Gestational VPA exposure enhances facial stimulation-evoked cerebellar mossy fiber–granule cell transmission via GluN2A-contanning NMDA receptor in offspring mice

Gestational VPA exposure enhances facial stimulation-evoked cerebellar mossy fiber–granule cell transmission via GluN2A-contanning NMDA receptor in offspring mice | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gestational VPA exposure enhances facial stimulation-evoked cerebellar mossy fiber–granule cell transmission via GluN2A-contanning NMDA receptor in offspring mice De-Lai Qiu, Yong-Xue Yuan, Yang Liu, Jing Zhang, Yan-Hua Bing, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3677267/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jul, 2024 Read the published version in Translational Psychiatry → Version 1 posted 10 You are reading this latest preprint version Abstract Valproic acid (VPA) is one of the most efficient antiepileptic drugs, which exposed during gestation has been employed as an animal model for autism spectrum disorder (ASD). Numerous studies have demonstrated that the dysfunction of synaptic transmission of cerebellar cortical circuitry contributes to the social deficits and repetitive behaviors of ASD. In this study, we investigated the effect of VPA exposure during pregnancy on the tactile stimulation-evoked cerebellar mossy fibers-granule cell (MF-GC) synaptic transmission in the urethane-anesthetized mice. Three-chamber test showed that VPA-exposed mice exhibited significant reduction of social interaction comparted with control group. In vivo electrophysiological recordings revealed that a paired-facial stimulation evoked MF-GC synaptic, N1 and N2. The evoked MF-GC synaptic responses in VPA-exposed mice exhibited a significant increase in area under the curve (AUC) of N1, amplitude and AUC of N2 than that of VPA-untreated mice. Cerebellar surface application of a selective N-methyl-D-aspartate (NMDA) receptors blocker, D-APV, significantly depressed the facial stimulation-evoked MF-GC synaptic transmission. In presence of D-APV, AUC of N1, amplitude and AUC of N2 in VPA-exposed mice were no significant than that of VPA-untreated mice. Notably, blockade of GluN2A- but not GluN2B- subunit-containing NMDA receptor, significantly depressed the MF-GC synaptic transmission, and decreased AUC of N1, amplitude and AUC of N2 in VPA-exposed mice to a similar level of that in VPA-untreated mice. In addition, the expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice. These results indicate gestational VPA exposure produces behaviors of ASD accompanied with an enhancement of the cerebellar MF-GC synaptic transmission by an increase of GluN2A-contanning NMDA receptor in offspring mice. Health sciences/Diseases/Psychiatric disorders/Autism spectrum disorders Biological sciences/Neuroscience autism spectrum disorder (ASD) cerebellar cortex sensory information mossy fiber-granule synaptic transmission in vivo electrophysiological recording N-methyl-D-aspartate receptors valproic acid (VPA) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Autisms spectrum disorder (ASD) is a group of neurodevelopmental disorders of neurological origin primarily affecting social cognition, which expresses social defects and repetitive behaviors including poor or no language development, enhanced sensory sensitivity, repetitive behaviors, attention abnormalities, and resistance to novel environments 1 . ASDs could be caused by various factors, such as genetic and environmental contributors. The early studies revealed that the motor impairments of ASD including impairments in tasks of visio-motor and manual dexterity, limb coordination requiring balance, agility and speed as well as in gait and ataxia 2 . The mechanisms of ASD are considered to be related to the alternation of molecules, synaptic transmission and neuronal circuit function, including the excitatory and inhibitory synaptic transmission 3 – 6 . Recent evidences suggest that cerebellar circuitry function may contribute to motor impairments of ASD 7 , 8 . Valproate acid (VPA) is a unique drug, which is one of the most prescribed available antiepileptic drugs in the treatment of many epileptic and nonepileptic diseases 9 – 11 . VPA is well known for its side effects, such as neural tube defects, facial abnormalities, reduced intelligence, and high risk of ASD 12 – 16 . The mechanisms of VPA enhances the risk of ASD was related to several reasons, such as increased acetylation of histone proteins 17 – 20 , and altered synaptic development, transmission and long-term plasticity 21 – 28 . It has been demonstrated that prenatal VPA exposure enhances N-metyl-D-aspartate (NMDA) receptor-mediated synaptic transmission and plasticity, which may contribute to explain the autism-like behaviors 21 . Prenatally exposure of VPA enhances NMDA receptor function by upregulating of NMDAR subunits GluN2A and GluN2B in the rat brain 21 . Behavior experiments show that pharmacological depression of the NMDA receptor function improves social deficits in VPA-exposed rats, and improves the social deficits and repetitive behavers in VPA-exposed mice 29 . NMDARs are found on the membrane of cerebellar granule cells (GCs) and parallel fiber boutons, which play critical roles in sensory information processing, synaptic plasticity, motor learning and memory, neuropathy and disorder of cerebellum 30 – 32 . The expression of GluN2A and GluN2C mRNA were demonstrated in cerebellar GCs during the second postnatal week, whereas the expression of GluN2B mRNA was transiently expressed in GCs during the first two postnatal weeks in rats 33 . Later, it has been demonstrated that GluN1 and GluN2 are expressed in granule cell somas and paralle fiber boutons 30 , 31 , and GluN2A and GluN2C subunits were found at the postsynaptic membrane of MF-GC synapses in adult mouse cerebellum 34 . We recently showed that NMDA receptors, especially GluN2A contribute to the facial stimulation-evoked MF-GC synaptic transmission, which indicated that the NMDA receptors play a critical role during the lateral sensory information synaptic transmission in the mouse cerebellar granular layer 35 . Collectively, prenatally exposure of VPA enhances NMDA receptor function in the rat brain 21 , and pharmacological depression of the NMDA receptor function improves the social deficits and repetitive behavers in VPA-exposed mice 26 , 29 , suggesting that abnormal of NMDA receptor function may be occurred in cerebellar GCs and involves the MF-GC synaptic transmission in ASD model mice. Therefore, we here investigated the effects of prenatally exposed to VPA on the sensory stimulation-evoked MF-GC synaptic transmission in the mouse cerebellar cortex in vivo. 2. Methods 2.1 Preparation of VPA-exposed mice Pregnant C57BL6/J female mice (n = 10) were administrated a single subcutaneous injection of sodium valproate (Sigma) in saline (600 mg/kg) or saline alone (control), at ambryonic day 13.5. All behavioral and electrophysiological experiments were performed on mice at 8-12-week-old. The experimental procedures were approved by the Animal Care and Use Committee of the Yanbian University and were in accordance with the animal welfare guidelines of the U.S. National Institutes of Health. The permit number is SYXK (Ji) 2011-006. All animals were housed under a 12-h light: 12-h dark cycle with free access to food and water in a colony room under constant temperature (24 ± 1°C) and humidity (50 ± 5%). 2.2 Behavioral test The three-chamber test of social interaction was demonstrated previously (Silverman et al., 2010). The apparatus consists of a central chamber and two side chambers (40 × 20 × 22 cm). The mice performed a task which was composed three 10-min sessions. First, a test mouse became accustomed to the center chamber. Second, a test mouse was allowed to explore all three chambers. Third, a stranger mouse was put in a small plastic cage in the left or right chamber, chosen randomly to avoid side preference. Then, the subject mouse was allowed to explore all three chambers and the cage. Stranger mice were habituated to the plastic cage in the three-chamber apparatus for 30 min 24 h before the test. Chamber and sniffing time were measured using Smart 3.0 software (Panlab, Harward Apparatus). The preference index (%) was calculated as (S − E)/(S + E) × 100, where S and E denote stranger and empty cage, respectively 29 . 2.3 In vivo electrophysiological recordings The anesthesia and surgical procedures have been described previously 36 . The mice were anesthetized with urethane (1.3 mg/kg body weight, i.p.), and were tracheotomized to avoid respiratory obstruction. A watertight chamber was created and a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Crus II. The cerebellum surface was superfused with oxygenated artificial cerebrospinal fluid (ACSF: 125 mM NaCl, 3 mM KCl, 1 mM MgSO 4 , 2 mM CaCl 2 , 1 mM NaH 2 PO 4 , 25 mM NaHCO 3 , and 10 mM d-glucose) with a peristaltic pump (Gilson Minipulse 3; Villiers, Le Bel, France) at 0.5 ml/min. Rectal temperature was monitored and maintained at 37.0 ± 0.2°C. Extracellular recordings from cerebellar granular layer were performed with an Axopatch-200B amplifier (Molecular Devices, Foster City, CA, USA). The potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.4 software (Molecular Devices). Recording electrodes were filled with ACSF and with resistances of 3–5 MΩ. The recordings from granular layer were performed at depths of 300–350 µm under the pia mater membrane. Facial stimulation was performed by air-puff of the ipsilateral whisker pad through a 12-gauge stainless steel tube connected with a pressurized injection system (Picospritzer® III; Parker Hannifin Co., Pine Brook, NJ, USA). The air-puff stimuli were controlled by a personal computer, and were synchronized with the electrophysiological recordings and delivered at 0.05 Hz via a Master 8 controller (A.M.P.I., Jerusalem, Israel) and Clampex10.4 software. For isolating MF-GC synaptic transmission, picrotoxin (100 µM) was added to ACSF during all recordings to prevent GABA A receptor-mediated inhibitory components. Paired-stimulation (10 ms, 60 psi) was used to evoke the MF-GC synaptic responses. Picrotoxin, sodium valproate and D-(-)-2-Amino-5-phosphonopentanoic Acid (D-APV) were bought from Sigma-Aldrich (Shanghai, China). PEAQX and TCN 237 were purchased from Tocris (Bristol, UK). The drugs were dissolved in ACSF, and applied directly onto the cerebellar surface by a peristaltic pump (0.5 ml/min). 2.4 Immunohistochemistry and imaging Mice (n = 6) were deeply anesthetized with an intraperitoneal injection of chloral hydrate (7%; 5 ml/kg), and then transcardially perfused by cold phosphate buffer (PBS; pH 7.4), followed by 4% paraformaldehyde (PFA, Sinopharm Chemical Reagent Co, China) PBS solution. Brain was post-fixed in PFA for 48 hours at 4°C, and washed with PBS. The cerebellum was separated from the brain with a razor blade, and was exposed to sucrose in PBS for more than 6 hours. After embedding in Tissue-Tek O.C.T. Compound (Beijing Zhong Shan Jin Qiao Biotechnology Co, China), the cerebellum was quickly frozen in − 80°C refrigerator for 2 hours. Then cerebellum was sectioned into 8 µm slices in the sagittal plane using a freezing microtome (CM1900, Leica, Germany). Sections were rewarming at 25°C for 30 minutes and then fixed with 4°C precooled acetone. Slices were stored at 4°C for immunohistochemical experiments. Microscope slides were permeabilized with 0.3% Triton X-100 in PBS, and then were blocked (10% donkey serum in PBS) and incubated in a primary antibody (rabbit anti-GluN2A, 1:50, abcam), followed by Alexa Fluor 488 donkey anti-rabbit (Life Tech, 1:1000) and 4′,6-diamidino-2-phenylindole (DAPI, 1:1000). The primary antibody was incubated overnight at 4°C. The secondary antibody and DAPI, were incubated for 2 hours at room temperature. Fluorescence images were acquired by confocal laser-scanning microscope (Nikon C2, Tokyo, Japan) 37 . The electrophysiological data were analyzed using Clampfit 10.4 software (Molecular Devices, Foster City, CA, USA). Using ImageJ software (V1.8.0) to analyze the average fluorescence intensity of GluN2A expression in the granular cell layer. Mean gray value = Integrate fluorescence intensity of the region/Area of the region. All data are expressed as the mean ± S.E.M. Differences between the mean values recorded under baseline (ACSF) and test conditions were evaluated with the paired Student’s t-test, whereas the mean values of VPA-exposed mice and VPA untreated mice were compared using one-way ANOA (SPSS software; Chicago, IL). P -values below 0.05 were considered statistically significant. 3. Results 3.1 VPA-exposed mice exhibited an impairment of social interaction In order to determine whether VPA-exposed mice expressed an impairment of social interaction, we performed the three-chamber test in which the relative preference of subject mouse for exploration of a stranger mouse trapped in a cage vs. an empty cage was compared. Our data showed that the VPA-exposed mice (8–12 weeks old) expressed a significant reduction of social interaction compared with VPA-unexposed (control) mice. The sniffing time to stranger cage in VPA-exposed mice was significantly reduced than that in VPA-unexposed mice (control) (F = 13.5, P = 0.0017, n = 10; Fig. 1 A), and the preference index based on sniffing time of VPA-exposed mice was significant shorter than that in control mice (F = 13.5, P = 0.0017, n = 10; Fig. 1 C). The chamber time to stranger cage in VPA-exposed mice was significantly reduced compared with VPA-unexposed mice (F = 14.1, P = 0.0015, n = 10; Fig. 2 D), and the preference index based on chamber time of VPA-exposed mice was also significant shorter than that in control mice (F = 13.5, P = 0.0017, n = 10; Fig. 1 E). These results indicated that VPA-exposed mice exhibited defeat of social interaction. 3.2 Properties of the facial stimulation-evoked MF-GC synaptic transmission in VPA-exposed mice In the presence of GABA A receptors antagonist, a paired air-puff stimulation of the ipsilateral whisker pad (10 ms, 50–60 psi, 50 ms interval) evoked a couple of negative components N1 and N2 in the cerebellar granular layer, which were identified as facial stimulation-evoked MF-GC synaptic transmission (Fig. 2 A) 35 – 37 (Zhang et al., 2020; Li et al., 2021; Lu et al., 2022). The mean amplitude of N1 in VPA-exposed mice was no significantly different than that in VPA-untreated mice (F = 0.21, P = 0.65, n = 10; Fig. 2 B), whereas the mean area under the curve (AUC) of N1 in VPA-exposed mice was significantly larger than that in VPA-untreated mice (F = 8.2, P = 0.01, n = 10; Fig. 2 C). Notably, the facial stimulation evoked a stronger N2 in VPA-exposed mice compared with VPA-untreated mice. The mean amplitude of the facial stimulation-evoked N2 in VPA-exposed mice was significantly higher than that in VPA-untreated mice (F = 10.52, P = 0.0045, n = 10; Fig. 2 D), and the mean AUC of N2 in VPA-exposed mice was significantly larger than that in VPA-untreated mice (F = 7.73, P = 0.001, n = 10; Fig. 2 E). These results indicate that VPA exposure produces a significantly enhancement of the facial stimulation-induced MF-GC synaptic transmission in vivo in mice. 3.3 VPA exposure enhanced the MF-GC synaptic transmission through NMDA receptor Since NMDA receptors contribute to the facial stimulation evoked by MF–GC synaptic transmission in the mouse cerebellar cortex 35 ,36 . We further examined the effect of a selective NMDARs antagonist, D-APV on the facial stimulation-evoked MF–GC synaptic transmission in VPA-exposed mice and VPA-untreated mice. Application of D-APV (250 µM) significantly depressed the facial stimulation-evoked MF–GC synaptic in both control (VPA-untreated) and VPA-exposed mice (P < 0.001 vs ACSF; Fig. 3 ). In the presence of D-APV, the mean value of AUC and half-width of N1 was significantly lower than that in ACSF in control and VPA-exposed mice (P < 0.001, n = 8; Fig. 3 B, C). Notably, the mean AUC of N1 in ACSF in VPA-exposed mice was significantly larger than that in control mice (P 0.05, n = 8; Fig. 3 B, C). In the presence of D-APV, the mean value of amplitude and AUC of N2 was significantly lower than that in ACSF in control and VPA-exposed mice (P < 0.001, n = 8; Fig. 3 D, E), and these values in ACSF of VPA-exposed mice was significantly larger than that of control mice (P 0.05, n = 8; Fig. 3 D, E). These results indicate that blocking NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in VPA-exposed mice. 3.4 Enhancement of the MF–GC synaptic transmission in VPA-exposed mice via the GluN2A-containing NMDA receptor In cerebellar cortex, GluN2A is expressed on the somas of GCs and the boutons of their axons 30 , 31 and contributes to facial stimulation-evoked MF–GC synaptic transmission in vivo in mice 35 ,36 (Zhang et al, 2020; Li et al., 2021). We then employed an antagonist of the GluN2A-containing NMDA receptor, PEAQX (10 µM) to study whether the enhancement of the facial stimulation-evoked MF–GC synaptic transmission was mediated by the GluN2A-containing NMDA receptor. Similar to D-APV, cerebellar surface perfusion of PEAQX (10 µM) significantly depressed the facial stimulation-evoked MF–GC synaptic in both control (VPA-untreated) and VPA-exposed mice (P < 0.001 vs ACSF; Fig. 4 ). In the presence of PEAQX, the mean value of AUC and half-width of N1 was significantly lower than that in ACSF in control and VPA-exposed mice (P < 0.001, n = 8; Fig. 4 B, C). The mean AUC of N1 in ACSF in VPA-exposed mice was significantly larger than that in control mice(P 0.05, n = 8; Fig. 4 B, C).The mean value of amplitude and AUC of N2 was significantly lower than that in ACSF in control and VPA-exposed mice (P < 0.001, n = 8; Fig. 4 D, E), and these values in ACSF of VPA-exposed mice was significantly larger than that of control mice (P 0.05, n = 8; Fig. 3 D, E). These results indicate that blocking GluN2A-containing NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in vivo in VPA-exposed mice. Moreover, we used a selective GluN2B antagonist, TCN237 (10 µM), to observe whether the enhancement of the facial stimulation-evoked MF–GC synaptic transmission was dependent on GluN2B-containing NMDA receptor 35 – 37 . Perfusion of TCN237 did not significantly change the parameters of the facial stimulation-evoked MF-GC synaptic transmission (Fig. 5 ). Cerebellar surface perfusion of TCN-237 (10 µM) did not change the facial stimulation-evoked MF–GC synaptic transmission in both control (VPA-untreated) and VPA-exposed mice (P > 0.001 vs ACSF; Fig. 5 ). In the presence of TCN237, the mean value of AUC and half-width of N1 was no significant different than that in ACSF of control and VPA-exposed mice (P > 0.001, n = 8; Fig. 5 B, C), but these value in ACSF and TCN237of VPA-exposed mice was significantly higher than that in control mice (P 0.001, n = 8; Fig. 5 D, E). In addition, these values of VPA-exposed mice in ACSF and TCN237 were significantly higher than that of control mice (P < 0.05, n = 8, n = 8; Fig. 5 D, E). These results indicate that blocking GluN2B-containing NMDA receptor does not prevent the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in the VPA-exposed mice. In addition, we observed whether the expression of GluN2A-containing NMDA receptor increased in cerebellar granular layer of VPA-exposed mice by confocal laser-scanning microscope. As shown in Fig. 6 , the GluN2A subunit-containing NMDA receptor immunoreactivity was expressed in granule cell layer of both VPA-treated and control mice (Fig. 6 A), and the mean expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice (Fig. 6 B). Collectively, the present results indicate that an increase of GluN2A-containing NMDA receptor in cerebellar granule cell layer of VPA-exposed mice, which produce an enhancement of the facial stimulation-evoked MF-GC synaptic transmission. 4. Discussion The main finding of this study is that VPA-exposed mice expressed a significant enhancement of the sensory stimulation-evoked MF-PC synaptic transmission than that of VPA-untreated mice. The enhancement of the MF-PC synaptic transmission in VPA-exposed mice is abolished by either blocking NMDA receptor or antagonizing NR2A containing NMDA receptor. These results indicate gestational VPA exposure produces behaviors of ASD accompanied with an enhancement of the cerebellar MF-GC synaptic transmission via GluN2A-contanning NMDA receptor in offspring mice. The results suggest abnormal of the sensory stimulation-evoked MF-GC synaptic transmission may contribute to the mechanisms of motor coordination and skill learning impairments in ASD model mice. Although VPA enhances the risk of ASDs is related to altered synaptic development, transmission and plasticity 21 – 28 , 38 , the neurobiological mechanisms underlying ASD development are unclear. Cerebellar cortical neurons receive sensory information from outside through mossy fiber-granule cell pathway, and generate command which related to motor coordination and motor learning 39 . Previous study has demonstrated that deficits in tasks of visio-motor and manual dexterity, limb coordination and speed in gait and ataxia in ASD patients and animal models 40 , which suggest that cerebellar circuitry function may contribute to the motor impairments of ASD. The present results showed that VPA-exposed mice expressed a defeat of social interaction, and exhibited a significantly enhancement of the facial stimulation-induced MF-GC synaptic transmission, suggesting that abnormal of the sensory information transmission at cerebellar MF-GC synapse in vivo in VPA-exposed ASD mice. In cerebellar cortex, NMDA receptors are found on the membrane of GCs and their axonal boutons, which play critical roles in modulation of MF-GC synaptic transmission and plasticity in cerebellar cortex 30 – 32 , 34 – 37 . Notably, our results showed that blocking NMDA receptor depressed the MF-GC synaptic transmission, and abolished the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in the VPA-induced ASD mice. These results indicate that the enhancement of the MF-GC synaptic transmission in ASD mice is mediated by NMDA receptor. The abnormal function of NMDA receptor has been demonstrated previously in several brain regions of VPA-exposed animals 21 , 23 , 28 , 29 . It has demonstrated that an abnormal increase in NMDA receptor function in the brains of rats prenatally exposed to VPA, which by upregulation of GluN2A and GluN2B subunits NMDA receptors, and increase of NMDA receptor mediated synaptic currents in the medial prefrontal cortex 21 , 23 , 28 . Moreover, pharmacological inhibition of NMDA receptor function in VPA-treated rats improves social deficits 26 , rescues both social deficits and repetitive behaviors 29 . Postnatal administration of low-dose NMDA receptor antagonist improves ASD-associated behaviors in the VPA-treated rats, indicating that prenatally exposure of VPA induced ASD-like behaviors in adult rats through suppression of NMDA receptor’s function 29 , 41 . Consistent previous reports 23 , 26 , 28 , 29 , the present results indicate that an enhancement of NMDA receptor function during the facial stimulation-evoked MF-GC synaptic transmission in VPA-treated mice. In cerebellar GCs, GluN2A and GluN2C mRNA were detected in cerebellar granule cells during the second postnatal week, whereas GluN2B mRNA was transiently expressed in GCs during the first 2 postnatal weeks in rats 33 . GluN2A has been detected on the somas of GCs and boutons of parallel fibers in cerebellar cortex of adult mice 31 , 33 . Our previous results showed that GluN2A, but GluN2B contributes to facial stimulation-evoked MF–GC synaptic transmission and long-term synaptic plasticity in vivo in mice 35 – 37 . The present results show that blockade of GluN2A-containing NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in vivo in VPA-exposed mice. These results suggest that the enhancement of the facial stimulation-evoked MF-GC synaptic transmission through GluN2A-containing NMDA receptor in cerebellar cortex of VPA-exposed mice. Importantly, the expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice, indicating that the enhancement of the facial stimulation-evoked MF-GC synaptic transmission through GluN2A-containing NMDA receptor in VPA-exposed mouse cerebellar cortex. The present results provide evidence for understanding the cellular and synaptic mechanisms of motor impairment in VPA-exposed animals. Declarations Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundations of China (32070986, 32171005, 32260195), the Major Projects of the Ministry of Science and Technology of China (2021ZD0202300), the Science and technology development plan project of Jilin Province, China (YDZJ202201ZYTS588), and the Application Foundation Project of Yanbian University (ydkj202321). References Cohen M.J., Meador K. J., Browning N., May R., Baker G.A., Clayton-Smith J., et al., (2013) Fetal antiepileptic drug exposure: adaptive and emotional behavioral functioning at age 6years. Epilepsy Behav 29: 308–315. Fatemi SH, Aldinger, K.A., Ashwood, P., Bauman, M.L., Blaha C.D., et al (2012) Consensus paper: pathological role of the cerebellum in autism. Cerebellum 11:777–807. Spooren, W., Lindemann, L., Ghosh, A., and Santarelli, L., (2012) Synapse dysfunction in autism: a molecular medicine approach to drug discovery in neurodevelopmental disorders. Trends Pharmacol Sci 33: 669–684. 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Lin, H.C., Gean, P.W., Wang, C.C., Chan, Y.H., and Chen, P.S., (2013) The amygdala excitatory/inhibitory balance in a valproate-induced rat autism model. PLoS ONE 8: e55248. Martin, H. G., and Manzoni, O. J. (2014) Late onset deficits in synaptic plasticity in the valproic acid rat model of autism. Front Cell Neurosci 8:23. Kang, J., and Kim, E., (2015) Suppression of NMDA receptor function in mice prenatally exposed to valproic acid improves social deficits and repetitive behaviors. Front Mol Neurosci 8:17 Glitsch, M., and Marty, A., (1999) Presynaptic effects of NMDA in cerebellar Purkinje cells and interneurons. J Neurosci 19(2): 511–519. Casado, M., Dieudonné, S., Ascher, P., (2000) Presynaptic N-methyl-D-aspartate receptors at the parallel fiber-Purkinje cell synapse. Proc Natl Acad Sci USA 97(21): 11593–7. Jin, W.Z., Liu, H., Wan, P., Chu, C.P., Qiu, D.L., (2016) Propofol enhances facial stimulation-evoked responses in the cerebellar granule cell layer via NMDA receptor activation in mice in vivo. Eur J Neurosci 788: 37–44. Akazawa, C., Shigemoto, R., Bessho, Y., Nakanishi, S., Mizuno, N., (1994) Differential expression of five N-methyl-D-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats. J Comp Neurol 347(1): 150–60. Yamada, K., Fukaya, M., Shimizu, H., Sakimura, K., Watanabe, M., (2001) NMDA receptor subunits GluRepsilon1, GluRepsilon3 and GluRzeta1 are enriched at the mossy fibre-granule cell synapse in the adult mouse cerebellum. Eur J Neurosci 13: 2025–3206. Zhang, X.Y., Zhang, G.J., Li, B.X., Bing, Y.H., Cui, B.R., Cui, L.N., et al., (2020) NMDARs contribute to the facial stimuli-evoked mossy fiber-granule cell synaptic transmission in vivo in mice. Neurosci Lett 736: 135285 Silverman, J. L., Yang, M., Lord, C., and Crawley, J. N., (2010) Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci 11: 490–502. 36. Li, B.X., Dong, G.H., Li, H.L., Zhang, J.S., Bing, Y.H., Chu, C.P., et al., (2021) Chronic Ethanol Exposure Enhances Facial Stimulation-Evoked Mossy Fiber–Granule Cell Synaptic Transmission via GluN2A Receptors in the Mouse Cerebellar Cortex. Front Syst Neurosci 657884. Lu, D., Wan, P., Liu, Y., Jin, X.H., Chu, C.P., Bing, Y.H., Qiu, D.L., (2022) Facial Stimulation Induces Long-Term Potentiation of Mossy Fiber-Granule Cell Synaptic Transmission via GluN2A-Containing N-Methyl-D-Aspartate Receptor/Nitric Oxide Cascade in the Mouse Cerebellum. Front Cell Neurosci16:863342. Chanda, S., Ang, C. E., Lee, Q. Y., Ghebrial, M., Haag, D., Shibuya, Y., et al., (2019) Direct reprogramming of human neurons identifies MARCKSL1 as a pathogenic mediator of valproic acid-induced teratogenicity. Cell Stem Cell 25: 103–119. Gao, Z., van Beugen, B.J., and De Zeeuw, C.I., (2012) Distributed synergistic plasticity and cerebellar learning. Nat Rev Neurosci 13(9): 619–35. Fatemi SH, Aldinger, K.A., Ashwood, P., Bauman, M.L., Blaha C.D., et al (2012) Consensus paper: pathological role of the cerebellum in autism. Cerebellum 11:777–807. Mohammadi, S., Asadi-Shekaari, M., Basiri, M., Parvan, M., Shabani, M., Nozari, M., (2020) Improvement of autistic-like behaviors in adult rats prenatally exposed to valproic acid through early suppression of NMDA receptor function. Psychopharmacology (Berl), 237(1): 199–208. Additional Declarations The authors have declared there is NO conflict of interest to disclose The authors declare that they have no conflict of interest. Cite Share Download PDF Status: Published Journal Publication published 03 Jul, 2024 Read the published version in Translational Psychiatry → Version 1 posted Editorial decision: revise 08 May, 2024 Review # 2 received at journal 03 Apr, 2024 Reviewer # 2 agreed at journal 01 Apr, 2024 Review # 1 received at journal 24 Mar, 2024 Reviewer # 1 agreed at journal 06 Mar, 2024 Reviewers invited by journal 08 Jan, 2024 Submission checks completed at journal 04 Dec, 2023 First submitted to journal 01 Dec, 2023 Unknown event 29 Nov, 2023 Editor assigned by journal 28 Nov, 2023 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-3677267","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":265947612,"identity":"3cc566a3-2dcb-42de-89bc-541bc171f23f","order_by":0,"name":"De-Lai Qiu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYDCC4wwMBxgqGBJAbAnitBwGaTlDqhYGxjZStPAdZt54mHfe4TyDA8wHb/Mw2OUR1CJ5mK3gMO+2w8UGB9iSrXkYkosJajE4zGMA0pK44QCPmTQPw4HEBuK0zAFp4f9GipYGsC1sxGkB+eXgnGPpxUCGseUcg2TCWviON2/+8KbGOg/IeHjjTYUdYS0gtwFxMwMDM4xNpJY64pSOglEwCkbByAQAbeQ/DXYmZZYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8825-4707","institution":"Jillin Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"De-Lai","middleName":"","lastName":"Qiu","suffix":""},{"id":265947613,"identity":"8712b2f9-bf0e-41c7-8ec7-01df7b962f08","order_by":1,"name":"Yong-Xue Yuan","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong-Xue","middleName":"","lastName":"Yuan","suffix":""},{"id":265947614,"identity":"92b0b02f-5af0-48b5-8df1-6974352360a9","order_by":2,"name":"Yang Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""},{"id":265947615,"identity":"d3bd7f42-4225-4df7-a10a-2b3dc4062ec5","order_by":3,"name":"Jing Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":265947616,"identity":"cefccad4-d644-4dbd-9df3-b1294f20516d","order_by":4,"name":"Yan-Hua Bing","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yan-Hua","middleName":"","lastName":"Bing","suffix":""},{"id":265947617,"identity":"1d182d19-4bfc-4e32-967c-12608d5bdbe6","order_by":5,"name":"Chao-Yue Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chao-Yue","middleName":"","lastName":"Chen","suffix":""},{"id":265947618,"identity":"44fb6b37-0d48-41a0-aa04-2888734e21ef","order_by":6,"name":"Guang-Gao Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guang-Gao","middleName":"","lastName":"Li","suffix":""},{"id":265947619,"identity":"bd4e97ea-8865-4cfe-8b47-8ffa40cea3b8","order_by":7,"name":"Chun-Ping Chu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-Ping","middleName":"","lastName":"Chu","suffix":""},{"id":265947620,"identity":"36fa902d-a623-475b-98ad-ab9279009313","order_by":8,"name":"Ming-Ji Yin","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ming-Ji","middleName":"","lastName":"Yin","suffix":""}],"badges":[],"createdAt":"2023-11-28 15:05:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3677267/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3677267/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41398-024-02990-0","type":"published","date":"2024-07-03T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49435049,"identity":"7b1dd9a7-3f74-4789-a455-15b7a09fbfb0","added_by":"auto","created_at":"2024-01-10 20:01:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":58145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVPA exposed mice exhibited ASD like social interaction in the three-chamber test. \u003c/strong\u003e(A) Representative traces showing the movements of control (VPA-untreated) and VPA-exposed mice during the social interaction in the three-chamber. (B) Summary of data showing the time spent sniffing stranger mouse or empty in control and VPA-exposed mice. (C) Bar graph showing the preference index based on sniffing time of (B). (D) Poled data showing the time spent in the chamber with stranger mouse or empty in control and VPA-exposed mice. (E) Bar graph showing the preference index based on chamber time of (D). n = 10 mice in each group. * P \u0026lt; 0.05 vs. empty; # \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.01.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/b70ffffb60429ba3234428e3.png"},{"id":49435048,"identity":"1a33ae67-78a9-427c-9cd6-6364f28bb1b4","added_by":"auto","created_at":"2024-01-10 20:01:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProperties of the cerebellar mossy-fiber-granule cell (MF-GC) synaptic transmission evoked by facial stimulation in VPA-exposed mice\u003c/strong\u003e. (A)Representative traces showing that the paired air-puff stimuli (10 ms, 60 psi) on the ipsilateral whisker pad-evoked MF-GC synaptic transmission in control and VPA treated mice. (B, C) Bar graphs showing the amplitude (B) and area under the curve (C) of N1. (D, E) summary of data showing the amplitude (B) and area under the curve (C) of N2. Note that the facial stimulation-evoked MF-GC synaptic transmission was significant enhanced in VPA-exposed mice. n = 10 mice in each group. # P \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.02.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/f905148bffd2d48e52a6ebc5.png"},{"id":49436538,"identity":"73595250-9eaf-4489-9927-ea0538b7e1f9","added_by":"auto","created_at":"2024-01-10 20:09:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40908,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of NMDARs blocker, D-APV on the facial stimulation-evoked MF–GC synaptic transmission in VPA-exposed mice.\u003c/strong\u003e (A) Representative traces showing that the paired air-puff stimuli (10 ms, 60 psi) on the ipsilateral whisker pad-evoked MF-GC synaptic transmission in control and VPA mice during treatments with ACSF, D-APV (250 μM), D-APV (250 μM), and washout. (B, C) Meran (± S.E.M) with individual data showing the amplitude(B) and half-width (C) of N1in treatments with ACSF, D-APV, and washout. (D, E) Mean value (± S.E.M) with individual data showing the amplitude (D) and area under the curve (E) of N2 during treatments of ACSF, D-APV, and washout. n = 8 in each group. * P \u0026lt; 0.05 vs. ACSF; # P \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.03.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/361927b55ddfe893f5acf0a5.png"},{"id":49435053,"identity":"c0cb9acd-128a-47e0-a6df-bc9a2158eed3","added_by":"auto","created_at":"2024-01-10 20:01:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of a selective NR2A antagonist, PEAQX (10 μM) on the facial stimulation-evoked MF–GC synaptic transmission in VPA exposed mice. \u003c/strong\u003e(A) Representative traces showing that the paired air-puff stimuli (10 ms, 60 psi) on the ipsilateral whisker pad-evoked MF-GC synaptic transmission in control and VPA mice in treatments of ACSF, PEAQX (10 μM), and washout. (B, C) Meran (± S.E.M) with individual data showing the amplitude (B) and half-width (C) of N1in treatments with ACSF, PEAQX, and washout. (D, E) Mean value (± S.E.M) with individual data showing the amplitude (D) and area under the curve (E) of N2 during treatments of ACSF, PEAQX, and washout. n = 8 in each group. * P \u0026lt; 0.05 vs. ACSF; # P \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.04.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/3e203d57a63fcae146cb5dda.png"},{"id":49435050,"identity":"bc54d82d-dc90-452c-ae46-c41c509b3014","added_by":"auto","created_at":"2024-01-10 20:01:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNR2B blockade did not change the facial stimulation-evoked MF–GC synaptic transmission in VPA exposed mice. \u003c/strong\u003e(A) Representative traces showing that the paired air-puff stimuli (10 ms, 60 psi) on the ipsilateral whisker pad-evoked MF-GC synaptic transmission in control and VPA mice in treatments of ACSF, TCN (10 μM), and washout. (B, C) Meran (± S.E.M) with individual data showing the amplitude (B) and half-width (C) of N1in treatments with ACSF, TCN, and washout. (D, E) Mean value (± S.E.M) with individual data showing the amplitude (D) and area under the curve (E) of N2 during treatments of ACSF, TCN, and washout. n = 8 in each group. # P \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.05.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/eb1912975254775b30dddb82.png"},{"id":49435052,"identity":"1b5dff7d-189c-44f4-a76c-6043d40a0bf1","added_by":"auto","created_at":"2024-01-10 20:01:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229084,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of GluN2A immunoreactivity in cerebellar granule cell layer was increased in VPA-exposed mice.\u003c/strong\u003e (A) Upper, Digital micrographs show the confocal image of DAPI (blue) in cerebellar lobule Crus II of WT (Left) and 5XFAD (Right) mice. DAPI is a blue nucleic acid dye that preferentially dyes the dsDNA of cells. (B) Higher magnifications of the boxed area in (A). Lower, Higher magnifications of the boxed area in (B) showing GluN2A subunit-containing NMDA receptor immunoreactivity expressed in GL (green; arrows). PCL, Purkinje cell layer; GCL, granule cell layer. (B) Mean gray values of GluN2A immunoreactivity in cerebellar lobule Crus II of control and VPA-exposed mice. * P \u0026lt; 0.05 vs. control.\u003c/p\u003e","description":"","filename":"OnlineFig.06.png","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/10657cc0656e60e245bb5bea.png"},{"id":59637900,"identity":"9ebb3cb8-e076-4d30-80c2-8cdb11ec4386","added_by":"auto","created_at":"2024-07-04 07:08:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1237491,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3677267/v1/068bbdff-59bf-405c-8d71-1ca3c3e0c01a.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose\nThe authors declare that they have no conflict of interest.","formattedTitle":"Gestational VPA exposure enhances facial stimulation-evoked cerebellar mossy fiber–granule cell transmission via GluN2A-contanning NMDA receptor in offspring mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAutisms spectrum disorder (ASD) is a group of neurodevelopmental disorders of neurological origin primarily affecting social cognition, which expresses social defects and repetitive behaviors including poor or no language development, enhanced sensory sensitivity, repetitive behaviors, attention abnormalities, and resistance to novel environments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. ASDs could be caused by various factors, such as genetic and environmental contributors. The early studies revealed that the motor impairments of ASD including impairments in tasks of visio-motor and manual dexterity, limb coordination requiring balance, agility and speed as well as in gait and ataxia \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The mechanisms of ASD are considered to be related to the alternation of molecules, synaptic transmission and neuronal circuit function, including the excitatory and inhibitory synaptic transmission \u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recent evidences suggest that cerebellar circuitry function may contribute to motor impairments of ASD\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eValproate acid (VPA) is a unique drug, which is one of the most prescribed available antiepileptic drugs in the treatment of many epileptic and nonepileptic diseases \u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. VPA is well known for its side effects, such as neural tube defects, facial abnormalities, reduced intelligence, and high risk of ASD \u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The mechanisms of VPA enhances the risk of ASD was related to several reasons, such as increased acetylation of histone proteins \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and altered synaptic development, transmission and long-term plasticity \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt has been demonstrated that prenatal VPA exposure enhances N-metyl-D-aspartate (NMDA) receptor-mediated synaptic transmission and plasticity, which may contribute to explain the autism-like behaviors \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Prenatally exposure of VPA enhances NMDA receptor function by upregulating of NMDAR subunits GluN2A and GluN2B in the rat brain \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Behavior experiments show that pharmacological depression of the NMDA receptor function improves social deficits in VPA-exposed rats, and improves the social deficits and repetitive behavers in VPA-exposed mice \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNMDARs are found on the membrane of cerebellar granule cells (GCs) and parallel fiber boutons, which play critical roles in sensory information processing, synaptic plasticity, motor learning and memory, neuropathy and disorder of cerebellum \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The expression of GluN2A and GluN2C mRNA were demonstrated in cerebellar GCs during the second postnatal week, whereas the expression of GluN2B mRNA was transiently expressed in GCs during the first two postnatal weeks in rats \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Later, it has been demonstrated that GluN1 and GluN2 are expressed in granule cell somas and paralle fiber boutons \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and GluN2A and GluN2C subunits were found at the postsynaptic membrane of MF-GC synapses in adult mouse cerebellum \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We recently showed that NMDA receptors, especially GluN2A contribute to the facial stimulation-evoked MF-GC synaptic transmission, which indicated that the NMDA receptors play a critical role during the lateral sensory information synaptic transmission in the mouse cerebellar granular layer \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Collectively, prenatally exposure of VPA enhances NMDA receptor function in the rat brain \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, and pharmacological depression of the NMDA receptor function improves the social deficits and repetitive behavers in VPA-exposed mice \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, suggesting that abnormal of NMDA receptor function may be occurred in cerebellar GCs and involves the MF-GC synaptic transmission in ASD model mice. Therefore, we here investigated the effects of prenatally exposed to VPA on the sensory stimulation-evoked MF-GC synaptic transmission in the mouse cerebellar cortex in vivo.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of VPA-exposed mice\u003c/h2\u003e \u003cp\u003ePregnant C57BL6/J female mice (n\u0026thinsp;=\u0026thinsp;10) were administrated a single subcutaneous injection of sodium valproate (Sigma) in saline (600 mg/kg) or saline alone (control), at ambryonic day 13.5. All behavioral and electrophysiological experiments were performed on mice at 8-12-week-old. The experimental procedures were approved by the Animal Care and Use Committee of the Yanbian University and were in accordance with the animal welfare guidelines of the U.S. National Institutes of Health. The permit number is SYXK (Ji) 2011-006. All animals were housed under a 12-h light: 12-h dark cycle with free access to food and water in a colony room under constant temperature (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Behavioral test\u003c/h2\u003e \u003cp\u003eThe three-chamber test of social interaction was demonstrated previously (Silverman et al., 2010). The apparatus consists of a central chamber and two side chambers (40 \u0026times; 20 \u0026times; 22 cm). The mice performed a task which was composed three 10-min sessions. First, a test mouse became accustomed to the center chamber. Second, a test mouse was allowed to explore all three chambers. Third, a stranger mouse was put in a small plastic cage in the left or right chamber, chosen randomly to avoid side preference. Then, the subject mouse was allowed to explore all three chambers and the cage. Stranger mice were habituated to the plastic cage in the three-chamber apparatus for 30 min 24 h before the test. Chamber and sniffing time were measured using Smart 3.0 software (Panlab, Harward Apparatus). The preference index (%) was calculated as (S\u0026thinsp;\u0026minus;\u0026thinsp;E)/(S\u0026thinsp;+\u0026thinsp;E) \u0026times; 100, where S and E denote stranger and empty cage, respectively \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In vivo electrophysiological recordings\u003c/h2\u003e \u003cp\u003eThe anesthesia and surgical procedures have been described previously \u003csup\u003e36\u003c/sup\u003e. The mice were anesthetized with urethane (1.3 mg/kg body weight, i.p.), and were tracheotomized to avoid respiratory obstruction. A watertight chamber was created and a 1-1.5 mm craniotomy was drilled to expose the cerebellar surface corresponding to Crus II. The cerebellum surface was superfused with oxygenated artificial cerebrospinal fluid (ACSF: 125 mM NaCl, 3 mM KCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, and 10 mM d-glucose) with a peristaltic pump (Gilson Minipulse 3; Villiers, Le Bel, France) at 0.5 ml/min. Rectal temperature was monitored and maintained at 37.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u0026deg;C.\u003c/p\u003e \u003cp\u003eExtracellular recordings from cerebellar granular layer were performed with an Axopatch-200B amplifier (Molecular Devices, Foster City, CA, USA). The potentials were acquired through a Digidata 1440 series analog-to-digital interface on a personal computer using Clampex 10.4 software (Molecular Devices). Recording electrodes were filled with ACSF and with resistances of 3\u0026ndash;5 MΩ. The recordings from granular layer were performed at depths of 300\u0026ndash;350 \u0026micro;m under the pia mater membrane.\u003c/p\u003e \u003cp\u003eFacial stimulation was performed by air-puff of the ipsilateral whisker pad through a 12-gauge stainless steel tube connected with a pressurized injection system (Picospritzer\u0026reg; III; Parker Hannifin Co., Pine Brook, NJ, USA). The air-puff stimuli were controlled by a personal computer, and were synchronized with the electrophysiological recordings and delivered at 0.05 Hz via a Master 8 controller (A.M.P.I., Jerusalem, Israel) and Clampex10.4 software. For isolating MF-GC synaptic transmission, picrotoxin (100 \u0026micro;M) was added to ACSF during all recordings to prevent GABA\u003csub\u003eA\u003c/sub\u003e receptor-mediated inhibitory components. Paired-stimulation (10 ms, 60 psi) was used to evoke the MF-GC synaptic responses. Picrotoxin, sodium valproate and D-(-)-2-Amino-5-phosphonopentanoic Acid (D-APV) were bought from Sigma-Aldrich (Shanghai, China). PEAQX and TCN 237 were purchased from Tocris (Bristol, UK). The drugs were dissolved in ACSF, and applied directly onto the cerebellar surface by a peristaltic pump (0.5 ml/min).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunohistochemistry and imaging\u003c/h2\u003e \u003cp\u003e Mice (n\u0026thinsp;=\u0026thinsp;6) were deeply anesthetized with an intraperitoneal injection of chloral hydrate (7%; 5 ml/kg), and then transcardially perfused by cold phosphate buffer (PBS; pH 7.4), followed by 4% paraformaldehyde (PFA, Sinopharm Chemical Reagent Co, China) PBS solution. Brain was post-fixed in PFA for 48 hours at 4\u0026deg;C, and washed with PBS. The cerebellum was separated from the brain with a razor blade, and was exposed to sucrose in PBS for more than 6 hours. After embedding in Tissue-Tek O.C.T. Compound (Beijing Zhong Shan Jin Qiao Biotechnology Co, China), the cerebellum was quickly frozen in \u0026minus;\u0026thinsp;80\u0026deg;C refrigerator for 2 hours. Then cerebellum was sectioned into 8 \u0026micro;m slices in the sagittal plane using a freezing microtome (CM1900, Leica, Germany). Sections were rewarming at 25\u0026deg;C for 30 minutes and then fixed with 4\u0026deg;C precooled acetone. Slices were stored at 4\u0026deg;C for immunohistochemical experiments.\u003c/p\u003e \u003cp\u003eMicroscope slides were permeabilized with 0.3% Triton X-100 in PBS, and then were blocked (10% donkey serum in PBS) and incubated in a primary antibody (rabbit anti-GluN2A, 1:50, abcam), followed by Alexa Fluor 488 donkey anti-rabbit (Life Tech, 1:1000) and 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, 1:1000). The primary antibody was incubated overnight at 4\u0026deg;C. The secondary antibody and DAPI, were incubated for 2 hours at room temperature. Fluorescence images were acquired by confocal laser-scanning microscope (Nikon C2, Tokyo, Japan)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe electrophysiological data were analyzed using Clampfit 10.4 software (Molecular Devices, Foster City, CA, USA). Using ImageJ software (V1.8.0) to analyze the average fluorescence intensity of GluN2A expression in the granular cell layer. Mean gray value\u0026thinsp;=\u0026thinsp;Integrate fluorescence intensity of the region/Area of the region. All data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Differences between the mean values recorded under baseline (ACSF) and test conditions were evaluated with the paired Student\u0026rsquo;s t-test, whereas the mean values of VPA-exposed mice and VPA untreated mice were compared using one-way ANOA (SPSS software; Chicago, IL). \u003cem\u003eP\u003c/em\u003e-values below 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 VPA-exposed mice exhibited an impairment of social interaction\u003c/h2\u003e \u003cp\u003eIn order to determine whether VPA-exposed mice expressed an impairment of social interaction, we performed the three-chamber test in which the relative preference of subject mouse for exploration of a stranger mouse trapped in a cage vs. an empty cage was compared. Our data showed that the VPA-exposed mice (8\u0026ndash;12 weeks old) expressed a significant reduction of social interaction compared with VPA-unexposed (control) mice. The sniffing time to stranger cage in VPA-exposed mice was significantly reduced than that in VPA-unexposed mice (control) (F\u0026thinsp;=\u0026thinsp;13.5, P\u0026thinsp;=\u0026thinsp;0.0017, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), and the preference index based on sniffing time of VPA-exposed mice was significant shorter than that in control mice (F\u0026thinsp;=\u0026thinsp;13.5, P\u0026thinsp;=\u0026thinsp;0.0017, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The chamber time to stranger cage in VPA-exposed mice was significantly reduced compared with VPA-unexposed mice (F\u0026thinsp;=\u0026thinsp;14.1, P\u0026thinsp;=\u0026thinsp;0.0015, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and the preference index based on chamber time of VPA-exposed mice was also significant shorter than that in control mice (F\u0026thinsp;=\u0026thinsp;13.5, P\u0026thinsp;=\u0026thinsp;0.0017, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These results indicated that VPA-exposed mice exhibited defeat of social interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Properties of the facial stimulation-evoked MF-GC synaptic transmission in VPA-exposed mice\u003c/h2\u003e \u003cp\u003eIn the presence of GABA\u003csub\u003eA\u003c/sub\u003e receptors antagonist, a paired air-puff stimulation of the ipsilateral whisker pad (10 ms, 50\u0026ndash;60 psi, 50 ms interval) evoked a couple of negative components N1 and N2 in the cerebellar granular layer, which were identified as facial stimulation-evoked MF-GC synaptic transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA)\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e(Zhang et al., 2020; Li et al., 2021; Lu et al., 2022). The mean amplitude of N1 in VPA-exposed mice was no significantly different than that in VPA-untreated mice (F\u0026thinsp;=\u0026thinsp;0.21, P\u0026thinsp;=\u0026thinsp;0.65, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), whereas the mean area under the curve (AUC) of N1 in VPA-exposed mice was significantly larger than that in VPA-untreated mice (F\u0026thinsp;=\u0026thinsp;8.2, P\u0026thinsp;=\u0026thinsp;0.01, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Notably, the facial stimulation evoked a stronger N2 in VPA-exposed mice compared with VPA-untreated mice. The mean amplitude of the facial stimulation-evoked N2 in VPA-exposed mice was significantly higher than that in VPA-untreated mice (F\u0026thinsp;=\u0026thinsp;10.52, P\u0026thinsp;=\u0026thinsp;0.0045, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and the mean AUC of N2 in VPA-exposed mice was significantly larger than that in VPA-untreated mice (F\u0026thinsp;=\u0026thinsp;7.73, P\u0026thinsp;=\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results indicate that VPA exposure produces a significantly enhancement of the facial stimulation-induced MF-GC synaptic transmission \u003cem\u003ein vivo\u003c/em\u003e in mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3 VPA exposure enhanced the MF-GC synaptic transmission through NMDA receptor\u003c/h2\u003e \u003cp\u003eSince NMDA receptors contribute to the facial stimulation evoked by MF\u0026ndash;GC synaptic transmission in the mouse cerebellar cortex \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,36\u003c/sup\u003e. We further examined the effect of a selective NMDARs antagonist, D-APV on the facial stimulation-evoked MF\u0026ndash;GC synaptic transmission in VPA-exposed mice and VPA-untreated mice. Application of D-APV (250 \u0026micro;M) significantly depressed the facial stimulation-evoked MF\u0026ndash;GC synaptic in both control (VPA-untreated) and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs ACSF; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the presence of D-APV, the mean value of AUC and half-width of N1 was significantly lower than that in ACSF in control and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Notably, the mean AUC of N1 in ACSF in VPA-exposed mice was significantly larger than that in control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8), but they are similar in the presence of D-APV (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). In the presence of D-APV, the mean value of amplitude and AUC of N2 was significantly lower than that in ACSF in control and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), and these values in ACSF of VPA-exposed mice was significantly larger than that of control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8), but they are also similar in the presence of D-APV (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). These results indicate that blocking NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in VPA-exposed mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Enhancement of the MF\u0026ndash;GC synaptic transmission in VPA-exposed mice via the GluN2A-containing NMDA receptor\u003c/h2\u003e \u003cp\u003eIn cerebellar cortex, GluN2A is expressed on the somas of GCs and the boutons of their axons \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003eand contributes to facial stimulation-evoked MF\u0026ndash;GC synaptic transmission in vivo in mice \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,36\u003c/sup\u003e(Zhang et al, 2020; Li et al., 2021). We then employed an antagonist of the GluN2A-containing NMDA receptor, PEAQX (10 \u0026micro;M) to study whether the enhancement of the facial stimulation-evoked MF\u0026ndash;GC synaptic transmission was mediated by the GluN2A-containing NMDA receptor. Similar to D-APV, cerebellar surface perfusion of PEAQX (10 \u0026micro;M) significantly depressed the facial stimulation-evoked MF\u0026ndash;GC synaptic in both control (VPA-untreated) and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs ACSF; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the presence of PEAQX, the mean value of AUC and half-width of N1 was significantly lower than that in ACSF in control and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). The mean AUC of N1 in ACSF in VPA-exposed mice was significantly larger than that in control mice(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8), but they are similar in the presence of PEAQX (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C).The mean value of amplitude and AUC of N2 was significantly lower than that in ACSF in control and VPA-exposed mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E), and these values in ACSF of VPA-exposed mice was significantly larger than that of control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8), but they are also similar in the presence of PEAQX (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). These results indicate that blocking GluN2A-containing NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in vivo in VPA-exposed mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMoreover, we used a selective GluN2B antagonist, TCN237 (10 \u0026micro;M), to observe whether the enhancement of the facial stimulation-evoked MF\u0026ndash;GC synaptic transmission was dependent on GluN2B-containing NMDA receptor \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Perfusion of TCN237 did not significantly change the parameters of the facial stimulation-evoked MF-GC synaptic transmission (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Cerebellar surface perfusion of TCN-237 (10 \u0026micro;M) did not change the facial stimulation-evoked MF\u0026ndash;GC synaptic transmission in both control (VPA-untreated) and VPA-exposed mice (P\u0026thinsp;\u0026gt;\u0026thinsp;0.001 vs ACSF; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the presence of TCN237, the mean value of AUC and half-width of N1 was no significant different than that in ACSF of control and VPA-exposed mice (P\u0026thinsp;\u0026gt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), but these value in ACSF and TCN237of VPA-exposed mice was significantly higher than that in control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). The mean value of amplitude and AUC of N2 was no significant different than that in ACSF of control and VPA-exposed mice (P\u0026thinsp;\u0026gt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). In addition, these values of VPA-exposed mice in ACSF and TCN237 were significantly higher than that of control mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;8, n\u0026thinsp;=\u0026thinsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, E). These results indicate that blocking GluN2B-containing NMDA receptor does not prevent the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in the VPA-exposed mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we observed whether the expression of GluN2A-containing NMDA receptor increased in cerebellar granular layer of VPA-exposed mice by confocal laser-scanning microscope. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the GluN2A subunit-containing NMDA receptor immunoreactivity was expressed in granule cell layer of both VPA-treated and control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), and the mean expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Collectively, the present results indicate that an increase of GluN2A-containing NMDA receptor in cerebellar granule cell layer of VPA-exposed mice, which produce an enhancement of the facial stimulation-evoked MF-GC synaptic transmission.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe main finding of this study is that VPA-exposed mice expressed a significant enhancement of the sensory stimulation-evoked MF-PC synaptic transmission than that of VPA-untreated mice. The enhancement of the MF-PC synaptic transmission in VPA-exposed mice is abolished by either blocking NMDA receptor or antagonizing NR2A containing NMDA receptor. These results indicate gestational VPA exposure produces behaviors of ASD accompanied with an enhancement of the cerebellar MF-GC synaptic transmission via GluN2A-contanning NMDA receptor in offspring mice. The results suggest abnormal of the sensory stimulation-evoked MF-GC synaptic transmission may contribute to the mechanisms of motor coordination and skill learning impairments in ASD model mice.\u003c/p\u003e \u003cp\u003eAlthough VPA enhances the risk of ASDs is related to altered synaptic development, transmission and plasticity \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, the neurobiological mechanisms underlying ASD development are unclear. Cerebellar cortical neurons receive sensory information from outside through mossy fiber-granule cell pathway, and generate command which related to motor coordination and motor learning \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Previous study has demonstrated that deficits in tasks of visio-motor and manual dexterity, limb coordination and speed in gait and ataxia in ASD patients and animal models \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, which suggest that cerebellar circuitry function may contribute to the motor impairments of ASD. The present results showed that VPA-exposed mice expressed a defeat of social interaction, and exhibited a significantly enhancement of the facial stimulation-induced MF-GC synaptic transmission, suggesting that abnormal of the sensory information transmission at cerebellar MF-GC synapse in vivo in VPA-exposed ASD mice.\u003c/p\u003e \u003cp\u003eIn cerebellar cortex, NMDA receptors are found on the membrane of GCs and their axonal boutons, which play critical roles in modulation of MF-GC synaptic transmission and plasticity in cerebellar cortex \u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Notably, our results showed that blocking NMDA receptor depressed the MF-GC synaptic transmission, and abolished the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in the VPA-induced ASD mice. These results indicate that the enhancement of the MF-GC synaptic transmission in ASD mice is mediated by NMDA receptor. The abnormal function of NMDA receptor has been demonstrated previously in several brain regions of VPA-exposed animals \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. It has demonstrated that an abnormal increase in NMDA receptor function in the brains of rats prenatally exposed to VPA, which by upregulation of GluN2A and GluN2B subunits NMDA receptors, and increase of NMDA receptor mediated synaptic currents in the medial prefrontal cortex\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Moreover, pharmacological inhibition of NMDA receptor function in VPA-treated rats improves social deficits \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, rescues both social deficits and repetitive behaviors\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Postnatal administration of low-dose NMDA receptor antagonist improves ASD-associated behaviors in the VPA-treated rats, indicating that prenatally exposure of VPA induced ASD-like behaviors in adult rats through suppression of NMDA receptor\u0026rsquo;s function \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Consistent previous reports\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the present results indicate that an enhancement of NMDA receptor function during the facial stimulation-evoked MF-GC synaptic transmission in VPA-treated mice.\u003c/p\u003e \u003cp\u003eIn cerebellar GCs, GluN2A and GluN2C mRNA were detected in cerebellar granule cells during the second postnatal week, whereas GluN2B mRNA was transiently expressed in GCs during the first 2 postnatal weeks in rats \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. GluN2A has been detected on the somas of GCs and boutons of parallel fibers in cerebellar cortex of adult mice \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Our previous results showed that GluN2A, but GluN2B contributes to facial stimulation-evoked MF\u0026ndash;GC synaptic transmission and long-term synaptic plasticity in vivo in mice \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The present results show that blockade of GluN2A-containing NMDA receptor depresses the MF-GC synaptic transmission, and abolishes the enhancement of the facial stimulation-evoked MF-GC synaptic transmission in vivo in VPA-exposed mice. These results suggest that the enhancement of the facial stimulation-evoked MF-GC synaptic transmission through GluN2A-containing NMDA receptor in cerebellar cortex of VPA-exposed mice. Importantly, the expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice, indicating that the enhancement of the facial stimulation-evoked MF-GC synaptic transmission through GluN2A-containing NMDA receptor in VPA-exposed mouse cerebellar cortex. The present results provide evidence for understanding the cellular and synaptic mechanisms of motor impairment in VPA-exposed animals.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundations of China (32070986, 32171005, 32260195), the Major Projects of the Ministry of Science and Technology of China (2021ZD0202300), the Science and technology development plan project of Jilin Province, China (YDZJ202201ZYTS588), and the Application Foundation Project of Yanbian University (ydkj202321).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCohen M.J., Meador K. 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Y., Ghebrial, M., Haag, D., Shibuya, Y., et al., (2019) Direct reprogramming of human neurons identifies MARCKSL1 as a pathogenic mediator of valproic acid-induced teratogenicity. Cell Stem Cell 25: 103\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, Z., van Beugen, B.J., and De Zeeuw, C.I., (2012) Distributed synergistic plasticity and cerebellar learning. Nat Rev Neurosci 13(9): 619\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFatemi SH, Aldinger, K.A., Ashwood, P., Bauman, M.L., Blaha C.D., et al (2012) Consensus paper: pathological role of the cerebellum in autism. Cerebellum 11:777\u0026ndash;807.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammadi, S., Asadi-Shekaari, M., Basiri, M., Parvan, M., Shabani, M., Nozari, M., (2020) Improvement of autistic-like behaviors in adult rats prenatally exposed to valproic acid through early suppression of NMDA receptor function. Psychopharmacology (Berl), 237(1): 199\u0026ndash;208.\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":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"autism spectrum disorder (ASD), cerebellar cortex, sensory information, mossy fiber-granule synaptic transmission, in vivo electrophysiological recording, N-methyl-D-aspartate receptors, valproic acid (VPA)","lastPublishedDoi":"10.21203/rs.3.rs-3677267/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3677267/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eValproic acid (VPA) is one of the most efficient antiepileptic drugs, which exposed during gestation has been employed as an animal model for autism spectrum disorder (ASD). Numerous studies have demonstrated that the dysfunction of synaptic transmission of cerebellar cortical circuitry contributes to the social deficits and repetitive behaviors of ASD. In this study, we investigated the effect of VPA exposure during pregnancy on the tactile stimulation-evoked cerebellar mossy fibers-granule cell (MF-GC) synaptic transmission in the urethane-anesthetized mice. Three-chamber test showed that VPA-exposed mice exhibited significant reduction of social interaction comparted with control group. \u003cem\u003eIn vivo\u003c/em\u003e electrophysiological recordings revealed that a paired-facial stimulation evoked MF-GC synaptic, N1 and N2. The evoked MF-GC synaptic responses in VPA-exposed mice exhibited a significant increase in area under the curve (AUC) of N1, amplitude and AUC of N2 than that of VPA-untreated mice. Cerebellar surface application of a selective N-methyl-D-aspartate (NMDA) receptors blocker, D-APV, significantly depressed the facial stimulation-evoked MF-GC synaptic transmission. In presence of D-APV, AUC of N1, amplitude and AUC of N2 in VPA-exposed mice were no significant than that of VPA-untreated mice. Notably, blockade of GluN2A- but not GluN2B- subunit-containing NMDA receptor, significantly depressed the MF-GC synaptic transmission, and decreased AUC of N1, amplitude and AUC of N2 in VPA-exposed mice to a similar level of that in VPA-untreated mice. In addition, the expression of GluN2A subunit-containing NMDA receptor immunoreactivity in granule cell layer of VPA-treated mice was significantly higher than that in control mice. These results indicate gestational VPA exposure produces behaviors of ASD accompanied with an enhancement of the cerebellar MF-GC synaptic transmission by an increase of GluN2A-contanning NMDA receptor in offspring mice.\u003c/p\u003e","manuscriptTitle":"Gestational VPA exposure enhances facial stimulation-evoked cerebellar mossy fiber–granule cell transmission via GluN2A-contanning NMDA receptor in offspring mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-10 20:01:15","doi":"10.21203/rs.3.rs-3677267/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-05-08T10:33:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-04-03T07:41:12+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-04-01T08:39:32+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-24T16:09:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-06T07:30:36+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-01-08T17:49:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-04T15:06:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Psychiatry","date":"2023-12-01T14:30:04+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2023-11-29T11:41:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-11-28T15:00:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"tp","sideBox":"Learn more about [Translational Psychiatry](http://www.nature.com/tp/)","snPcode":"41398","submissionUrl":"https://mts-tp.nature.com/cgi-bin/main.plex","title":"Translational Psychiatry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e3bc2b5-b57a-4518-964f-340a42a95119","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28019114,"name":"Health sciences/Diseases/Psychiatric disorders/Autism spectrum disorders"},{"id":28019115,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2024-07-04T07:08:43+00:00","versionOfRecord":{"articleIdentity":"rs-3677267","link":"https://doi.org/10.1038/s41398-024-02990-0","journal":{"identity":"translational-psychiatry","isVorOnly":false,"title":"Translational Psychiatry"},"publishedOn":"2024-07-03 04:00:00","publishedOnDateReadable":"July 3rd, 2024"},"versionCreatedAt":"2024-01-10 20:01:15","video":"","vorDoi":"10.1038/s41398-024-02990-0","vorDoiUrl":"https://doi.org/10.1038/s41398-024-02990-0","workflowStages":[]},"version":"v1","identity":"rs-3677267","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3677267","identity":"rs-3677267","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}