VPA-exposed Offspring Research Articles

Continuous exercise training rescues hippocampal long-term potentiation in the VPA rat model of Autism: Uncovering sex-specific effects

Long‐term potentiation (LTP) impairment has been reported in many studies of autistic models. The aim of the present study was to investigate the effects of interval training (IT) and continuous training (CT) exercises on LTP in the hippocampal dentate gyrus (DG) neurons of valproic acid (VPA) rat model of autism. To induce an autism-like model, pregnant rats were injected 500 mg/kg NaVPA (intraperitoneal) on the embryonic day 12.5. IT and CT aerobic exercises started on postnatal day 56 in the offspring. Four weeks after IT and/or CT exercises, the offspring were urethane-anesthetized and placed into a stereotaxic apparatus for surgery, electrode implantation, and field potential recording. In the DG region, excitatory post synaptic potentials (EPSP) slope and population spike (PS) amplitude were measured. Sex differences in LTP were evident for control rats but not for VPA-exposed offspring. LTP was significantly smaller in VPA-exposed male offspring compared with control male rats. In contrast to males, there was no difference between VPA-exposed female offspring and control female rats. Interestingly, we observed a sex difference in the response to exercise between VPA-exposed male and female offspring. CT exercise training (but not IT) increased LTP in VPA-exposed male offspring. Both IT and CT exercise trainings had no effect on intact LTP in VPA-exposed female offspring. Our work suggests that there may be differences in the benefits of exercise interventions based on sex, and CT exercise training could be more beneficial for LTP improvements.

Mice prenatally exposed to valproic acid do not show autism-related disorders when fed with polyunsaturated fatty acid-enriched diets

Dietary supplementations with n-3 polyunsaturated fatty acid (PUFA) have been explored in autism spectrum disorder (ASD) but their efficiency and potential in ameliorating cardinal symptoms of the disease remain elusive. Here, we compared a n-3 long-chain (LC) PUFA dietary supplementation (n-3 supp) obtained from fatty fish with a n-3 PUFA precursor diet (n-3 bal) obtained from plant oils in the valproic acid (VPA, 450 mg/kg at E12.5) ASD mouse model starting from embryonic life, throughout lactation and until adulthood. Maternal and offspring behaviors were investigated as well as several VPA-induced ASD biological features: cerebellar Purkinje cell (PC) number, inflammatory markers, gut microbiota, and peripheral and brain PUFA composition. Developmental milestones were delayed in the n-3 supp group compared to the n-3 bal group in both sexes. Whatever the diet, VPA-exposed offspring did not show ASD characteristic alterations in social behavior, stereotypies, PC number, or gut microbiota dysbiosis while global activity, gait, peripheral and brain PUFA levels as well as cerebellar TNF-alpha levels were differentially altered by diet and treatment according to sex. The current study provides evidence of beneficial effects of n-3 PUFA based diets, including one without LCPUFAs, on preventing several behavioral and cellular symptoms related to ASD.

Chronic inhibition of astrocytic aquaporin-4 induces autistic-like behavior in control rat offspring similar to maternal exposure to valproic acid

Social communication and interaction deficits, memory impairment, and anxiety-like behavior are characterized in many people identified with autism spectrum disorder (ASD). A thorough understanding of the specific aspects that contribute to the deficiencies associated with ASD can aid research into the etiology of the disorder while also providing targets for more effective intervention. As part of the ASD pathophysiology, alterations in synaptogenesis and abnormal network connections were seen in high-order brain areas, which control social behavior and communication. The early emergence of microglia during nervous system development may contribute to synaptic dysfunction and the pathobiology of ASD. Since aquaporin-4 (AQP4) appears to be required for the basic procedures of synapse activation, certain behavioral and cognitive impairments as well as disturbance in water homeostasis might likely arise from AQP4 deficiency. Here, through the measurement of the water content of the hippocampus and behavioral experiments we aim to explore the contribution of astrocytic AQP4 to the autism-like behavior induced by prenatal valproic acid (VPA) exposure and whether inhibition of AQP4 per se can induce autistic-like behavior in control rats.Microinjection of TGN-020 (10 µM, i.c.v), a specific AQP4 inhibitor, for 7 successive days before behavioral tasks from postnatal day 28 to 35 revealed that inhibition of AQP4 in the control offspring caused lower social interaction and locomotor activity, higher anxiety, and decreased ability to recognize novel objects, very similar to the behavioral changes observed in offspring prenatally exposed to VPA. However, VPA-exposed offspring treated with TGN-020, showed no further remarkable behavioral impairments than those detected in the autistic-like rats. Furthermore, both control offspring treated with TGN-020 and offspring exposed to VPA had a considerable accumulation of water in their hippocampi. But AQP4 inhibition did not affect the water status of the autistic-like rats.The findings of this study revealed that control offspring exhibited similar hippocampal water retention and behavioral impairments that were observed in maternal VPA-exposed offspring following inhibition of astrocytic AQP4, whereas, in autistic-like rats, it did not produce any significant change in water content and behaviors. Findings suggest that AQP4 deficiency could be associated with autistic disorder and may be a potential pharmaceutical target for treating autism in the future.

Adolescent swimming exercise following maternal valproic acid treatment improves cognition and reduces stress-related symptoms in offspring mice: Role of sex and brain cytokines

Valproic acid (VPA) treatment during pregnancy is a risk factor for developing autism spectrum disorder, cognitive deficits, and stress-related disorders in children. No effective therapeutic strategies are currently approved to treat or manage core symptoms of autism. Active lifestyles and physical activity are closely associated with health and quality of life during childhood and adulthood. This study aimed to evaluate whether swimming exercise during adolescence can prevent the development of cognitive dysfunction and stress-related disorders in prenatally VPA-exposed mice offspring. Pregnant mice received VPA, afterwards, offspring were subjected to swimming exercise. We assessed neurobehavioral performances and inflammatory cytokines (interleukin-(IL)6, tumor-necrosis-factor-(TNF)α, interferon-(IFN)γ, and IL-17A) in the hippocampus and prefrontal cortex of offspring. Prenatal VPA treatment increased anxiety-and anhedonia-like behavior and decreased social behavior in male and female offspring. Prenatal VPA exposure also increased behavioral despair and reduced working and recognition memory in male offspring. Although prenatal VPA increased hippocampal IL-6 and IFN-γ, and prefrontal IFN-γ and IL-17 in males, it only increased hippocampal TNF-α and IFN-γ in female offspring. Adolescent exercise made VPA-treated male and female offspring resistant to anxiety-and anhedonia-like behavior in adulthood, whereas it only made VPA-exposed male offspring resistant to behavioral despair, social and cognitive deficits in adulthood. Exercise reduced hippocampal IL-6, TNF-α, IFN-γ, and IL-17, and prefrontal IFN-γ and IL-17 in VPA-treated male offspring, whereas it reduced hippocampal TNF-α and IFN-γ in VPA-treated female offspring. This study suggests that adolescent exercise may prevent the development of stress-related symptoms, cognitive deficits, and neuroinflammation in prenatally VPA-exposed offspring mice.

Rescue of social deficits by early-life melatonin supplementation through modulation of gut microbiota in a murine model of autism

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a rapidly increasing global prevalence. Early unstable and immature microbiota are often observed in ASD patients, resulting in neurobehavioral dysfunction. Since the establishment of stable gut microbiota in early life falls into the same critical time window as neurodevelopment, manipulations of the gut microbiota during early life could become a promising strategy for ASD. Melatonin is an endogenous hormone and can restore gut microbial dysbiosis under various disease conditions. Here, we explored the effects of melatonin supplementation during early life on the gut microbiota of the offspring and the subsequent impact on ASD-associated behaviors. Using the valproic acid (VPA) - induced mouse model of autism, we found that melatonin supplementation during late gestation and early postnatal development rescued the social deficits of the offspring. In addition, melatonin restored gut microbial dysbiosis in the VPA-exposed offspring, which was characterized by the significant upregulation of Akkermansia spp. Furthermore, supplementation of Akkermansia spp. alleviated the social deficits induced by VPA exposure via activating the dopaminergic neurons in the ventral tegmental area. These findings discover a novel mechanism underlying the gut microbiota regulation of social behaviors and provide the biological basis for developing gut microbiota-based therapeutics for ASD

Neuronal differentiation reporter mice as a new methodology for detecting in vivo developmental neurotoxicity

Current in vivo developmental neurotoxicity (DNT) tests are not performed routinely for chemical risk assessment because they are time and resource intensive and require many animals. Therefore, new methodologies are required that can detect and evaluate the DNT potential of chemicals in a more simple, quantitative, and objective manner. Toward this end, we generated transgenic mice expressing reporter genes (luciferase and lacZ) under the control of the rat synapsin 1 promoter (Syn-Rep mice) and evaluated their usefulness as a DNT detection tool. Brain luciferase expression levels in Syn-Rep mice increased dramatically from just before to after birth, peaked early in the postnatal period, subsequently decreased sharply, and then remained low after weaning. This pattern is analogous to the generally recognized temporal changes in synapse numbers in the developing mammal brain. To evaluate further the responsiveness of Syn-Rep mice during DNT induction, we administered valproic acid (VPA), a reference DNT-inducing chemical, to pregnant mice and evaluated its effect on reporter gene expression in the developing brains of Syn-Rep pups. In vivo luminescence in the brains of VPA-exposed pups was significantly lower than in controls from postnatal days 4 to 13. Moreover, luciferase activity in the prefrontal cortexes of 8-week-old VPA-exposed offspring was significantly lower than in controls, reflecting the reduced number of neurons in the prefrontal cortex. These results suggest that the Syn-Rep mice are potentially useful tools for streamlined detection of chemical-induced DNT in the developing mammalian brain.

Continuous Exposure to Alpha-Glycosyl Isoquercitrin from Gestation Ameliorates Disrupted Hippocampal Neurogenesis in Rats Induced by Gestational Injection of Valproic Acid.

This study examined the ameliorating effect of alpha-glycosyl isoquercitrin (AGIQ), an antioxidant, on disrupted hippocampal neurogenesis in the dentate gyrus (DG) in a rat model of autism spectrum disorder induced by prenatal valproic acid (VPA) exposure. Dams were intraperitoneally injected with 500mg/kg VPA on gestational day 12. AGIQ was administered in the diet at 0.25 or 0.5% to dams from gestational day 13 until weaning at postnatal day (PND) 21 and then to pups until PND 63. At PND 21, VPA-exposed offspring showed decreased numbers of type-2a and type-3 neural progenitor cells (NPCs) among granule cell lineage subpopulations. AGIQ treatment at both doses rescued the reduction in type-3 NPCs. AGIQ upregulated Reln and Vldlr transcript levels in the DG at 0.5% and ≥ 0.25%, respectively, and increased the number of reelin+ interneurons in the DG hilus at 0.5%. AGIQ at 0.25% and/or 0.5% also upregulated Ntrk2, Cntf, Igf1, and Chrnb2. At PND 63, there were no changes in the granule cell lineage subpopulations in response to VPA or AGIQ. AGIQ at 0.25% increased the number of FOS+ granule cells, accompanied by Gria2 and Gria3 upregulationand increasing trend in the number of FOS+ granule cells at 0.5%. There was no definitive evidence of VPA-induced oxidative stress in the hippocampus throughout postnatal life. These results indicate that AGIQ ameliorates the VPA-induced disruption of hippocampal neurogenesis at weaning involving reelin, BDNF-TrkB,CNTF, and IGF1 signaling, and enhances FOS-mediated synaptic plasticity in adulthood, potentially through AMPA-receptor upregulation. The ameliorating effects of AGIQ may involve direct interactions with neural signaling cascades rather than antioxidant capacity.

In utero Exposure to Valproic-Acid Alters Circadian Organisation and Clock-Gene Expression: Implications for Autism Spectrum Disorders.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder characterised by restrictive patterns of behaviour and alterations in social interaction and communication. Up to 80% of children with ASD exhibit sleep-wake cycle disturbances, emphasising the pressing need for novel approaches in the treatment of ASD-associated comorbidities. While sleep disturbances have been identified in ASD individuals, little has been done to assess the contribution of the circadian system to these findings. The objective of this study is to characterise circadian behaviour and clock-gene expression in a valproic acid (VPA)-induced animal model of autism to highlight perturbations potentially contributing to these disturbances. Male and female VPA-exposed offspring underwent circadian challenges, including baseline light-dark cycles, constant dark/light and light pulse protocols. Baseline analysis showed that VPA-exposed males, but not females, had a greater distribution of wheel-running behaviour across light-dark phases and a later activity offset (p < 0.0001), while controls showed greater activity confinement to the dark phase (p = 0.0256). Constant light analysis indicated an attenuated masking response and an increase in the number of days to reach arrhythmicity (p < 0.0001). A 1-h light pulse (150 lux) at CT 15 after 6 days of constant dark showed that both sexes exposed to VPA exhibited a lesser phase-shift when compared to controls (p = 0.0043). Immunohistochemical and western-blot assays reveal no alterations in retinal organisation or function. However, immunohistochemical assay of the SCN revealed altered expression of BMAL1 expression in VPA-exposed males (p = 0.0016), and in females (p = 0.0053). These findings suggest alterations within the core clockwork of the SCN and reduced photic-entrainment capacity, independent of retinal dysfunction. The results of this study shed light on the nature of circadian dysregulation in VPA-exposed animals and highlights the urgent need for novel perspectives in the treatment of ASD-associated comorbidities.

Correlation among gut microbiota, fecal metabolites and autism-like behavior in an adolescent valproic acid-induced rat autism model

This study aims to understand the relationship between fecal metabolites and gut microbiota in an adolescent valproic acid-induced rat autism model (VPA-exposed offspring). We analyzed the fecal samples of VPA-exposed offspring using 16S rRNA gene sequencing and untargeted metabolomics. Autism-like behavior was evaluated by a three-chamber sociability test and a self-grooming test. Based on these data, we analyzed the association among fecal metabolites, gut microbiota and autism-like behavior. Behavioral tests showed that VPA-exposed offspring displayed typical autism-like behavior. Forty-nine named differential fecal metabolites and 14 enriched KEGG pathways were identified between the VPA and control groups. Five fecal metabolites may be used as characteristic metabolites. The richness and diversity of gut microbiota did not differ between the two groups, while the overall composition of gut microbiota was significantly different. Candidatus_Saccharimonas, Desulfovibrio, [Eubacterium]_xylanophilum_group and Ruminococcus_2 were the characteristic genera of VPA-exposed offspring. Correlation analysis revealed a tight relationship among gut microbiota, fecal metabolites and autistic behavior in VPA-exposed offspring. This study illustrates that specific alterations in gut microbiota and fecal metabolites may be regarded as characteristics of VPA-exposed offspring. The characteristic gut microbiota and fecal metabolites as well as their relationship may play a crucial role in autism-like behavior caused by prenatal exposure to VPA.

Abnormal spatiotemporal expression pattern of progranulin and neurodevelopment impairment in VPA-induced ASD rat model

Some environmental risk factors have been proven to contribute to the etiology of autism spectrum disorder (ASD). Exposure to the antiepileptic drug valproic acid (VPA) during pregnancy significantly increases the risk of ASD in humans, and consequently is utilized as a validated animal model of ASD in rodents; however, the precise molecular and cellular mechanisms remain ill-defined. In the present study, we investigated the effect of prenatal VPA exposure on the spatiotemporal dynamics of Progranulin (PGRN) expression, neuronal apoptosis, synapse density, and AKT/GSK-3β pathway activation in the brains of VPA-exposed offspring. Results from behavioral tests were consistent with prior studies showing impaired sociability, restricted interests and increased repetitive behaviors in VPA rats at postnatal days 28–32. Our data also indicated that VPA exposure resulted in abnormal dynamics of PGRN expression in different brain regions at the different development stages. The temporal and spatial patterns of PGRN expression were consistent with the spatiotemporal regularity of abnormalities, which observed in apoptosis-related protein levels, neuron numbers, dendritic spine density, synapse-related protein levels, and AKT/GSK-3β phosphorylation in VPA rats. It suggests that prenatal VPA exposure may affect the spatiotemporal regularity of neuronal apoptosis and synaptic development/regression via interfering with the spatiotemporal process of PGRN expression and downstream AKT/GSK-3β pathway activation. This may be a potential mechanism of the abnormal neuroanatomical changes and ASD-like behaviors in VPA-induced ASD.

Targeting the inhibition of fatty acid amide hydrolase ameliorate the endocannabinoid-mediated synaptic dysfunction in a valproic acid-induced rat model of Autism

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by social interaction impairment, stereotypical/repetitive behaviors and emotional deregulation. The endocannabinoid (eCB) system plays a crucial role in modulating the behavioral traits that are typically core symptoms of ASD. The major molecular mechanisms underlying eCB-dependent long-term depression (eCB-LTD) are mediated by group 1 metabotropic glutamate receptor (mGluR)-induced removal of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Recently, modulation of anandamide (AEA), one of the main endocannabinoids in the brain, has been reported to alter social behaviors in genetic models of ASD. On this basis, we investigated the effects of treatment and the synaptic mechanism underlying AEA-mediated signaling in prenatal exposure to valproic acid (VPA) in rats. We found that the social deficits, repetitive behaviors and abnormal emotion-related behaviors in VPA-exposed offspring were improved after treatment with an inhibitor of AEA degrading enzyme, URB597. Using an integrative approach combing electrophysiological and cellular mechanisms, the results showed that the impaired eCB-LTD, abnormal mGluR-mediated LTD (mGluR-LTD) and decreased removal of AMPAR subunits GluA1 and GluA2 were reversed by URB597 in the prefrontal cortex (PFC) of VPA-exposed offspring. Taken together, these results provide the first evidence that rescue of the ASD-like phenotype by URB597 is mediated by enhancing the mechanism of removal of AMPAR subunits GluA1/2 underlying AEA signaling in the PFC in a VPA-induced model of ASD.

Deep Brain Stimulation Modified Autism-Like Deficits via the Serotonin System in a Valproic Acid-Induced Rat Model.

Deep brain stimulation (DBS) is known to be a promising treatment for resistant depression, which acts via the serotonin (5-hydroxytryptamine, 5-HT) system in the infralimbic prefrontal cortex (ILPFC). Previous study revealed that dysfunction of brain 5-HT homeostasis is related to a valproate (VPA)-induced rat autism spectrum disorder (ASD) model. Whether ILPFC DBS rescues deficits in VPA-induced offspring through the 5-HT system is not known. Using VPA-induced offspring, we therefore explored the effect of DBS in autistic phenotypes and further investigated the underlying mechanism. Using combined behavioral and molecular approaches, we observed that applying DBS and 5-HT1A receptor agonist treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reversed sociability deficits, anxiety and hyperactivity in the VPA-exposed offspring. We then administered the selective 5-HT1A receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate (WAY 100635), following which the effect of DBS in terms of improving autistic behaviors was blocked in the VPA-exposed offspring. Furthermore, we found that both 8-OH-DPAT and DBS treatment rescued autistic behaviors by decreasing the expressions of NR2B subunit of N-methyl-D-aspartate receptors (NMDARs) and the β3 subunit of γ-aminobutyric acid type A receptors (GABAAR) in the PFC region. These results provided the first evidence of characteristic behavioral changes in VPA-induced offspring caused by DBS via the 5-HT system in the ILPFC.

D-Cycloserine Ameliorates Autism-Like Deficits by Removing GluA2-Containing AMPA Receptors in a Valproic Acid-Induced Rat Model.

Valproic acid (VPA)-exposed rat offspring have demonstrated autism spectrum disorder (ASD) phenotypes and impaired N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD) in the lateral nucleus of the amygdala. NMDAR partial agonist D-cycloserine (DCS) has been reported to act as a cognitive enhancer by increasing the NMDAR response to improve autistic-like phenotypes in animals. However, the mechanism of DCS in alleviating the ASD is still unknown. Using combined behavioral, electrophysiological, and molecular approaches, we found that DCS administration rescued social interaction deficits and anxiety/repetitive-like behaviors observed in VPA-exposed offspring. In the amygdala synapses, DCS treatment reversed the decreased paired pulse ratio (PPR) and the impaired NMDAR-dependent LTD, increased the frequency and amplitude of miniature excitatory post-synaptic currents (mEPSCs), and resulted in a higher dendritic spine density at the amygdala synapses in the VPA-exposed offspring. Moreover, we found that DCS facilitated the removal of GluA2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (GluA2/AMPARs) by inducing NMDAR-dependent LTD in the VPA-exposed offspring. We further established that the effects of DCS treatment, including increased GluA2/AMPAR removal and rescues of impaired social behavior, were blocked by Tat-GluA23Y, a GluA2-derived peptide that disrupted regulation of AMPAR endocytosis. These results provided the first evidence that rescue of the ASD-like phenotype by DCS is mediated by the mechanism of GluA2/AMPAR removal in VPA-exposed rat offspring.

The transgenerational inheritance of autism-like phenotypes in mice exposed to valproic acid during pregnancy.

Autism spectrum disorder (ASD) is a heterogeneously pervasive developmental disorder in which various genetic and environmental factors are believed to underlie its development. Recently, epigenetics has been suggested as a novel concept for ASD aetiology with a proposition that epigenetic marks can be transgenerationally inherited. Based on this assumption of epigenetics, we investigated the transgenerational inheritance of ASD-like behaviours and their related synaptic changes in the VPA animal model of ASD. The first generation (F1) VPA-exposed offspring exhibited autistic-like impaired sociability and increased marble burying. They also showed increased seizure susceptibility, hyperactivity and decreased anxiety. We mated the VPA-exposed F1 male offspring with naïve females to produce the second generation (F2), and then similarly mated the F2 to deliver the third generation (F3). Remarkably, the autism-like behavioural phenotypes found in F1 persisted to the F2 and F3. Additionally, the frontal cortices of F1 and F3 showed some imbalanced expressions of excitatory/inhibitory synaptic markers, suggesting a transgenerational epigenetic inheritance. These results open the idea that E/I imbalance and ASD-like behavioural changes induced by environmental insults in mice can be epigenetically transmitted, at least, to the third generation. This study could help explain the unprecedented increase in ASD prevalence.

Alleviation of N-Methyl-D-Aspartate Receptor-Dependent Long-Term Depression via Regulation of the Glycogen Synthase Kinase-3β Pathway in the Amygdala of a Valproic Acid-Induced Animal Model of Autism.

The amygdala plays crucial roles in socio-emotional behavior and cognition, both of which are abnormal in autism spectrum disorder (ASD). Valproic acid (VPA)-exposed rat offspring have demonstrated ASD phenotypes and amygdala excitatory/inhibitory imbalance. However, the role of glutamatergic synapses in this imbalance remains unclear. In this study, we used a VPA-induced ASD-like model to assess glutamatergic synapse-dependent long-term depression (LTD) and depotentiation (DPT) in the amygdala. We first confirmed that the VPA-exposed offspring exhibited sociability deficits, anxiety, depression-like behavior, and abnormal nociception thresholds. Then, electrophysiological examination showed a significantly decreased paired-pulse ratio in the amygdala. In addition, both NMDA-dependent LTD and DPT were absent from the amygdala. Furthermore, we found that the levels of glycogen synthase kinase3β (GSK-3β) phosphorylation and β-catenin were significantly higher in the amygdala of the experimental animals than in the controls. Local infusion of phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the amygdala reversed the increased phosphorylation level and impaired social behavior. Taken together, the results suggested that NMDA receptor-related synaptic plasticity is dysfunctional in VPA-exposed offspring. In addition, GSK-3β in the amygdala is critical for synaptic plasticity at the glutamatergic synapses and is related to social behavior. Its role in the underlying mechanism of ASD merits further investigation.

Early physical and motor development of mouse offspring exposed to valproic acid throughout intrauterine development

Clinical research has identified developmental delay and physical malformations in children prenatally exposed to the antiepileptic drug (AED) valproic acid (VPA). However, the early signs of neurodevelopmental deficits, their evolution during postnatal development and growth, and the dose effects of VPA are not well understood. The present study aimed to examine the influence of maternal exposure to a wide dose range (50, 100, 200 and 400mg/kg/day) of VPA during breeding and gestation on early physical and neuromotor development in mice offspring. Body weight gain, eye opening, the surface righting reflex (SRR) and tail suspension test (TST) were examined in the offspring at postnatal days 5, 10 and 15. We observed that: (1) all tested doses of VPA reduced the body weight of the offspring and the timing of eye opening; (2) offspring exposed to VPA displayed immature forms of righting and required more time to complete the SRR; (3) latency for the first immobilization in the TST is shorter in offspring exposed to higher doses of VPA; however, mice in all groups exposed to VPA exhibited atypical changes in this parameter during the examined period of maturation; (4) irregularities in swinging and curling activities were observed in animals exposed to higher doses of VPA. This study points to delayed somatic development and postponed maturation of the motor system in all of the offspring prenatally exposed to VPA, with stronger effects observed at higher doses. The results implicate that the strategy of continuous monitoring of general health and achievements in motor milestones during the early postnatal development in prenatally VPA-exposed offspring, irrespectively of the dose applied, could help to recognize early developmental irregularities.

MeCP2 Modulates Sex Differences in the Postsynaptic Development of the Valproate Animal Model of Autism.

Males are predominantly affected by autism spectrum disorders (ASD) with a prevalence ratio of 5:1. However, the underlying pathological mechanisms governing the male preponderance of ASD remain unclear. Recent studies suggested that epigenetic aberrations may cause synaptic dysfunctions, which might be related to the pathophysiology of ASD. In this study, we used rat offspring prenatally exposed to valproic acid (VPA) as an animal model of ASD. We found male-selective abnormalities in the kinetic profile of the excitatory glutamatergic synaptic protein expressions linked to N-methyl-D-aspartate receptor (NMDAR), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and metabotropic glutamate receptor 5 (mGluR5) pathways in the prefrontal cortex of the VPA-exposed offspring at postnatal weeks 1, 2, and 4. Furthermore, VPA exposure showed a male-specific attenuation of the methyl-CpG-binding protein 2 (MeCP2) expressions both in the prefrontal cortex of offspring and in the gender-isolated neural progenitor cells (NPCs). In the gender-isolated NPCs culture, higher concentration of VPA induced an increased glutamatergic synaptic development along with decreased MeCP2 expression in both genders suggesting the role of MeCP2 in the modulation of synaptic development. In the small interfering RNA (siRNA) knock-down study, 50 pmol of Mecp2 siRNA inhibited the MeCP2 expression in male- but not in female-derived NPCs with concomitant induction of postsynaptic proteins such as PSD95. Taken together, we suggest that the male-inclined reduction of MeCP2 expression is involved in the abnormal development of glutamatergic synapse and male preponderance in the VPA animal models of ASD.

Activation of mGluR2/3 underlies the effects of N-acetylcystein on amygdala-associated autism-like phenotypes in a valproate-induced rat model of autism.

Autism-like phenotypes in male valproate (VPA)-exposed offspring have been linked to high glutamatergic neurotransmission in the thalamic-amygdala pathway. Glial cystine/glutamate exchange (system Xc−), which exchanges extracellular cystine for intracellular glutamate, plays a significant role in the maintenance of extracellular glutamate. N-acetylcysteine (NAC) is a cystine prodrug that restores extracellular glutamate by stimulating system Xc−. In this study, we examined the effects of NAC on autism-like phenotypes and neurotransmission in the thalamic–amygdala synapses, as well as the involvement of metabotropic glutamate receptors 2/3 (mGluR2/3). Valproate-treated rats received a single intraperitoneal injection of 500 mg/kg NaVPA on E12.5. On postnatal day 21 (P21), NAC or saline was administered once daily for 10 days. From day 8 to 10, NAC was given 1/2 h prior to behavioral testing. Chronic administration of NAC restored the duration and frequency of social interaction and ameliorated anxiety-like behaviors in VPA-exposed offspring. In amygdala slices, NAC treatment normalized the increased frequency of mEPSCs and decreased the paired pulse facilitation (PPF) induced by VPA exposure. The effects of NAC on social interaction and anxiety-like behavior in the VPA-exposed offspring were blocked after intra-amygdala infusion of mGluR2/3 antagonist LY341495. The expressions of mGluR2/3 protein and mGluR2 mRNA were significantly lower in the VPA-exposed offspring. In contrast, the mGluR3 mRNA level did not differ between the saline- and VPA-exposed offspring. These results provide the first evidence that the disruption of social interaction and enhanced presynaptic excitatory transmission in VPA-exposed offspring could be rescued by NAC, which depends on the activation of mGluR2/3.

Altered gut microbiota and activity in a murine model of autism spectrum disorders

Autism spectrum disorder (ASD) is a heterogeneous group of complex neurodevelopmental disorders with evidence of genetic predisposition. Intestinal disturbances are reported in ASD patients and compositional changes in gut microbiota are described. However, the role of microbiota in brain disorders is poorly documented. Here, we used a murine model of ASD to investigate the relation between gut microbiota and autism-like behaviour. Using next generation sequencing technology, microbiota composition was investigated in mice in utero exposed to valproic acid (VPA). Moreover, levels of short chain fatty acids (SCFA) and lactic acid in caecal content were determined. Our data demonstrate a transgenerational impact of in utero VPA exposure on gut microbiota in the offspring. Prenatal VPA exposure affected operational taxonomic units (OTUs) assigned to genera within the main phyla of Bacteroidetes and Firmicutes and the order of Desulfovibrionales, corroborating human ASD studies. In addition, OTUs assigned to genera of Alistipes, Enterorhabdus, Mollicutes and Erysipelotrichalis were especially associated with male VPA-exposed offspring. The microbial differences of VPA in utero-exposed males deviated from those observed in females and was (i) positively associated with increased levels of caecal butyrate as well as ileal neutrophil infiltration and (ii) inversely associated with intestinal levels of serotonin and social behaviour scores. These findings show that autism-like behaviour and its intestinal phenotype is associated with altered microbial colonization and activity in a murine model for ASD, with preponderance in male offspring. These results open new avenues in the scientific trajectory of managing neurodevelopmental disorders by gut microbiome modulation.

5-HT1A-receptor agonist modified amygdala activity and amygdala-associated social behavior in a valproate-induced rat autism model

Accumulating evidence suggests that dysfunction of the amygdala is related to abnormal fear processing, anxiety, and social behaviors noted in autistic spectrum disorders (ASDs). In addition, studies have shown that disrupted brain serotonin homeostasis is linked to ASD. With a valproate (VPA)-induced rat ASD model, we investigated the possible role of amygdala serotonin homeostasis in autistic phenotypes and further explored the underlying mechanism. We first discovered that the distribution of tryptophan hydroxylase immunoreactivity in the caudal raphe system was modulated on postnatal day (PD) 28 of the VPA-exposed offspring. Then, we found a significantly higher serotonin transporter availability in the amygdala of the VPA-exposed offspring on PD 56 by using single photon emission computed tomography and computed tomography co-registration following injection of (123)I-labeled 2-((2-(dimethylamino)methyl)phenyl)thio)-5-iodophenylamine((123)I[ADAM]). Furthermore, treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, increased social interaction and improved fear memory extinction in the VPA-exposed offspring. 8-OH-DPAT treatment also reversed the characteristics of miniature excitatory post-synaptic currents as well as paired pulse facilitation observed in lateral amygdala slices. These results provided further evidence to support the role of the amygdala in characteristic behavioral changes in the rat ASD model. The serotonergic projections that modulate the amygdala function might play a certain role in the development and treatment of behavioral symptoms exhibited in individuals with ASD.