent vulnerability of the developing brain to VPA exposure, revealing distinct mechanisms of disruption in prenatal and postnatal administration. They underscore the need to minimize exposure risks during late gestation and early postnatal periods, which are crucial for neurodevelopment.\u003c/p\u003e","manuscriptTitle":"Stage-Dependent Disruptions in Neurogenesis and Neurotrophins’ Production Following Prenatal and Postnatal Valproic Acid Exposure: Implications for Autism Spectrum Disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 19:47:42","doi":"10.21203/rs.3.rs-5920890/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-22T15:21:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T14:56:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-03T17:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177458589837528264187856375635632980336","date":"2025-04-10T10:11:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294551470053124957975818722603076559518","date":"2025-04-05T16:04:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-05T15:48:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T08:58:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Neurobiology","date":"2025-03-29T20:27:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cemn","sideBox":"Learn more about [Cellular and Molecular Neurobiology](https://www.springer.com/journal/10571)","snPcode":"10571","submissionUrl":"https://submission.nature.com/new-submission/10571/3","title":"Cellular and Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"78800dd1-e060-44dc-b2c9-806c0515e548","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-10T16:02:43+00:00","versionOfRecord":{"articleIdentity":"rs-5920890","link":"https://doi.org/10.1007/s10571-025-01623-4","journal":{"identity":"cellular-and-molecular-neurobiology","isVorOnly":false,"title":"Cellular and Molecular Neurobiology"},"publishedOn":"2025-11-05 15:57:52","publishedOnDateReadable":"November 5th, 2025"},"versionCreatedAt":"2025-04-10 19:47:42","video":"","vorDoi":"10.1007/s10571-025-01623-4","vorDoiUrl":"https://doi.org/10.1007/s10571-025-01623-4","workflowStages":[]},"version":"v1","identity":"rs-5920890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5920890","identity":"rs-5920890","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}