Lacticaseibacillus rhamnosus GR-1 prevents autism-like behaviors by reshaping the maternal and offspring microbiome

Lacticaseibacillus rhamnosus GR-1 prevents autism-like behaviors by reshaping the maternal and offspring microbiome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lacticaseibacillus rhamnosus GR-1 prevents autism-like behaviors by reshaping the maternal and offspring microbiome Ruili Yang, Jinchun Xu, Yi Xu, Chengqing Huang, Feng Zhu, Tian Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5930312/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in npj Biofilms and Microbiomes → Version 1 posted 11 You are reading this latest preprint version Abstract As a prevalent neurodevelopmental disease, whether ASD (autism spectrum disorder) can be ameliorated by the early use of a single microbe remains unknown. Here we report that Lacticaseibacillus rhamnosus GR-1 (LGR-1) prevented the occurrence of autism-like behaviors when administered exclusively to the pregnant mice, as evidenced by the improved behaviors and restored E/I balance in the prefrontal cortex of male pups. In parallel, the offspring microbiome was reshaped by LGR-1 treatment, mediated by the vertical transmission of maternal microbiome, with its roles validated by microbiota transplant and cross-fostering. In addition to gut commensals, the LGR-1-shaping vaginal microbiota also contributed to the establishment of “beneficial” microbiome. Regarding key taxa in offspring, Akkermansia muciniphila was influenced by LGR-1 and exerted effect on the ensuing behavior, through modulating immune pathways related to IL-17-producing cell population. In conclusion, a single microbe applied in utero protects offspring from autism-like behaviors via microbiome transmission, shedding light on the microbe-based avenue to mitigate the risk of ASD. Biological sciences/Microbiology/Communities/Microbiome Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Autism spectrum disorder (ASD) is a complex neurodevelopmental condition primarily characterized by deficits in sociability and stereotyped behaviors 1 . The worldwide prevalence of ASD continues to rise in recent years, with estimates ranging between 1 ~ 3% according to various sources 2 – 4 , which affects males four times more than females 5 . In addition to core symptoms, a significant proportion of individuals with ASD also experience gastrointestinal (GI) issues 6 , 7 . Consequently, there is a growing interest in the relationship between the gut microbiome and ASD 8 , 9 . By transferring microbiota from ASD patients to germ-free animals, the recipients exhibit autistic symptoms 10 . This evidence is a testament to the importance of gut microbiome in the pathophysiology of ASD. The maternal status is closely associated with the occurrence of ASD in offspring. For instance, a high-fat diet in dams can induce social and synaptic deficits in their pups 11 . Maternal immune activation serves as a routine strategy to generate murine models with autism-like symptoms, presumably through IL-17 (Interleukin 17) stimulation 12 . Notably, immune cell activation by gut bacteria is a prime cause of IL-17 release in this context 13 , suggesting a causal link between maternal microbiome and ASD. This correlation was further substantiated by findings that maternal dysbiosis, either introduced by antibiotics or fecal transplants, is sufficient to trigger the neurological abnormalities in offspring, including deficits in social interaction 14 , 15 . Despite the implication of maternal microbiome, how it can be orchestrated to mitigate autistic symptoms remains less understood. The current advances might allow for a promising microbe-based intervention scheme, especially given that there are only two drugs (risperidone and aripiprazole) approved by FDA for the treatment of ASD. While risperidone is effective in controlling hyperactivity and disruptive behaviors 16 , its function in addressing the core symptoms of ASD is still under debate 17 . Moreover, most medications are based on a “curative strategy”, designed after the symptoms have occurred. Considering the significant merits of preventive measures, the maternal microbiome could be viewed as the potential target for prophylactic intervention. A growing body of literature suggests that lactobacillus strains could be used to combat autism-like symptoms. Hsiao et al. 18 discovered that the oral intake of Bacteroides fragilis improved gut permeability and alleviated ASD-related defects. Another species, Limosilactobacillus reuteri , was potent in mitigating the social abnormalities and stereotypies in a variety of autism models 6 , 19 . According to our previous study, Clostridium butyricum alleviates social subordinance in mice with intestinal dysbiosis 20 , suggesting that a specific bacterium may impose a desirable effect on social behaviors. While these microbes show promise in repairing autism-related damage, no strains have been identified to play a strictly (prenatally) preventive role in the prototypic autism models. Lacticaseibacillus rhamnosus GR-1 (formerly known as Lactobacillus rhamnosus GR-1, LGR-1) is a strain first isolated from the female urogenital tract, and later found to exhibit probiotic effect when orally administered to the intestine 21 . Our prior work showed that LGR-1 could improve spatial memory 22 , proposing it a candidate strain for protection against other brain-related behaviors. In this study, we investigated the prophylactic role of LGR-1 in ameliorating autism-like deficits and attempted to understand the underlying mechanisms, with a focus on the generational inheritance of the gut microbiome. This study could help us find a new way to address autism-related adversities by early interfering with the microbiome of the pregnant mothers. Results Prenatal administration of LGR-1 mitigates the autism-like behaviors. Valproic acid (VPA) can trigger autism through injection on embryonic day (ED) 12.5 6 . In this study, both the VPA-induced and idiopathic mice, namely BTBR T + Itpr3 tf /J (BTBR), were used as animal models for autism. To investigate if LGR-1 could alleviate the related deficits, this strain was administered daily to pregnant dams from conception until parturition. The intake was immediately discontinued after birth, and the animal behaviors were examined at postnatal week 8 (PNW8, Fig. 1 a). Firstly, a three-chamber test was adopted to evaluate the social performance of the subject mice (Fig. 1 b). According to the results, LGR-1 significantly prevented mice from autism-like lesions, as evidenced by their preference to interact with another mouse (or novel conspecific) over an inanimate object (or old one) ( P = 0.011 for sociability, P = 0.005 for social novelty; Fig. 1 c-e; Supplementary video 1–4). Thus, social performance was improved through the prenatal use of LGR-1. Remarkably, the prophylactic effect appears to be strain-specific, as a similar outcome was not observed with the routine functional partner of LGR-1, Limosilactobacillus reuteri RC14 (LRC14), or another common probiotic strain, Lacticaseibacillus rhamnosus GG (LGG; Fig. 1 d and e). Moreover, the LGR-1 strain itself may be pivotal for intervention, as its supernatant did not offer protection (Fig. 1 d and e). The efficacy of LGR-1 was consistent across the models used, with both environment-induced (VPA) and idiopathic (BTBR) mice ( P = 0.002 for sociability, P = 0.012 for social novelty; Fig. 1 f and g) benefitting from LGR-1 pretreatment. Marble burying test is a paradigm used to assess repetitive behavior, another core symptom of ASD. This test revealed that stereotypies were attenuated in both VPA-induced ( P < 0.001) and BTBR mice ( P = 0.004) following LGR-1 pretreatment (Fig. 1 h). Besides, a reciprocal interaction assay yielded similar results: the interaction duration and number of contacts between mouse pairs were enhanced after prenatal addition of LGR-1 (Supplementary Fig. 1a and b). Interestingly, postnatal administration of LGR-1 also led to a varying degree of social improvements (Supplementary Fig. 1c), but not stereotypies (Supplementary Fig. 1d). E/I imbalance is a hallmark feature in some ASD patients, indicating abnormal synaptic transmission and cortical circuitry 10 , 23 . To investigate this phenomenon in the present condition, we recorded miniature excitatory post-synaptic currents (mEPSC) and miniature inhibitory post-synaptic currents (mIPSC) in the medial prefrontal cortex. Frequency analysis revealed that BTBR mice displayed a decreased mIPSC while maintaining constant mEPSC, suggesting an increased E/I ratio (Fig. 1 i and j, Supplementary Fig. 1e and f). This deviation was ameliorated by LGR-1, as observed in the changes of mIPSC frequency ( P = 0.032; Fig. 1 j). The synaptic fluctuation was accompanied by the concurrent reprogramming of cortical gene expression (Supplementary Fig. 1g), which showed the enrichment of MAPK signaling among others (Supplementary Fig. 1h), in response to LGR-1. Given the correlation between microbiome and autism, we next examined the gut microbiome of pups pre-treated with LGR-1. Revealed by the principal component analysis, the in utero addition of LGR-1 steered the microbiome towards a state closer to the untreated mice, compared to that of the socially-deficient conspecifics (Fig. 1 k). Meanwhile, the supplementation with either LGR-1 supernatant or LGG resulted in the divergent alterations of microbial community. Together, prenatal treatment of LGR-1 improves autism-like behaviors and restores the microbiome. Prenatal treatment alters offspring microbiome via vertical transmission. We next sought to understand how prenatal treatment of LGR-1 could cause considerable changes in the offspring microbiome. Gut microbiome can be vertically transmitted from mothers to infants 24 , offering a route for generational influences on microbiome compositions. Therefore, we first examined the maternal microbiome’s response to LGR-1 treatment. As shown in Fig. 2 a, LGR-1 profoundly modified the microbiome of pregnant dams within the BTBR inbred colonies, potentially laying the groundwork for the transmission hypothesis. Next, we investigated vertical inheritance of microbiome in the context of LGR-1 intervention using metagenomics. Regarding bacterial species shared between dams and pups, mice tend to harbor similar species with their own mothers rather than with other dams receiving the same treatment (Supplementary Fig. 2a). This tendency remained consistent across treatments and was pronounced for the most abundant species of the pups (Supplementary Fig. 2b). Subsequently, the StrainPhlAn was used to identify the bacterial strains present in at least 16 samples and to analyze their genetic distance among samples. As revealed by the data, the genetic distances of “intra-pair” (dam-pup) were significantly lower than “inter-pair” ( P = 0.003; Fig. 2 b), suggesting a skewed tendency of microbiome transmission. The distribution of the normalized genetic distance (nGD) aligned with the same patterns (Fig. 2 c). Based on the evidence of generational transmission in the studied settings, the reprogrammed offspring microbiome (Fig. 1 k) might be a consequence of the premeditated maternal microbiome. These trends were consistent when comparisons between mothers and pups were expanded to those between treatment groups (Supplementary Fig. 2c and d). Although vertical inheritance occurs regardless of treatment, it is still crucial to determine whether the addition of LGR-1 can alter the transmission rate of specific taxa. As shown in Fig. 2 d, LGR-1 led to a variety of transmission drifts, either towards intra-pair or inter-pair, depending on the specific strain under study. This indicates that LGR-1 serves as a robust driving force, capable of reshuffling the transmission trajectory, and subsequently accounting for a restructured microbiome in offspring mice. Phylogenetic analysis provided a comprehensive picture of taxa with the highest abundance. A subset of microbes displayed distinct abundance levels in the presence or absence of LGR-1. Among the taxa identified by StrainPhlAn, nTR (normalized transmission rate) was then calculated, indicating variable degrees of transmission across all samples or LGR-1-treated samples (Fig. 2 e). As two representative taxa due to their high prevalence among groups, the phylogenetic trees of Muribaculum intestinale and Lactobacillus prophage Lj928 were shown in Supplementary Fig. 2e and f, with a focus on the mother-offspring pairs and treatment-group pairs. Besides, we also showcase the phylogenetic similarities of certain reference strains, underpinning their preferences in either generation (Supplementary Fig. 2g). These microbes exemplify the existence and plasticity of vertical transmission. Collectively, microbiome transmission persists and evolves in the context of LGR-1 intervention. The orchestrated microbiome mediates the prophylactic effect of LGR-1. To investigate whether microbiome variation is pivotal for social interaction, FMT (fecal microbiota transplant) 25 was conducted in either dams or pups (Fig. 3 a). The effectiveness of the transplant was validated through 16S rRNA sequencing, indicating that recipients from the LGR-1-treated donors harbored microbiome with close proximity to their donors rather than the socially-deficient conspecifics (Fig. 3 b). In terms of behavior, mice engrafted with LGR-1-treated microbiome exhibited marked improvement in sociability ( P < 0.001 for mFMT, P < 0.001 for oFMT; Fig. 3 c) and social novelty ( P = 0.002 for mFMT, P < 0.001 for oFMT; Fig. 3 d). Due that maternal microbiome transfer also brought the comparable benefits to their offspring, maternal microbiome remodeling is not the isolated event but has vertical influence on social behaviors. Considering the temporal separation of intervention (pregnancy) and effect (postweaning), alternative routes may come into play at different stages. IL-17A was previously implicated in the crosstalk between maternal microbiome and fetal brain, promoting the autistic-like phenotype 12 . In the current settings, however, we found no significant variations in the IL-17A levels in maternal serum of autistic models or LGR-1-pretreated mice (Supplementary Fig. 3a), suggesting that IL-17A might not be the prime target for LGR-1 on dams. Additionally, given the possibility of direct inheritance of LGR-1 by the offspring, our metagenomic data did not find evidence of Limosilactobacillus rhamnosus in some LGR-1-pretreated pups (Supplementary Fig. 3b). Furthermore, when LGR-1 was supplemented during lactation, the pup microbiome was not altered in a manner comparable to the in utero treatment (Supplementary Fig. 3c-e). Next, we cross-fostered pups with dams from different groups (Fig. 3 e). Along with the restructured microbiome (Fig. 3 f), most pups exhibited social properties resembling their foster mothers, both in sociability (Fig. 3 g) and social novelty (Fig. 3 h). The data underscored the significance of maternal microbiome in shaping social performance and identified lactation as a critical period for conveying the benefits of early LGR-1 use. However, it’s worth noting that not all phenotypes were consistent with those of foster dams, as exemplified by the sociability of pups with their biological mothers remedied with LGR-1 (Fig. 3 g). This might suggest that maternal microbiome should not be considered as the sole contributor to the offspring behavior, and that LGR-1 does not function solely through orchestrating maternal microbiome. In summary, our findings indicate a mechanistic link between maternal microbiome and the behavioral benefits brought by LGR-1. Vaginal microbiota modulated by LGR-1 contributes to the lasting effect of the microbiome. We next attempted to decipher other LGR-1-related factors responsible for the altered offspring microbiome and behavior. As LGR-1 originally inhabits in the urogenital system 26 , and vaginal bacteria are considered the seeding microbiome of newborns 27 , roles of vaginal microbiota were then investigated in the studied remediation. Firstly, maternal vaginal microbiota was sequenced prior to parturition in response to LGR-1 addition. As revealed by Fig. 4 a, VPA injection dampened the dominant status of lactobacillus, a tide further turned by the continuous impact of LGR-1. In the aspect of compositions, LGR-1 caused a dominant presence of Lb. rhamnosus in the murine vagina (Fig. 4 b), a finding consistent with the notion of vagina-rectal communication 28 . To further test the importance of vaginal microbiome, we interrupted the LGR-1 intake at E14 (Embryonic day 14), a week preceding delivery (Fig. 4 c). As a result, the test mice were unable to normally socialize with the resident mice in a three-chamber trial, albeit administered with LGR-1 ( P = 0.07; Fig. 4 d). In the event of social novelty, the halted treatment also abrogated the benefits brought by LGR-1 ( P = 0.879; Fig. 4 e). By analyzing the offspring microbiome, we found that pausing LGR-1 led to a disparate bacterial community in pups’ intestine (Fig. 4 f). Therefore, the LGR-1-modifying vaginal microbiota contributes to the offspring’s intestinal microbiome, as well as the resulting social behavior. By adding LGR-1 during lactation (Supplementary Fig. 3c), the impact of vaginal microbiota on the offspring’s microbiota can be artificially bypassed. In this case, LGR-1 lost its ability to rescue the social deficits ( P = 0.014 for sociability with a preference to the inanimate object, P = 0.871 for social novelty), in contrast with the prenatal administration (Fig. 4 g and h). Along with the taxa distribution at the phylum and genus level (Supplementary Fig. 3d and e), PCA also indicates that LGR-1 led to the distinct offspring microbiota when added in lactation instead of gestation (Fig. 4 i), during which no roles of vaginal microbiota were involved. In summary, the LGR-1-modifying vaginal microbiota plays part in the offspring’s microbiome and social benefits. LGR-1 has impact on behavior by modulating Akkermansia in offspring. To further delineate the compositional properties of “good” microbiome modified by LGR-1, the taxa with the highest fold changes were ranked and shown in Fig. 5 a. In this matrix, only taxa with the “reversal mode”, which abundance was partially restored by LGR-1, were incorporated. In contrast to the LGR-1-pretreated group, Akkermansia muciniphila (AKK) was highly enriched in the VPA-exposed mice. In light of its importance on the central nervous system 29 , AKK was selected for further studies. Of note, the offspring abundance of AKK displayed a trend inverse to that of the maternal Lb. rhamnosus in the dam-pup pairs revealed by the metagenomic sequencing (Fig. 5 b). Moreover, AKK was found lower in the offspring of the recipients engrafted with the LGR-1-modifying microbiome ( P = 0.005; Fig. 5 c), suggesting a marked generational correlation. Besides, other candidate lactobacillus strains did not generate the comparable AKK alterations (Supplementary Fig. 4a). Next, we investigate if AKK is associated with the autism-like behavior. Tetracycline can be used to decrease AKK in murine intestine 30 . Our results showed that administering tetracycline at a dose of 3 g/l indeed altered the microbiome of BTBR mice, reducing AKK levels close to zero (Fig. 5 d). Consequently, AKK elimination drove the mice to behave more socially and less repetitively than their defective conspecifics, reversing the animal performance to a varying extent ( P < 0.001 for sociability, P = 0.049 for stereotypies between “BTBR + Tet” and “BTBR”; Fig. 5 e and f). In contrast, the addition of AKK did not yield the recovery effect (Fig. 5 e and f, Supplementary Fig. 4b-d). Additionally, tetracycline acted in a dose-dependent manner (Supplementary Fig. 4e), likely due to its complicated impact on the overall microbiome. AKK was then administered to the male pups pretreated with LGR-1 at early life (Fig. 5 g). As a result, social deficits were partly reproduced by the postweaning effect of this microbe ( P < 0.001 for sociability, P = 0.313 for social novelty; Fig. 5 h and i), indicating that the LGR-1-AKK axis may play roles in modulating autism-like behaviors. The inability to cause lesions to sociability (Fig. 5 h) might indicate a dominant role of LGR-1 over AKK in this functional aspect. Collectively, the LGR-1’s social effect is related to the level of AKK in offspring. AKK influences autism-like behavior through the immune-brain pathway. Finally, to interrogate how AKK influences autism-like symptoms, we attempted to dissect the gut-brain axis involved in the studied intervention. The metagenomic sequencing data showed that LGR-1 modified the abundance of a list of carbohydrate-active enzymes (Supplementary Fig. 5a) and increased the gene copies of butyrate-related enzymes in microbial genomes (Supplementary Fig. 5b), suggesting potential roles of the microbe-derived metabolites. Based on it, we first performed the metabolomic analysis targeting the short chain fatty acids. As revealed by the results (Fig. 6 a), the addition of AKK reversed the LGR-1-mediated enhancement of 2-methylbutyrate and isovaleric acid. Since these BCFAs (branched-chain fatty acids) were recently found to exert anti-inflammatory effects 31 , the immune route was further examined with respect to its roles in the studied gut-brain axis. Immune infiltration, indicative of a compromised intestinal barrier, was observed in PE-stained colon tissues of BTBR mice. This lesion was mitigated by the prophylactic addition of LGR-1 (Fig. 6 b). Notably, when AKK was further administered to the offspring mice, there appeared a marked propagation of goblet cells, resulting in a new form of aberrations. This observation could be relevant to the mucin-degrading property of AKK 32 , while goblet cells are known to produce mucins to compensate for the loss. To further examine the immune status beyond the host-microbe interface, we determined the changes of T helper cells in spleens in response to the LGR-1/AKK treatment. According to the flow cytometric data, LGR-1 inhibited the differentiation of CD4 + IL-17 + cells in BTBR mice, which was then partly reversed by supplementing Akkermansia strains (p = 0.011, Fig. 6 c, d), an observation different from the changes in pregnant dams 12 . In addition, the differentiated Th1 cells (CD4 + IFNγ + ) were also decreased with the prenatal use of LGR-1, but no significant influence was detected after postnatal colonization of AKK (p = 0.447, Supplementary Fig. 5c, d). Consistent with the gut-associated and systemic immunity, brain immunity was also sensitive to signals from gut microbiota. In the prefrontal cortex, the microglial activation in BTBR mice was partly inhibited by the prenatal addition of LGR-1, and the favorable effect was then postnatally attenuated by AKK (Fig. 6 e, f), suggesting that immune route was intensively involved in the studied gut-brain crosstalk. Microglial activity is tightly associated with neuronal function. Consequently, the prefrontal E/I ratio was measured in response to treatment of LGR-1 and AKK (Fig. 6 g, h; Supplementary Fig. 5e-h). In alignment with previous findings, the frequency of mIPSC was increased by prenatal administration of LGR-1, a change subsequently reversed by postnatal AKK (p = 0.035, Fig. 6 h). As mEPSC remained unaltered throughout this process, the E/I balance was assumed to be re-disturbed upon AKK’s colonization. To further explore the influence of CD4 + IL17 + cells on the studied gut-brain communication, an antibody binding to IL-17A, namely Secukinumab, was used to abrogate the functioning of IL-17A (Fig. 6 i). When Secukinumab was intraperitoneally injected into the mice treated with the prenatal LGR-1 and postnatal AKK, the behavioral impairment caused by AKK was significantly attenuated, as evidenced by the repetitive (p < 0.001, Fig. 6 j) and social behaviors (p < 0.001, Fig. 6 k). This provides a piece of evidence that IL-17A plays essential roles in conveying the gut-derived signals into the brain, in the context of LGR-1/AKK intervention. Taken together, AKK has impact on autism through the immune-brain pathways. Discussion Can autism be prevented in pregnancy? Here we report that prenatal intake of LGR-1 reduced the risk of autism-like symptoms. According to the previous findings, either autism-relevant traits 33 or gut microbiome 24 persisted across generations, which implied the significance of maternal microbiome on the autistic behaviors. This was further supported by the finding that ASD-like symptoms were induced in the offspring by applying antibiotics to the maternal microbiome 14 . A strategy with opposite directions was used here to confer benefits on the autistic-like animals, discovering that a specific strain, LGR-1, is potent in improving the relevant social deficits when applied at early life. Moreover, vertical transmission of microbiome was found to play important roles in this preventative paradigm, emphasizing the generational axis of LGR-1/AKK (Fig. 7 ). To our knowledge, this represents a new instance of using a single probiotic strain to, at least in part, prevent the occurrence of autism-like behaviors. Given the obvious merits of prophylactic approach, this could provide an intriguing choice for combatting the autism-related complications. Pregnancy is a critical period for the onset of autism. Viral infection in pregnancy, usually accompanied by immune activation, has been employed in mice to establish autistic models 18 , 34 . This may pinpoint the likeliness of preventing autism as early as pregnancy. Accordingly, this notion has been examined in several studies: resveratrol corrected the ASD-like abnormalities caused by VPA 35 ; antibiotic treatment before and during pregnancy attenuated the social deficits caused by maternal immune activation 36 . Despite these advances, this strategy is still at its infancy, scarcely involving the use of the microbe-based methods. Notably, the gut microbiome in gestation is highly dynamic and unique, rendering it susceptible to intervention with a premeditated orientation 37 . Accordingly, the present research shows that maternal microbiome can also be targeted for the positive moderation, generating the desirable behavioral outcomes rather than negative effects. However, it remains a challenging task to define a “good maternal microbiota”: although LGR-1 can induce a collection of changes among commensals, it did not necessarily generate a singular/exclusive combination to alleviate the autism-related performance. The “ bona fide ” determinant could be the overall balance of the maternal bacteria during childbirth, accounting for the “normal” seeding and the early-life development of offspring microbiome. A variety of probiotic strains have been suggested to improve autistic symptoms, exemplified by Bacteroides fragilis 18 , and Limosilactobacillus reuteri 11 . While these microbes show robust activities in ameliorating autism-related behavior, the current study uses a single bacterial strain preemptively to achieve the prophylactic effect. Importantly, this conferred protection varies depending on the specific strain used (Fig. 1 d and e), indicating the remarkable strain selectivity. It’s intriguing to speculate that this phenomenon might be associated with the unique properties of LGR-1, a strain initially isolated from distal urethra and then known to be probiotic in the gastrointestinal tract 26 . Nonetheless, the key structural determinant that empowers LGR-1 to modulate social behavior remains poorly understood, warranting future investigations. But it appears unlikely to ascribe the behavioral benefits to the inheritance of LGR-1 per se , as its host species Lb. rhamnosus was not detected in the feces of certain offspring, and LGR-1 did not improve mice behaviors during lactation (Fig. 4 g and h). Based on this analysis, we propose that the molecular determinant of LGR-1 is likely utilized to moderate the composition of maternal commensals, as well as to reprogram their tendency of being transmitted to the next generation. The time window is critical in determining the effect of an intervention. For instance, exposure to taurin improved the social performance of BTBR mice 10 . But this only occurred during pregnancy and lactation, while administration at later life did not yield the posivie effect. In the present study, LGR-1 was administered exclusively during the prenatal period, achieving early-life intervention of autism-like complications. Contrarily, the main effect was elicted at the postnatal stage including lactation, which may be attributed to the ongoing microbiome-reshaping impact of the maternal microbiome, as demonstrated by cross-fostering trails (Fig. 3 g and h). Therefore, while the prenatal route can not be entirely ruled out, the effective period of LGR-1 is supposed to separate from its initial colonization. Given the current evidence, no definitive conclusions can be drawn as to whether other strains could exert a prophylactic effect via the similar mechanisms to LGR-1. The maternal gut is the largest source of commensal bacteria living in the GI tract of offspring 38 . The mechanisms driving this “vertical tranmission” are largely unclear, but it might be related to the persistence of maternal bacteria and their better adaptation to the intestinal niche compared to microbes acquired from other sources 24 . Considering the existence of microbiota inheritance, it provides a route to use maternal intervention to improve the offspring symptoms. This was achieved in this study, with its importance best elucidated in the maternal FMT trials (Fig. 3 c and d). However, it remains challenging to accurately predict the transmission profile for each taxonomic unit, due to the remarkable variations of the physiological settings. Furthermore, transmission is a well-defined process associated with the strain nature, rather than its relative abundance in a microbial community, an observation (data not shown) consistent with the previous report 39 . On the other side, microbiome transmission also has relevance with vaginal microbiota, as the majority of strain transmission occurred in babies delivered vaginally (74.39%), at a higher frequency than those delivered by C-section (12.56%) 40 . LGR-1 retains its capacity to modulate vaginal microbiota. Indeed, LGR-1 is the first strain thought to replenish the vaginal microbiota as the exogenous probiotic 41 . This capability is linked to its effect on social behavior, due to the importance of vaginal microbiota on the prevalence of ASD 42 , 43 . This notion was further substantiated by the present finding that LGR-1 acted, in part, by modulating the composition of vaginal microbiota. According to our data, the maternal vaginal microbiota, coupled with their gut microbiome, impacted the formation of offspring microbiome, probably by modulating their seeding and early-life development, which may serve as the crucial events relating to the autism-like phenotypes. This proposition also aligns with the theory that the pioneering microbes can drive the development of subsequent microbiota, which coincide with a critical time window of neurodevelopment 40 , 44 . Therefore, LGR-1 should be viewed as a unique strain, with modulatory activity on both populations of microbes present in the pregnant dams, constituting its major specificity of behavioral remediation. Akkermansia muciniphila is widely regarded as the next-generation beneficial microbe. It’s interesting to notice that this microbe is impacted by the “first generation probiotic”, lactobacillus (Fig. 5 a-c), through unknown routes in separate generations. AKK can be delivered to the progeny by vertical inheritance, a finding consistent with a previous study 30 . Remarkably, the two microbes showed opposite trends in abundance, indicating that LGR-1 might curb the ability of AKK to transmit and thrive in the next generation. Another intriguing observation here is the unexpected high existence of AKK in the autism-like animals, which did not adhere to the positive effect of this “beneficial microbe”. In fact, this is not a rare case, as AKK was shown to be enhanced, albeit with conflicting data, in the intestines of both animal models and some ASD patients 10 , 45 , 46 , which is supposedly associated with the mucin-degrading activity of AKK. In a general sense, AKK can facilitate the turnover of mucins, but its excessive growth might lead to the injuries of the gut barrier. This hypothesis was supported by our finding that, upon AKK administration, an excess of goblet cells were observed at the gut-bacteria interface (Fig. 6 a), suggesting a compensatory response towards damage. Another possibility is the involvement of AKK with inflammation. While AKK is traditionally considered anti-inflammatory, it also promotes the maturation of leukocytes, and participates in the chemotaxis and complement cascade 47 . In an instance, AKK exacerbated the salmonella-mediated gut inflammation 48 . Based on its discrete performance, AKK’s action is not predisposed, but likely dependent on the physiological context and dosage used. Besides, AKK is unlikely to act independently to influence behavior, but help building a specific microbiome that serve its function. While the focus of this study is the generational mechanism of prophylactic effect, the detailed gut-brain routes of Akkermansia , in terms of the T cell trafficking or oxytocinergic system, should be explicitly dissected in the following investigations. This study has some limitations: (I) one of the challenges with the gut-brain axis is the difficulty of establishing the cause-and-effect relationship. In this case, while LGR-1 is shown to alleviate ASD-like symptoms, considerations of other upstream factors such as diet and gene cannot be excluded; (II) while animal models are useful to demonstrate key characteristics of ASD, they may not fully reflect the complexity of human behavior and brain circuits. Therefore, clinical trials are required in future to validate the effects of LGR-1 on human subjects; (III) ASD is no longer considered the dysfunction of a specialized brain area but the wide-ranging reorganization. Gut microbiome is quite unlikely to affect all brain areas of interest in a desired manner, which means it probably cannot resolve all the ASD-related aberrations; (IV) the molecular basis for strain specificity remains unclear, which may be associated with the key determinants of LGR-1 in modulating social behavior. Further research is needed to address these difficulites. Collectively, given the lack of amenable medications of ASD, gut microbiome has been increasingly appreciated to remediate autism-related deficits. In this study, manipulating the maternal microbiome using a single strain, LGR-1, is effective in attenuating autism-like behaviors in newborn animals. This strategy leverages the non-genetic yet inheritable feature of gut microbiome to maneuver generational effect. In conclusion, the microbe-based preventive approach is tested here, offering a promising avenue to address the challenges of ASD-like complications by tailoring the microbiome of pregnant mothers. Methods Study design. The objective of this study is to assess the prophylactic effect of LGR-1 on autism-like behaviors, and to define the underlying generational mechanisms focusing on vertical transmission of gut microbiome. Both VPA-exposed and idiopathic BTBR mice were used to establish the autism-like models. For the major paradigm, LGR-1 was fed to the pregnant mice by oral gavage at a dose of 10 9 /mice/d, starting from conception till parturition. The control groups were treated with saline/solvent following the same paradigm. Secukinumab was intraperitoneally injected into the mice at a dose of 10 mg/kg at 10 and 6 days prior to behavioral trials. Three-chamber test and reciprocal social interaction were used to assess the sociability of the test mice, and marble burying trial was used to assess their reciprocal behaviors. The behavioral tests were only conducted when male mice reached adulthood (7–9 weeks old). Electrophysiological recordings were used to assess the E/I balance in the prefrontal cortex. Microbiome samples were collected prior to the behavioral trials and then subjected for metagenomic sequencing for assessment of vertical transmission, or 16S-rRNA sequencing for other assessments. Mice with obvious health problems were excluded from the relevant test and analysis, and no data was excluded unless the mice refused to explore in the arena, too aggressive or always tended to climb upwards along the wall or cages during the behavioral test. Sample size was determined by the availability of pregnant dams and reagents and was not predetermined, with at least two different litters used in each treatment group. The order of measurements was randomized towards treatment groups. In all experiments, data obtained in individual mice was considered as one biological replicate. A minimum of three biological replicates were used in each experiment. Behavioral experiments were replicated at least twice with independent cohort of mice, along with most microbiome sequencing and other analysis. Data collection was not halted until the experiments were completed. Mice. C57BL/6J mice were housed under SPF (specific pathogen-free) conditions at a 12-hour light/dark cycle per day and had access to food and water ad libitum . These mice were originally obtained from the animal facility of Anhui Medical University and bred in-house. The VPA-treated mice were generated by administering a single dose (400 mg/kg) of sodium valproate (CSNpharm, Chicago, USA) to the pregnant C57BL/6J mice at embryonic day 12.5 (E12.5) 49 . The BTBR T + Itpr3 tf /J (BTBR) inbred mice, known to display an inherent autistic-like phenotype, were purchased from The Jackson Laboratory (Bar Harbor, USA). They were housed in the cycle of 14-hour light and 10-hour dark with ad libitum access to food and water. Each dam gave birth to an average 8 ~ 9 offspring. The total male offspring were randomly regrouped 3 ~ 6/cage at weaning, without cohousing non-littermates. For transmission studies, only one litter of offspring was reserved for each dam, and one male pup was randomly selected from it to establish the mother-pup pair, the feces of which were later used for metagenomic sequencing. At least two litters of male offspring were used for each treatment group in other experiments. Female mice were not used here due to the male prevalence of ASD, as well as the behavioral misrepresentation for females in the VPA-exposed 50 and BTBR models 51 . Throughout the trial, body weight and food/water consuming of mice were monitored, with any unhealthy or aberrant animals culled prior to test. All experiments were conducted following protocols approved by the Institutional Animal Care and Use Committee of Hefei University of Technology. Bacterial strains and culture. Lacticaseibacillus rhamnosus GR-1, GG and Limosilactobacillus reuteri RC-14 were obtained from our laboratory stocks. Cultures were maintained on MRS medium (Huankai, Guangzhou, China) in an anerobic chamber (HYQX-Ⅱ, Yuejin, Shanghai, China) with a gas mix of 10% hydrogen, 5% carbon dioxide and 85% nitrogen. The purity of the cultures was monitored by plating with serial dilutions. For inoculation, the lactobacillus strains were grown at 37 o C for 12 h. The bacteria were collected by centrifugation at 8 000 g for 5 minutes, washed twice with sterile PBS, and then suspended at a final concentration of 10 9 CFU/ml for further use. For LGR-1, the culture supernatant was filtered through 0.22 µm membrane filter and used as parabiotic. Akkermansia muciniphila DSM 22959 was purchased from Biobw (Beijing, China) and maintained in Brain Heart Infusion medium (Sigma-Aldrich, Shanghai, China) supplemented with 0.25 g/l mucin (Sigma-Aldrich, Shanghai, China). AKK was grown in the anerobic chamber for 48 h to obtain the bacterial samples. Bacterial supplementation. The pregnant dams received Lacticaseibacillus rhamnosus GR-1, GG, or Limosilactobacillus reuteri RC-14 at a concentration of 1 × 10 9 CFU per mouse/day, and the oral gavage was carried out solely during gestation. According to the metagenomic data (PRJNA1033296), the relative abundance of Lb. rhamnosus in the VPA-exposed dams was increased from 5.33 × 10 − 8 to 5.55 × 10 − 7 after LGR-1 treatment. For other paradigms, the postnatal administration of LGR-1 was conducted from weaning till the behavioral test (gavage through pups), or from birth to the first week of lactation respectively (gavage through dams). Male offspring received Akkermansia muciniphila DSM 22959 prepared under anaerobic conditions at a concentration of 1 × 10 10 CFU per mouse/day through daily oral gavage during PNW4-7. All bacterial supplementation was controlled by saline gavage. The bacterial viability was randomly checked and confirmed by plate counting. Drug treatment. For antibiotic administration, tetracycline hydrochloride (Bio Basic, Markham, Canada) was dissolved in 1% sucrose, resulting in final concentrations of 1.5 (L), 3 (M) and 10 g/l (H) respectively. The drug solution was sterilized with a 0.22 µm filter, and then supplied to the BTBR mice during PNW6 and 7 in their drinking water renewed every three days. For IL-17A signaling inhibition, Secukinumab (Ambeed, Beijing China) was dissolved in 0.9% saline, and i.p. injected into the mice treated with prenatal LGR-1 and postnatal AKK at a dosage of 10 mg/kg. The injection was conducted twice, specifically on 6 and 10 days before behavioral trials. Three-chamber trial. All behavioral tests were conducted on 7- to 9-week-old mice. Three-chamber trial was performed as previously described 52 . Briefly, the subject mouse was first habituated for 10 minutes in an empty 60 × 40 × 23 cm Plexiglass area formed by three interconnected chambers. Subsequently, an age/gender matched conspecific (Mouse 1) was placed into the left chamber, and the moving tracks of the subject mouse were recorded in the left and right chamber (inanimate object; Empty) in the following 10-min session (Supplementary videos were made from a bird’s-eye view). Sociability was then assessed by measuring the time spent by test mouse interacting with Mouse 1 compared to the inanimate chamber. Social novelty was evaluated in the third 10-min session, whereas a stranger (Mouse 2) was introduced to the right chamber, and the time distribution of the subject mouse was recorded and analyzed by ANY-maze (Stoelting, Wood Dale, USA) using the same methods. The arena was cleaned up between sessions. The human observers and analyzers were blinded to the treatment group. Marble burying test. Marble burying test was performed as previously described 11 . Briefly, the subject mouse was put into a 50 × 30 cm Plexiglass arena with a 5 cm-thick corncob bedding, with the surface covered by 24 regularly-spaced royal blue marbles. The mouse was allowed to freely explore inside the arena for 10 min. Subsequently, the mouse was removed, and marbles with at least two-thirds of their diameter obscured by bedding were counted as buried. The ratio of buried marbles was calculated to assess the severity of repetitive behavior. Fecal microbiota transplant. Fresh fecal samples were collected from donor mice and homogenized on ice in sterile PBS under aseptic conditions. The resulting slurry was spun at 700 g for 1 min at 4 o C. The supernatants were then collected for oral gavage in a dosage of 0.2 ml per recipient mouse. For offspring transplant, donor samples were collected from 8-week-old male mice exhibiting autistic traits or those treated with LGR-1 prenatally. The transplant was then performed on the 5-week-old mice with social deficits daily and continued for 3 weeks. For maternal fecal transplant, the pregnant dams approaching delivery were used as donors, and their fecal samples were transferred to the defective mothers throughout pregnancy. Cross-fostering experiment. Pups were cross-fostered at birth with the conspecifics born on the same day. The whole litters were removed from their biological mothers and gently introduced to a new cage with the foster mother. Pups from the socially-deficient mothers were cross-fostered to the LGR-1-intervening mothers, and vice versa. This experiment was controlled by cross-fostering pups to another mother within the same treatment group. 16S rRNA sequencing and analysis. 16S rRNA sequencing was performed as previously described 20 . Briefly, the fresh feces from male mice and female dams were collected before behavioral assays and delivery, respectively. Vaginal samples were collected by swabbing the vaginal sidewalls four times from pregnant dams on days approaching delivery, ensuring full contact with the swabs. All samples were then homogenized, subjected to DNA extraction using the HiPure Stool DNA Kit B (Magen Biotech, Guangzhou, China). The 16S rDNA V4 region was then amplified, subjected to library construction, and sequenced in the IonS5 TM XL platform (Thermo Fisher Scientific, Beijing, China). Data analysis was performed using the QIIME 2 (Quantitative Insights into Microbial Ecology) 53 . Chimeric sequences were detected using UCHIME algorithm, and reads were clustered into OTUs using the Uparse software (v7.0.1001, http://drive5.com/uparse/ ), with cutoff value set as 97% in similarity. Abundance data was normalized according to the sample with the fewest reads. In-house Perl scripts in QIIME 2 were used to analyze alpha-diversity (within samples) and beta-diversity (among samples). R (version 4.3.1) with the built-in function “prcomp” was used to perform Principal Component Analysis (PCA). Statistical significance of sample groupings was determined by adonis method. The graphical presentation was conducted using OriginLab 2024b. All 16S rRNA sequencing data are publicly available (PRJNA1032734, 1033214, 1033221, 1033224, 1146173 and 1033320). Metagenomic sequencing and analysis. Metagenomic sequencing was used to detect the microbiome transmission during LGR-1 intervention. A total amount of 1 µg DNA per sample was used as input material for library generation using NEBNext Ultra™ DNA Library Prep Kit for Illumina (NEB, Ipswich, USA). Briefly, the DNA sample was fragmented by sonication to a size of 350 bp, which was further end-polished, A-tailed and ligated with the full-length adaptor for Illumina sequencing, followed by PCR amplification. The resulting PCR products were purified using the AMPure XP system (Beckman Coulter Diagnostics, Brea, USA). The libraries were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified with qPCR. Amplified and barcoded libraries were then pooled in approximately equimolar ratios before being sequenced on an Illumina Hiseq platform 4000, generating pair-end reads. The raw reads (6377.41 ± 36.59 Mbp, Mean ± SEM) were examined, with low-quality and unwanted reads (0.168 ± 0.017%, Mean ± SEM) filtered by Bowtie2.2.4 ( http://bowtiebio.sourceforge.net/bowtie2/index.shtml ). MEGAHIT (v1.0.4-beta) was employed to assemble the clean data. For gene prediction, MetaGeneMark (V2.10, http://topaz.gatech.edu/GeneMark/ ) was run to filter out sequences shorter than 100 nucleotides from the predicted results with default parameters. Subsequently, non-redundant, unique initial genes (Unigene) were derived with the assistance of the CD-HIT software (V4.5.8, http://www.bioinformatics.org/cd-hit ). Taxonomy prediction was performed using DIAMOND software (V0.9.9, https://github.com/bbuchfink/diamond/ ), via blasting the Unigenes against sequences of Bacteria extracted from the NR database (Version: 2018-01-02, https://www.ncbi.nlm.nih.gov/ ). The functional gene pathways were profiled by blasting Unigenes against functional databases including KEGG ( http://www.kegg.jp ) and CAZy ( http://www.cazy.org ). The best Blast Hit was used for subsequent analysis. To perform further data-mining at the species- and strain-level, StrainPhlAn (an integral part of MetaPhlAn, https://github.com/biobakery/MetaPhlAn ) was employed to identify a tandem of conserved and unique markers, enabling the retrieval of taxonomic profiles and inter-sample genetic distances by detecting single nucleotide polymorphisms (SNPs) for each identified taxon. 12 taxa were then recovered with a minimum prevalence of 16 across all samples. The genetic distance for each taxon was then derived according to the RAxML algorithm. For one specified taxon, genetic distances between the tested dam-pup pairs were counted, while normalized genetic distances (nGDs) were calculated as data normalized by the median of all genetic distances. To depict the strain distribution within samples and groups, some reference strains of the subject species were incorporated into phylogenetic analysis and comparisons. The cutoff value for strain similarity is set as the lowest 15% of all genetic distances incorporated. The maximum likelihood phylogenetic tree of the top 100 abundant species was established by PhyloPhlAn ( https://github.com/biobakery/phylophlan ). All relevant phylogenetic data was subjected to iTOL (interactive tree of life) 54 for graphical presentation. In terms of microbiome transmission rate, nTR (normalized transmission rate) was calculated as the ratio of inter-pair nGDs to the total (inter-pair plus intra-pair) nGDs. The metagenomic data is available at PRJNA1033296. Immunohistochemistry. Immunohistochemistry was performed as previously described with some modifications 20 . Briefly, mice were anesthetized and perfused with 10 ml 0.9% sodium chloride followed by 30 ml 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The separated brain tissues were post-fixed in 4% PFA at 4 o C for 24 h, and then cryoprotected in 30% sucrose till tissue deposition. Coronal slices (40 µm thick) were obtained from frozen tissue with a sliding blade microtome and rinsed in PBS containing 0.7% of Triton X-100 (PBSTX; Aladdin Scientific) for 8 h to penetrate the plasma membrane. Slices were blocked with 10% fetal bovine serum (FBS) for 2 h and then incubated in primary antibodies overnight. Primary antibodies were visualized using second antibodies rotating in the dark for 1 h at 4 o C. Subsequently, the nucleus was stained using DAPI Staining solution (1:5000; Biosharp, Hefei, China), rinsed and mounted with Antifade Mounting Medium (Beyotime Biotechnology, Shanghai, China). Fluorescent imaging and data acquisition were performed on a Nikon C2 Confocal Microscope. The primary antibody used here is Rb Anti-Iba1 (Abcam, AB_2636859) with a dilution of 1:400, and the second antibody is Cy3-goat-anti-rabbit IgG (1:200; Proteintech, AB_2890957). Metabolomic analysis targeting SCFAs (short-chain fatty acids). The stock solution of individual SCFAs (Novogene, Beijing, China) were mixed and prepared in SCFA-free matrix to obtain a series of SCFA calibrators. The fecal samples of various treatment groups were first resuspended with liquid nitrogen, homogenized with methanol (80%) and centrifuged at 11000 g for 10 min to remove the protein. The supernatant was then subjected to derivatization, dilution with 80% methanol and homogenization with 5 µl mixed internal standard solution. An ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) system (Vanquish Flex UHPLC-TSQ Altis, Thermo, Dreieich, Germany) was used to quantify SCFAs. Separation was performed on a C18 column (2.1 × 100 mm, 1.7 µm, Waters, Milford, USA). The mobile phase consisted of 10 mM ammonium acetate in water (solvent A) and acetonitrile: isopropanol (1:1). The mass spectrometer was operated in negative multiple reaction mode (MRM). Parameters were set as follows: ionspray voltage (-4500 V), sheath gas (35 psi), ion source temp (550°C), auxiliary gas (50 psi) and collision gas (55 psi). H&E staining. Hematoxylin and eosin (H&E) staining was performed as described previously 22 . Briefly, the colons were dissected from the anesthetized mice, with fat removed using aseptic forceps. The tissues were then fixed in 4% paraformaldehyde solution at 4 o C for 48 h, followed by soaking in liquid paraffin at 65 o C for 1 h and air-dried. The slices (5 µm thick) were obtained using a microtome, and subjected to gradient dehydration with 75, 80, 95 and 100% ethanol. The tissue sections were stained with H&E and observed with a light microscope (Eclipse 80i; Nikon, Tokyo, Japan). Flow cytometry. Spleens were dissected from the sacrificed mice and rinsed in Hanks' Balanced Salt Solution (HBSS; Thermo Fisher Scientific, Beijing, China). The excised and fat-free tissues were incubated with collagenase D (Thermo Fisher Scientific, Beijing, CHina), strained and filtered to prepare cell suspensions. For flow cytometry, the cells were stained with Zombie Aqua (1:1000; BioLegend), Anti-Mouse CD16/32 (TruStain FcX™, 1:200; BioLegend, AB_1574973), CoraLite594-conjugated-Anti-Mouse CD3 (1:200; Proteintech, 17A2, AB_3064914), and BV421-conjugated-Anti-Mouse CD4 (1:200; Proteintech, RM4-4, AB_3064915). For intracellular staining, cells were treated with PMA (50 ng/ml; Thermo Fisher Scientific), ionomycin (500 ng/ml; Thermo Fisher Scientific) and Brefeldin A solution (1Х; BioLegend) for restimulation. The cells were then permeabilized and stained with PE-conjugated-Anti-Mouse IFNγ (1:200; Proteintech, XMG1.2, AB_2883916) and PerCP-Cy5.5-conjugated IL-17A antibody (1:200; BioLegend, eBio17B7, AB_2565780) using Intracellular Fixation/Permeabilization Buffer (E-CK-A109; Elabscience). Flow cytometric analysis was performed on an FACSAria III (BD Biosciences, New Jersey, USA). All data were re-analyzed using FlowJo software (BD Biosciences, New Jersey, USA). Electrophysiological recordings. mEPSC recordings were performed as described previously with some modifications 20 . Briefly, coronal slices of mPFC were prepared (300 µm thick) and transferred to oxygenated artificial cerebrospinal fluid (ACSF), containing 119 mM NaCl, 2.5 mM KCl, 1 mM NaH 2 PO 4 , 1.3 mM MgCl 2 , 2.5 mM CaCl 2 , 26.2 mM NaHCO 3 and 11 mM glucose. The incubation was performed at 34 o C for 30 min and subsequently at 27 o C for 1 h. Afterwards, slices were placed in a recording chamber perfused constantly with ACSF at a flow rate of 2 ml/min. After 5 minutes of equilibration, the recording was initiated by identifying layer V pyramidal neurons in mPFC based on cell size and morphology. Patch pipettes (3 ~ 6 MΩ) were filled with an internal solution containing 110 mM potassium gluconate, 40 mM KCl, 10 mM HEPES, 3 mM MgATP, 0.5 mM Na 2 GTP and 0.2 mM EGTA. ACSF was supplemented with 1 µM tetrodotoxin and 250 µM picrotoxin to record mEPSC. In terms of mIPSC, the internal solution was replaced with 140 mM CsCl, 2 mM MgCl 2 , 5 mM EGTA, 10 mM HEPES, 0.36 mM Na 3 GTP and 4.39 mM Na 2 ATP. ACSF was added with 1 µM tetrodotoxin, 10 µM CNQX and 50 µM APV to sequester and record mIPSC. Recordings were low-pass filtered at 2 kHz and acquired using an Multiclamp 700B amplifier (Molecular Devices, San Jose, USA) in conjunction with pClamp 11 software (Molecular Devices, San Jose, USA). Only cells with access resistance 100 MΩ were selected for analysis. Visual inspection of detected signals was allowed to reject noise artifacts. Statistical analysis. Data were represented as mean ± SEM from at least three biological replicates with at least two independent experiments. Unless otherwise stated, statistical analyses performed include Student’s t-test, one-way ANOVA with Tukey post hoc analysis, or two-way ANOVA with Sidak post hoc analysis. The number of samples ( n ) and P values were presented in the figure legends. P values were FDR-corrected when multiple comparisons (> 3) were performed. Analysis was performed and presented using GraphPad’s Prism (version 8.0) and OriginLab (version 2024b). Paired t-test was used to compare nGDs for the specified taxon. For the three-chamber experimental data, paired t test (two groups, Mann-Whitney U test for not-normally distributed data) or two-way ANOVA (> 3 groups) with post hoc Sidak comparisons were performed to analyze the same mouse’s traces in the left and right chambers. Unpaired t-test was applied in other comparisons between two groups. One-way ANOVA was performed when 3 or more treatment groups were involved in the statistics except the 3-chamber trial. All statistical analysis for 16S rRNA data was performed with QIIME 2. Alpha- and beta- diversity were analyzed with the in-house Perl scripts, while PCA was performed using the R software (version 4.3.1) with the built-in function “prcomp”. For metagenomic analysis, DIAMOND was conducted to indicate enrichment in pathways of KEGG and CAZy. StrainPhlAn was used to detect species or strains based on SNPs across most treatment samples. The genetic distances were deduced based on RAxML algorithm, which were further normalized and compared through paired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001 were considered statistically significant. Declarations Competing interests The authors declare no competing interests. Author Contribution H.L.W. and Y.X. conceived project and designed the study; R.Y., J.X. and Y.X. conducted most experiments and analyzed the sequencing data; C.H. performed the electrophysiological trials and the relevant analysis; F.Z. and T.W. conducted some animal trails and did the relevant analysis; R.K. aided in the animal studies; J.X. aided in the data visualization; X.G. conducted the functional test of LGR-1 and supervised the research; Y.X. and R.Y. summarized data and generated figures; Y.X. and H.L.W. wrote the manuscript. All authors reviewed and approved the manuscript. Acknowledgements This work was supported by National Natural Science Foundation of China (no. 81673624), Fundamental Research Funds for the Central Universities (no. JZ2020HGTB0053), and Anhui Provincial Key Research and Development Plan (no. 201904e01020001). We thank Dr. V. Pedicord and Dr. S. Suyama from University of Cambridge for the constructive advice to improve this manuscript; Prof. H. Tao, Prof. G. Liu and Dr. Z. Liu from Hefei University of Technology for the utility of animal facility; Dr. N. Bi for the technological advice for the electrophysiological recordings. Data Availability Metagenomic and 16S rRNA-seq results have been deposited in the ncbi database, assigned with accession number of PRJNA1033296 (metagenomics), PRJNA1033320, 1032734, 1033214, 1033221, 1146173 and 1033224 (16S rRNA-seq); Unprocessed data is deposited in the Mendeley database assigned with URL of “https://doi.org/10.17632/85vrrttt4y.1”. Correspondence and requests for materials should be addressed to Yi Xu or Hui-Li Wang. References Cryan JF, et al. The Microbiota-Gut-Brain Axis. Physiol Rev 99, 1877–2013 (2019). Maenner MJ, et al. Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2020. MMWR Surveill Summ 72, (2023). Shao E, et al. TAU ablation in excitatory neurons and postnatal TAU knockdown reduce epilepsy, SUDEP, and autism behaviors in a Dravet syndrome model. Sci Transl Med 14, eabm5527 (2022). Wang T, et al. De novo genic mutations among a Chinese autism spectrum disorder cohort. Nat Commun 7, 13316 (2016). Zhang Y, et al. Genetic evidence of gender difference in autism spectrum disorder supports the female-protective effect. Transl Psychiatry 10, 4 (2020). Sgritta M, et al. Mechanisms Underlying Microbial-Mediated Changes in Social Behavior in Mouse Models of Autism Spectrum Disorder. Neuron 101, 246–259 e246 (2019). Lou M, et al. Deviated and early unsustainable stunted development of gut microbiota in children with autism spectrum disorder. Gut, (2021). Yap CX, et al. Autism-related dietary preferences mediate autism-gut microbiome associations. Cell 184, 5916–5931 e5917 (2021). Coley-O'Rourke EJ, Hsiao EY. Microbiome alterations in autism spectrum disorder. Nature Microbiology 8, 1615–1616 (2023). Sharon G, et al. Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. Cell 177, 1600–1618 e1617 (2019). Buffington SA, Di Prisco GV, Auchtung TA, Ajami NJ, Petrosino JF, Costa-Mattioli M. Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring. Cell 165, 1762–1775 (2016). Choi GB, et al. The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. Science 351, 933–939 (2016). Kim S, et al. Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring. Nature 549, 528–532 (2017). Lebovitz Y, et al. Lactobacillus rescues postnatal neurobehavioral and microglial dysfunction in a model of maternal microbiome dysbiosis. Brain Behav Immun 81, 617–629 (2019). Qi Z, et al. A Novel and Reliable Rat Model of Autism. Front Psychiatry 12, 549810 (2021). Alsayouf HA, Talo H, Biddappa ML, De Los Reyes E. Risperidone or Aripiprazole Can Resolve Autism Core Signs and Symptoms in Young Children: Case Study. Children (Basel) 8, (2021). Lord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. Lancet 392, 508–520 (2018). Hsiao EY, et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451–1463 (2013). Tabouy L, et al. Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders. Brain Behav Immun 73, 310–319 (2018). Wang T, et al. Gut microbiota shapes social dominance through modulating HDAC2 in the medial prefrontal cortex. Cell Rep 38, 110478 (2022). Petrova MI, Reid G, Ter Haar JA. Lacticaseibacillus rhamnosus GR-1, a.k.a. Lactobacillus rhamnosus GR-1: Past and Future Perspectives. Trends Microbiol 29, 747–761 (2021). Gu X, et al. Probiotic Lactobacillus rhamnosus GR-1 supplementation attenuates Pb-induced learning and memory deficits by reshaping the gut microbiota. Front Nutr 9, 934118 (2022). Yu Y, et al. Changes to gut amino acid transporters and microbiome associated with increased E/I ratio in Chd8(+/-) mouse model of ASD-like behavior. Nat Commun 13, 1151 (2022). Ferretti P, et al. Mother-to-Infant Microbial Transmission from Different Body Sites Shapes the Developing Infant Gut Microbiome. Cell Host Microbe 24, 133–145 e135 (2018). De Palma G, et al. Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice. Sci Transl Med 9, (2017). Petrova MI, Reid G, Ter Haar JA. Lacticaseibacillus rhamnosus GR-1, a.k.a. Lactobacillus rhamnosus GR-1: Past and Future Perspectives. Trends Microbiol 29, 747–761 (2021). Dominguez-Bello MG, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 107, 11971–11975 (2010). Reid G, et al. Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 35, 131–134 (2003). Blacher E, et al. Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature 572, 474–480 (2019). Ansaldo E, et al. Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis. Science 364, 1179–1184 (2019). Taormina VM, Unger AL, Schiksnis MR, Torres-Gonzalez M, Kraft J. Branched-Chain Fatty Acids-An Underexplored Class of Dairy-Derived Fatty Acids. Nutrients 12, (2020). Derrien M, Vaughan EE, Plugge CM, de Vos WM. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol 54, 1469–1476 (2004). Meltzer A, Van de Water J. The Role of the Immune System in Autism Spectrum Disorder. Neuropsychopharmacology 42, 284–298 (2017). Shin Yim Y, et al. Reversing behavioural abnormalities in mice exposed to maternal inflammation. Nature 549, 482–487 (2017). Juybari KB, et al. Sex dependent alterations of resveratrol on social behaviors and nociceptive reactivity in VPA-induced autistic-like model in rats. Neurotoxicol Teratol 81, 106905 (2020). Estes ML, McAllister AK. Brain, Immunity, Gut: "BIG" Links between Pregnancy and Autism. Immunity 47, 816–819 (2017). Nuriel-Ohayon M, Neuman H, Koren O. Microbial Changes during Pregnancy, Birth, and Infancy. Front Microbiol 7, 1031 (2016). Li W, et al. Vertical Transmission of Gut Microbiome and Antimicrobial Resistance Genes in Infants Exposed to Antibiotics at Birth. J Infect Dis 224, 1236–1246 (2021). Yassour M, et al. Strain-Level Analysis of Mother-to-Child Bacterial Transmission during the First Few Months of Life. Cell Host Microbe 24, 146–154 e144 (2018). Shao Y, et al. Stunted microbiota and opportunistic pathogen colonization in caesarean-section birth. Nature 574, 117–121 (2019). Petrova MI, et al. Comparative Genomic and Phenotypic Analysis of the Vaginal Probiotic Lactobacillus rhamnosus GR-1. Front Microbiol 9, 1278 (2018). Bokulich NA, et al. Antibiotics, birth mode, and diet shape microbiome maturation during early life. Sci Transl Med 8, 343ra382 (2016). Curran EA, et al. Research review: Birth by caesarean section and development of autism spectrum disorder and attention-deficit/hyperactivity disorder: a systematic review and meta-analysis. J Child Psychol Psychiatry 56, 500–508 (2015). Sharon G, Sampson TR, Geschwind DH, Mazmanian SK. The Central Nervous System and the Gut Microbiome. Cell 167, 915–932 (2016). Alamoudi MU, Hosie S, Shindler AE, Wood JL, Franks AE, Hill-Yardin EL. Comparing the Gut Microbiome in Autism and Preclinical Models: A Systematic Review. Front Cell Infect Microbiol 12, 905841 (2022). Liu F, Li J, Wu F, Zheng H, Peng Q, Zhou H. Altered composition and function of intestinal microbiota in autism spectrum disorders: a systematic review. Transl Psychiatry 9, 43 (2019). Everard A, et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 110, 9066–9071 (2013). Belzer C, de Vos WM. Microbes inside–from diversity to function: the case of Akkermansia. ISME J 6, 1449–1458 (2012). Lim JS, Lim MY, Choi Y, Ko G. Modeling environmental risk factors of autism in mice induces IBD-related gut microbial dysbiosis and hyperserotonemia. Mol Brain 10, 14 (2017). Kazlauskas N, Seiffe A, Campolongo M, Zappala C, Depino AM. Sex-specific effects of prenatal valproic acid exposure on sociability and neuroinflammation: Relevance for susceptibility and resilience in autism. Psychoneuroendocrinology 110, 104441 (2019). DiLiberto E, Phatarpekar S, Theodorakis K, Chadman KK. Does the stranger mouse strain matter to female BTBR mice? Behavioural Brain Research 437, 114132 (2023). Reed MD, et al. IL-17a promotes sociability in mouse models of neurodevelopmental disorders. Nature 577, 249–253 (2020). Caporaso JG, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 335–336 (2010). Ciccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P. Toward automatic reconstruction of a highly resolved tree of life. Science 311, 1283–1287 (2006). Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterials.pdf VideoS1.mp4 VideoS2.mp4 VideoS3.mp4 VideoS4.mp4 Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in npj Biofilms and Microbiomes → Version 1 posted Editorial decision: Revision requested 19 Mar, 2025 Reviews received at journal 15 Mar, 2025 Reviews received at journal 10 Mar, 2025 Reviews received at journal 08 Mar, 2025 Reviewers agreed at journal 26 Feb, 2025 Reviewers agreed at journal 23 Feb, 2025 Reviewers agreed at journal 21 Feb, 2025 Reviewers invited by journal 21 Feb, 2025 Editor assigned by journal 15 Feb, 2025 Submission checks completed at journal 06 Feb, 2025 First submitted to journal 30 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5930312","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":411887711,"identity":"4bb95794-40ba-420d-b212-e8783c00fd02","order_by":0,"name":"Ruili Yang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Ruili","middleName":"","lastName":"Yang","suffix":""},{"id":411887712,"identity":"96a5c4fe-2f00-4148-9acd-c05f4eea5146","order_by":1,"name":"Jinchun Xu","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jinchun","middleName":"","lastName":"Xu","suffix":""},{"id":411887714,"identity":"b055d71e-58c7-419a-aa19-ac9a8eaa3a96","order_by":2,"name":"Yi Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYJCCAx8b5MAMCaK1HJzZYMzAQ5IWZl6StBjcyDE8bLvDIHE/A/PB2zwMdnkEtUjOyDE4nHvGILGHgS3ZmochuZigFn4JkJa2P0AtPGbSPAwHEhsIaWEDabFsA9nC/404LWBbGMFaeNiI0yLZ86zgYG+bgXHPYTZjyzkGyYS1GBxP3vzhZ5uBbHt788MbbyrsCGthYOAwgNDMYBMIqwcC9gdEKRsFo2AUjIIRDAAyxzfxVwiMpQAAAABJRU5ErkJggg==","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xu","suffix":""},{"id":411887718,"identity":"b6aa4eb8-60b2-4a20-9133-3548f7890ea9","order_by":3,"name":"Chengqing Huang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chengqing","middleName":"","lastName":"Huang","suffix":""},{"id":411887720,"identity":"77d08331-833e-43af-a567-c104441d9241","order_by":4,"name":"Feng Zhu","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Zhu","suffix":""},{"id":411887721,"identity":"7ffd7ab1-5618-41a4-b49f-dc0778e5f5bf","order_by":5,"name":"Tian Wang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tian","middleName":"","lastName":"Wang","suffix":""},{"id":411887724,"identity":"e0e67a97-a2d1-455f-aa1a-296435f3828f","order_by":6,"name":"Rui Kong","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Kong","suffix":""},{"id":411887725,"identity":"56ee0a85-6b19-43a5-bc68-7fde37bcd435","order_by":7,"name":"Jie Xiao","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xiao","suffix":""},{"id":411887726,"identity":"aa04119b-e353-4d36-b209-3dcb27c23f5e","order_by":8,"name":"Xiaozhen Gu","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Xiaozhen","middleName":"","lastName":"Gu","suffix":""},{"id":411887727,"identity":"d6458be5-ad94-4900-a7d4-4841e13e8a73","order_by":9,"name":"Hui-Li Wang","email":"","orcid":"","institution":"Hefei University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Hui-Li","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-01-30 12:38:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5930312/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5930312/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41522-025-00808-5","type":"published","date":"2025-09-24T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75703230,"identity":"beb55300-c902-481f-8cb5-8b0633b1c660","added_by":"auto","created_at":"2025-02-07 09:49:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1128430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrenatal administration of LGR-1 mitigates the autism-like behaviors. a\u003c/strong\u003e Scheme of experimental design for the treatment of LGR-1. Pregnant mice received LGR-1 by oral gavage till parturition at a dose of 10\u003csup\u003e9\u003c/sup\u003e/mouse/d. Behavioral test was performed at postnatal week 8 (PNW8). \u003cstrong\u003eb\u003c/strong\u003e Schematic of the 3-chamber social interaction task. \u003cstrong\u003ec \u003c/strong\u003eRepresentative exploratory trajectories of mice in the 3-chamber test. (\u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003eg)\u003c/strong\u003e Social behavior in LGR-1-pretreated mice as assessed in 3-chamber test (\u003cem\u003en\u003c/em\u003e = 5-16). A VPA-exposed model (\u003cstrong\u003ed \u003c/strong\u003eand \u003cstrong\u003ee\u003c/strong\u003e) and an idiopathic BTBR model (\u003cstrong\u003ef\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e) were used to evaluate the effect of prenatal LGR-1 intake on autism-like behaviors. Sociability (\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e) was measured by the propensity of the subject mouse to spend time with another mouse (Mouse 1), as compared to time spent alone in empty chamber (Empty), while social novelty was represented by the propensity to spend time with a novel mouse (Mouse 2), rather than a familiar one (Mouse 1). \u003cstrong\u003eh\u003c/strong\u003e Repetitive behavior in LGR-1 treated mice as assessed in marble burying test (\u003cem\u003en\u003c/em\u003e = 8-14 per group). Marble burying index was calculated according to the percentage of marbles buried in the 10-min test. Only marbles obscured at least two-thirds with bedding were counted. (\u003cstrong\u003ei\u003c/strong\u003e and \u003cstrong\u003ej\u003c/strong\u003e) Frequency of mEPSC (\u003cstrong\u003ei\u003c/strong\u003e) and mIPSC (\u003cstrong\u003ej\u003c/strong\u003e) in neurons residing in medial prefrontal cortex (\u003cem\u003en\u003c/em\u003e = 17-28). BTBR mice pretreated with LGR-1 were subjected to electrophysiological recordings at PNW8-9, and the representative waveforms and histograms were exhibited with respect to each treatment group. \u003cstrong\u003ek\u003c/strong\u003e Principal component analysis (PCA) for intestinal microbiome of offspring prenatally exposed by respective strains or culture supernatant (\u003cem\u003en\u003c/em\u003e = 5-8 per trial). LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; LRC14, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e RC14; LGG, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG; VPA/V, VPA treatment; B, BTBR. All data are given as mean ± SEM. One-way ANOVA was used to compare data in marble burying test and electrophysiological recording; for three-chamber test involving two groups, statistical significance was determined by paired t test for normally distributed data and Mann-Whitney U test for not-normally distributed data; for three-chamber test involving more than two groups, two-way ANOVA with post hoc Sidak comparisons were conducted. ns, \u003cem\u003eP \u0026gt;\u003c/em\u003e 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Figure1medium.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/32aea134cb5116d7689b28c0.jpg"},{"id":75704439,"identity":"e506de1f-a7c8-4d9f-993b-e48f42663865","added_by":"auto","created_at":"2025-02-07 09:57:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1524439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePrenatal treatment alters offspring microbiome via vertical transmission. a \u003c/strong\u003ePrincipal component analysis (PCA) for maternal microbiome supplemented with LGR-1 during pregnancy (\u003cem\u003en = \u003c/em\u003e5-7). \u003cstrong\u003eb\u003c/strong\u003e Genetic distances between intra-pair and inter-pair. 12 microbial strains were recovered by StrainPhlAn from metagenomic data, with a minimum prevalence of 16 samples. The genetic distance for each strain was then derived according to RAxML algorithm. Intra-pair refers to the mother-pup pair, and inter-pair means a pair formed by each mother with unrelated offspring within the same treatment group. Treatment groups are denoted by different colors (\u003cem\u003en = \u003c/em\u003e12 mother-pup pairs).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e) nGDs from the microbes recovered. nGD is the data normalized by the median of all genetic distances, and the proportion of nGDs was shown as histograms (\u003cstrong\u003ec\u003c/strong\u003e). The average nGDs of intra-pair and inter-pair were indicated by the respective dashed lines. For scatter (\u003cstrong\u003ed\u003c/strong\u003e), the same microbe was connected by a dashed arrow, to compare the nGDs with (VG) and without (VPA) LGR-1 treatment. Taxa are colored by phylum (Firmicutes, red; Bacteroidetes, green). \u003cstrong\u003ee\u003c/strong\u003e Maximum likelihood phylogenetic tree analysis of the top 100 enriched species. Phylogenetic analysis was performed using PhyloPhlAn, and branches were colored by phylum (Firmicutes, green; Bacteroidetes, yellow; Actinobacteria, red; Proteobacteria, blue). The log\u003csub\u003e10\u003c/sub\u003e of relative abundance of each species were shown in blue (VG) and red (VPA), respectively. Species profiled by StrainPhlAn were shown in bars, with the coverage of all samples (green) and VG group (brown). nTR (normalized transmission rate) was calculated as the ratio of inter-pair nGD with the total nGDs. LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; Control/C, untreated group; VPA/V, VPA treatment. All data are given as mean ± SEM. Paired t test was used to determine the statistical significance of GDs between intra-pair and inter-pair. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01.\u003c/p\u003e","description":"","filename":"Figure2medium.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/383da6a46011fe6b55ff02b8.jpg"},{"id":75703228,"identity":"fcbf06c6-4a11-4ac8-8814-bf0a6e187e9f","added_by":"auto","created_at":"2025-02-07 09:49:31","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":856123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe orchestrated microbiome mediates the prophylactic effect of LGR-1. a\u003c/strong\u003e Schematic of experimental design for the FMT (fecal microbiota transplant) trials. The VPA treated dams (throughout pregnancy) received the microbiome from LGR-1 treated dams (approaching delivery), and the same paradigm was also applied to pups (donor: 8 weeks old; recipient: from PNW6 to PNW8). The FMT procedure was controlled using VPA treated donors. m, mother; o, offspring. \u003cstrong\u003eb \u003c/strong\u003ePCA analysis for the offspring microbiome with various treatments of microbiota transfer (\u003cem\u003en\u003c/em\u003e = 6). (\u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e) Social behavior in FMT treated mice as assessed in 3-chamber test (\u003cem\u003en\u003c/em\u003e = 7-10). \u003cstrong\u003ee\u003c/strong\u003e Schematic of experimental design for the cross-fostering trials. After birth, male pups were cross-fostered by dams from other groups till weaning. \u003cstrong\u003ef \u003c/strong\u003ePCA analysis for the microbiome from offspring cross-fostered (\u003cem\u003en\u003c/em\u003e = 6-7). (\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e) Sociability and social novelty displayed in the cross-fostered mice as assessed in 3-chamber test (\u003cem\u003en\u003c/em\u003e = 8-15). The combinations of dams and pups during lactation were indicated below. LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; Model/VPA/V, VPA treatment. All data are given as mean ± SEM. For three-chamber test, two-way ANOVA with post hoc Sidak comparisons were conducted to test the statistical significance. ns, \u003cem\u003eP \u0026gt;\u003c/em\u003e 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Figure3medium.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/b8cf3e6a5687267d97c240dd.jpg"},{"id":75703233,"identity":"61a80b88-94f1-4e38-9763-a4859e9b2aa4","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1157954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVaginal microbiota modulated by LGR-1 contributes to the lasting effect of the microbiome.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Vaginal microbiome as depicted by the composition of \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eEnterococcus\u003c/em\u003e and \u003cem\u003eStreptococcus\u003c/em\u003e. The ring encircling the belt area indicates the representative colors of each group or genus, and the outermost circle shows the proportion of microbes in each group, referring to the various community state types (\u003cem\u003en\u003c/em\u003e = 3-4). \u003cstrong\u003eb\u003c/strong\u003e Species proportion within the genus of \u003cem\u003eLactobacillus\u003c/em\u003e in the vagina of dams in response to VPA and LGR-1 treatment. \u003cstrong\u003ec\u003c/strong\u003e Scheme of experimental design for the halted treatment of LGR-1. LGR-1 intake ceased at E14, approximately one week prior to parturition (\u003cem\u003en\u003c/em\u003e = 7-9). (\u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ee\u003c/strong\u003e) Sociability (\u003cstrong\u003ed\u003c/strong\u003e) and social novelty (\u003cstrong\u003ee\u003c/strong\u003e) displayed in mice with LGR-1 intake halted one week prior to parturition. The behavioral performance was assessed in 3-chamber test. \u003cstrong\u003ef\u003c/strong\u003e PCA analysis for the offspring microbiome reared by dams with halted treatment of LGR-1 during gestation (\u003cem\u003en\u003c/em\u003e = 6-7). (\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh\u003c/strong\u003e) Sociability (\u003cstrong\u003eg\u003c/strong\u003e) and social novelty (\u003cstrong\u003eh\u003c/strong\u003e) displayed in mice with LGR-1 intake during the first week of lactation. The social performance was assessed in 3-chamber test (\u003cem\u003en\u003c/em\u003e = 8-9). \u003cstrong\u003ei\u003c/strong\u003e PCA analysis for the murine microbiome treated with LGR-1 during lactation (\u003cem\u003en\u003c/em\u003e = 5-6). LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; VPA/V, VPA treatment. All data are given as mean ± SEM. For three-chamber test involving two groups, statistical significance was determined by paired t test for normally distributed data and Mann-Whitney U test for not-normally distributed data; for three-chamber test involving more than two groups, two-way ANOVA with post hoc Sidak comparisons were conducted. ns, \u003cem\u003eP \u0026gt;\u003c/em\u003e 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Figure4medium.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/22f754afbc94bd21bc2d927c.jpg"},{"id":75703239,"identity":"873ec358-084f-4fb7-ae73-36a4e4ddd601","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":993951,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLGR-1 has impact on behavior by modulating \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAkkermansia\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in offspring.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e Differentially regulated taxa in response to prenatal LGR-1 treatment, relative to the autism model established by VPA. The taxa with the “reversal mode”, that is, LGR-1 led to a direction toward (rather than away from) the untreated group, were presented. The taxa were ranked from low to high by their absolute fold change (VG versus VPA, x axis; \u003cem\u003en\u003c/em\u003e = 15). \u003cstrong\u003eb \u003c/strong\u003eThe relative abundance of maternal \u003cem\u003eLb. rhamnosus\u003c/em\u003e and offspring \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e (AKK) in 12 dam-pup pairs, as revealed by metagenomic analysis. The normalized log\u003csub\u003e2\u003c/sub\u003e value was indicated by the symbol size, and their ranks were shown from high to low by the color intensities. Red, pup; blue, dam. \u003cstrong\u003ec\u003c/strong\u003e Relative abundance of AKK as measured in the FMT-treated group (\u003cem\u003en\u003c/em\u003e = 12). The VPA treated dams (throughout pregnancy) received the microbiome from LGR-1 treated dams (approaching delivery). The FMT procedure was controlled using VPA treated donors. m, maternal. \u003cstrong\u003ed\u003c/strong\u003e PCA analysis for the microbiome from pups postnatally treated with tetracycline (3 g/l) from PNW6 till behavioral test (\u003cem\u003en\u003c/em\u003e = 6-8). The impact on the AKK abundance was indicated in the insert. \u003cstrong\u003ee \u003c/strong\u003eSociability displayed in the \u003cem\u003eAkkermansia\u003c/em\u003e- or tetracycline-treated mice as assessed in 3-chamber test (\u003cem\u003en\u003c/em\u003e = 9-11). \u003cstrong\u003ef \u003c/strong\u003eRepetitive behavior in the \u003cem\u003eAkkermansia\u003c/em\u003e- or tetracycline-treated mice as assessed in marble burying test (\u003cem\u003en\u003c/em\u003e = 9-10 per group). Marble burying index was calculated according to the percentage of marbles buried in the 10-min test. Only marbles obscured more than two-thirds with bedding were counted. \u003cstrong\u003eg\u003c/strong\u003e Scheme of experimental design for the postnatal treatment of AKK on the LGR-1-pretreated BTBR mice. AKK was administered to the male pups from PNW4 to PNW7 at dose of 10\u003csup\u003e10\u003c/sup\u003e/mouse/d (\u003cem\u003en\u003c/em\u003e = 6-8). (\u003cstrong\u003eh\u003c/strong\u003e and \u003cstrong\u003ei\u003c/strong\u003e) Sociability (\u003cstrong\u003eh\u003c/strong\u003e) and social novelty (\u003cstrong\u003ei\u003c/strong\u003e) displayed in mice with postnatal AKK administered into the mice prenatally treated with LGR-1. The social performance was assessed in the 3-chamber test. LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; VPA/V, VPA treatment; B, BTBR; AKK, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e; Tet, tetracycline. All data are given as mean ± SEM. For three-chamber test, two-way ANOVA with post hoc Sidak comparisons were conducted to test the statistical significance. Unpaired t test was performed to compare the AKK level in \u003cstrong\u003ec\u003c/strong\u003e. One-way ANOVA was used to compare data in marble burying test and AKK level in \u003cstrong\u003ed\u003c/strong\u003e. ns, \u003cem\u003eP \u0026gt;\u003c/em\u003e 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Figure5medium.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/2a14a23ab62c96f8ef6496cb.jpg"},{"id":75703267,"identity":"e69cad57-92b4-465d-bbcb-b5995b098af0","added_by":"auto","created_at":"2025-02-07 09:49:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1503336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAKK influences autism-like behavior through the immune-brain pathway. a\u003c/strong\u003e Metabolomic analysis targeting short-chain fatty acids (\u003cem\u003en\u003c/em\u003e = 5-6). The fecal samples from each treatment group were subjected to the UHPLC-MS analysis. BG, BTBR mice treated with LGR-1 prenatally; BGA, BG mice treated with AKK postnatally.\u003cstrong\u003e b \u003c/strong\u003eRepresentative histological images of colon tissues by H\u0026amp;E staining (\u003cem\u003en \u003c/em\u003e= 3 mice per group). Scale bar, 100 μm. The arrows indicate the infiltrated immune cells or goblet cells. Flow cytometric analysis of systemic immunity in spleens of 8-week-old male mice (n = 4 mice per group).\u003cstrong\u003e (c\u003c/strong\u003e and \u003cstrong\u003ed)\u003c/strong\u003e Representative histogram (\u003cstrong\u003ec\u003c/strong\u003e) and pseudocolor plots (\u003cstrong\u003ed\u003c/strong\u003e) of splenic IL-17A-expressing T-helper cells were shown. \u003cstrong\u003ee\u003c/strong\u003e Immunostaining of Iba1 (red) and nucleus (blue) in the medial prefrontal cortex of 8-week-old mice. The scale bar represents 100 μm; the arrows indicate some typical microglial cells. \u003cstrong\u003ef\u003c/strong\u003e Iba1\u003csup\u003e+\u003c/sup\u003e cells calculated from immunostaining slices (11-17 slices per group). The values were normalized against the BG group. (\u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003eh)\u003c/strong\u003e Frequency of mEPSC (\u003cstrong\u003eg\u003c/strong\u003e) and mIPSC (\u003cstrong\u003eh\u003c/strong\u003e) in neurons residing in medial prefrontal cortex (\u003cem\u003en\u003c/em\u003e = 9-28). Mice were subjected to electrophysiological recordings at PNW8-9, and the representative waveforms and histograms were exhibited. \u003cstrong\u003ei\u003c/strong\u003e Scheme of experimental design for the Secukinumab injection on the LGR-1-pretreated BTBR mice that were postnatally treated by AKK. Secukinumab was intraperitoneally injected into the mice at a dose of 10 mg/kg, and the injection was performed on 10 and 6 days prior to behavioral assessment, respectively (\u003cem\u003en\u003c/em\u003e = 6). \u003cstrong\u003ej \u003c/strong\u003eRepetitive behavior as assessed in marble burying test. Marble burying index was calculated according to the percentage of marbles buried in the 10-min test. Only marbles obscured more than two-thirds with bedding were counted.\u003cstrong\u003e k\u003c/strong\u003e Social novelty displayed in mice with Secukinumab treatment, as assessed in the 3-chamber test.\u003cstrong\u003e \u003c/strong\u003eControl, C57BL/6J mice; LGR-1/G, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1; AKK/A, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e. All data are given as mean ± SEM. One-way ANOVA was used to compare data in flow cytometry, immunostaining and electrophysiological recording. Paired t test was used in the statistics of marble burying index. Two-way ANOVA with post hoc Sidak comparisons were conducted to test the statistical significance in the three-chamber trial. ns, \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/4499e79aeebe6059bbd949c0.jpg"},{"id":75703245,"identity":"bd0e15a4-c431-488a-b6ed-e64db23ec6ed","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1977943,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of the prophylactic effect of LGR-1 on mouse models of autism. \u003c/strong\u003eThe exclusive administration of LGR-1 during gestation reshapes the intestinal and vaginal microbiomes of pregnant dams, which contributes to the establishment and development of a “beneficial” microbiome in their offspring. Notably, a discernable vertical transmission of gut microbiome was observed during this process, indicating the postanal stage as the critical temporal window for the specialized maternal microbiome to exert effect. By possessing the microbiome characterized by the reduced levels of \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, the socially-deficient pups exhibited an improvement in social interaction, a decrease in repetitive behaviors, and a restoration of excitatory/inhibitory (E/I) balance.\u003c/p\u003e","description":"","filename":"Figure7model.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/70501d05424bb4b1d98b6dc2.jpg"},{"id":92430481,"identity":"b0e881a3-ab45-46cf-b309-751a429343e8","added_by":"auto","created_at":"2025-09-29 16:05:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10478410,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/4c6980f7-b8f4-4326-a1c4-879dbfcfa651.pdf"},{"id":75704440,"identity":"affab1ae-3e8e-4059-be3b-a7c680deda5a","added_by":"auto","created_at":"2025-02-07 09:57:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1511980,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/69e05102cc93d9b0dae6401d.pdf"},{"id":75703232,"identity":"d604a0f7-a332-4c6d-8f6a-d67c388869b5","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2864136,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/dfed5d19badbe83d650c36fd.mp4"},{"id":75703254,"identity":"b0e5570c-6679-44e2-ad14-4a2d365015bf","added_by":"auto","created_at":"2025-02-07 09:49:33","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2915997,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/827035faccf6a6c9a74af5a4.mp4"},{"id":75703241,"identity":"53f1704f-3dcb-4218-9a35-8b77f9686e20","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2881109,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/d1d972dbae943b8f9886ef0a.mp4"},{"id":75703246,"identity":"084935a0-6e26-4779-a84d-2680f0147858","added_by":"auto","created_at":"2025-02-07 09:49:32","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3840160,"visible":true,"origin":"","legend":"","description":"","filename":"VideoS4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5930312/v1/a9fafab5bcbc93ccbdde053a.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lacticaseibacillus rhamnosus GR-1 prevents autism-like behaviors by reshaping the maternal and offspring microbiome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutism spectrum disorder (ASD) is a complex neurodevelopmental condition primarily characterized by deficits in sociability and stereotyped behaviors\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The worldwide prevalence of ASD continues to rise in recent years, with estimates ranging between 1\u0026thinsp;~\u0026thinsp;3% according to various sources\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, which affects males four times more than females\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In addition to core symptoms, a significant proportion of individuals with ASD also experience gastrointestinal (GI) issues\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Consequently, there is a growing interest in the relationship between the gut microbiome and ASD\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. By transferring microbiota from ASD patients to germ-free animals, the recipients exhibit autistic symptoms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This evidence is a testament to the importance of gut microbiome in the pathophysiology of ASD.\u003c/p\u003e \u003cp\u003eThe maternal status is closely associated with the occurrence of ASD in offspring. For instance, a high-fat diet in dams can induce social and synaptic deficits in their pups\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Maternal immune activation serves as a routine strategy to generate murine models with autism-like symptoms, presumably through IL-17 (Interleukin 17) stimulation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Notably, immune cell activation by gut bacteria is a prime cause of IL-17 release in this context\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, suggesting a causal link between maternal microbiome and ASD. This correlation was further substantiated by findings that maternal dysbiosis, either introduced by antibiotics or fecal transplants, is sufficient to trigger the neurological abnormalities in offspring, including deficits in social interaction\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Despite the implication of maternal microbiome, how it can be orchestrated to mitigate autistic symptoms remains less understood.\u003c/p\u003e \u003cp\u003eThe current advances might allow for a promising microbe-based intervention scheme, especially given that there are only two drugs (risperidone and aripiprazole) approved by FDA for the treatment of ASD. While risperidone is effective in controlling hyperactivity and disruptive behaviors\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, its function in addressing the core symptoms of ASD is still under debate\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Moreover, most medications are based on a \u0026ldquo;curative strategy\u0026rdquo;, designed after the symptoms have occurred. Considering the significant merits of preventive measures, the maternal microbiome could be viewed as the potential target for prophylactic intervention.\u003c/p\u003e \u003cp\u003eA growing body of literature suggests that lactobacillus strains could be used to combat autism-like symptoms. Hsiao et al.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e discovered that the oral intake of \u003cem\u003eBacteroides fragilis\u003c/em\u003e improved gut permeability and alleviated ASD-related defects. Another species, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e, was potent in mitigating the social abnormalities and stereotypies in a variety of autism models\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. According to our previous study, \u003cem\u003eClostridium butyricum\u003c/em\u003e alleviates social subordinance in mice with intestinal dysbiosis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, suggesting that a specific bacterium may impose a desirable effect on social behaviors. While these microbes show promise in repairing autism-related damage, no strains have been identified to play a strictly (prenatally) preventive role in the prototypic autism models.\u003c/p\u003e \u003cp\u003e \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1 (formerly known as \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e GR-1, LGR-1) is a strain first isolated from the female urogenital tract, and later found to exhibit probiotic effect when orally administered to the intestine\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our prior work showed that LGR-1 could improve spatial memory\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, proposing it a candidate strain for protection against other brain-related behaviors. In this study, we investigated the prophylactic role of LGR-1 in ameliorating autism-like deficits and attempted to understand the underlying mechanisms, with a focus on the generational inheritance of the gut microbiome. This study could help us find a new way to address autism-related adversities by early interfering with the microbiome of the pregnant mothers.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePrenatal administration of LGR-1 mitigates the autism-like behaviors.\u003c/b\u003e Valproic acid (VPA) can trigger autism through injection on embryonic day (ED) 12.5\u003csup\u003e6\u003c/sup\u003e. In this study, both the VPA-induced and idiopathic mice, namely BTBR T\u003csup\u003e+\u003c/sup\u003e Itpr3\u003csup\u003etf\u003c/sup\u003e/J (BTBR), were used as animal models for autism. To investigate if LGR-1 could alleviate the related deficits, this strain was administered daily to pregnant dams from conception until parturition. The intake was immediately discontinued after birth, and the animal behaviors were examined at postnatal week 8 (PNW8, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Firstly, a three-chamber test was adopted to evaluate the social performance of the subject mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). According to the results, LGR-1 significantly prevented mice from autism-like lesions, as evidenced by their preference to interact with another mouse (or novel conspecific) over an inanimate object (or old one) (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.011 for sociability, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.005 for social novelty; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-e; Supplementary video 1\u0026ndash;4). Thus, social performance was improved through the prenatal use of LGR-1. Remarkably, the prophylactic effect appears to be strain-specific, as a similar outcome was not observed with the routine functional partner of LGR-1, \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e RC14 (LRC14), or another common probiotic strain, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GG (LGG; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e). Moreover, the LGR-1 strain itself may be pivotal for intervention, as its supernatant did not offer protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e). The efficacy of LGR-1 was consistent across the models used, with both environment-induced (VPA) and idiopathic (BTBR) mice (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.002 for sociability, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.012 for social novelty; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef and g) benefitting from LGR-1 pretreatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMarble burying test is a paradigm used to assess repetitive behavior, another core symptom of ASD. This test revealed that stereotypies were attenuated in both VPA-induced (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001) and BTBR mice (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.004) following LGR-1 pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh). Besides, a reciprocal interaction assay yielded similar results: the interaction duration and number of contacts between mouse pairs were enhanced after prenatal addition of LGR-1 (Supplementary Fig.\u0026nbsp;1a and b). Interestingly, postnatal administration of LGR-1 also led to a varying degree of social improvements (Supplementary Fig.\u0026nbsp;1c), but not stereotypies (Supplementary Fig.\u0026nbsp;1d).\u003c/p\u003e \u003cp\u003eE/I imbalance is a hallmark feature in some ASD patients, indicating abnormal synaptic transmission and cortical circuitry\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To investigate this phenomenon in the present condition, we recorded miniature excitatory post-synaptic currents (mEPSC) and miniature inhibitory post-synaptic currents (mIPSC) in the medial prefrontal cortex. Frequency analysis revealed that BTBR mice displayed a decreased mIPSC while maintaining constant mEPSC, suggesting an increased E/I ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei and j, Supplementary Fig.\u0026nbsp;1e and f). This deviation was ameliorated by LGR-1, as observed in the changes of mIPSC frequency (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.032; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej). The synaptic fluctuation was accompanied by the concurrent reprogramming of cortical gene expression (Supplementary Fig.\u0026nbsp;1g), which showed the enrichment of MAPK signaling among others (Supplementary Fig.\u0026nbsp;1h), in response to LGR-1.\u003c/p\u003e \u003cp\u003eGiven the correlation between microbiome and autism, we next examined the gut microbiome of pups pre-treated with LGR-1. Revealed by the principal component analysis, the \u003cem\u003ein utero\u003c/em\u003e addition of LGR-1 steered the microbiome towards a state closer to the untreated mice, compared to that of the socially-deficient conspecifics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). Meanwhile, the supplementation with either LGR-1 supernatant or LGG resulted in the divergent alterations of microbial community.\u003c/p\u003e \u003cp\u003eTogether, prenatal treatment of LGR-1 improves autism-like behaviors and restores the microbiome.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePrenatal treatment alters offspring microbiome via vertical transmission.\u003c/b\u003e We next sought to understand how prenatal treatment of LGR-1 could cause considerable changes in the offspring microbiome. Gut microbiome can be vertically transmitted from mothers to infants\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, offering a route for generational influences on microbiome compositions. Therefore, we first examined the maternal microbiome\u0026rsquo;s response to LGR-1 treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, LGR-1 profoundly modified the microbiome of pregnant dams within the BTBR inbred colonies, potentially laying the groundwork for the transmission hypothesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigated vertical inheritance of microbiome in the context of LGR-1 intervention using metagenomics. Regarding bacterial species shared between dams and pups, mice tend to harbor similar species with their own mothers rather than with other dams receiving the same treatment (Supplementary Fig.\u0026nbsp;2a). This tendency remained consistent across treatments and was pronounced for the most abundant species of the pups (Supplementary Fig.\u0026nbsp;2b).\u003c/p\u003e \u003cp\u003eSubsequently, the StrainPhlAn was used to identify the bacterial strains present in at least 16 samples and to analyze their genetic distance among samples. As revealed by the data, the genetic distances of \u0026ldquo;intra-pair\u0026rdquo; (dam-pup) were significantly lower than \u0026ldquo;inter-pair\u0026rdquo; (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.003; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), suggesting a skewed tendency of microbiome transmission. The distribution of the normalized genetic distance (nGD) aligned with the same patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Based on the evidence of generational transmission in the studied settings, the reprogrammed offspring microbiome (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek) might be a consequence of the premeditated maternal microbiome. These trends were consistent when comparisons between mothers and pups were expanded to those between treatment groups (Supplementary Fig.\u0026nbsp;2c and d).\u003c/p\u003e \u003cp\u003eAlthough vertical inheritance occurs regardless of treatment, it is still crucial to determine whether the addition of LGR-1 can alter the transmission rate of specific taxa. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, LGR-1 led to a variety of transmission drifts, either towards intra-pair or inter-pair, depending on the specific strain under study. This indicates that LGR-1 serves as a robust driving force, capable of reshuffling the transmission trajectory, and subsequently accounting for a restructured microbiome in offspring mice.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis provided a comprehensive picture of taxa with the highest abundance. A subset of microbes displayed distinct abundance levels in the presence or absence of LGR-1. Among the taxa identified by StrainPhlAn, nTR (normalized transmission rate) was then calculated, indicating variable degrees of transmission across all samples or LGR-1-treated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). As two representative taxa due to their high prevalence among groups, the phylogenetic trees of \u003cem\u003eMuribaculum intestinale\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e prophage Lj928 were shown in Supplementary Fig.\u0026nbsp;2e and f, with a focus on the mother-offspring pairs and treatment-group pairs. Besides, we also showcase the phylogenetic similarities of certain reference strains, underpinning their preferences in either generation (Supplementary Fig.\u0026nbsp;2g). These microbes exemplify the existence and plasticity of vertical transmission.\u003c/p\u003e \u003cp\u003eCollectively, microbiome transmission persists and evolves in the context of LGR-1 intervention.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe orchestrated microbiome mediates the prophylactic effect of LGR-1.\u003c/b\u003e To investigate whether microbiome variation is pivotal for social interaction, FMT (fecal microbiota transplant)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e was conducted in either dams or pups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The effectiveness of the transplant was validated through 16S rRNA sequencing, indicating that recipients from the LGR-1-treated donors harbored microbiome with close proximity to their donors rather than the socially-deficient conspecifics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In terms of behavior, mice engrafted with LGR-1-treated microbiome exhibited marked improvement in sociability (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 for mFMT, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 for oFMT; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec) and social novelty (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.002 for mFMT, \u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 for oFMT; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Due that maternal microbiome transfer also brought the comparable benefits to their offspring, maternal microbiome remodeling is not the isolated event but has vertical influence on social behaviors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering the temporal separation of intervention (pregnancy) and effect (postweaning), alternative routes may come into play at different stages. IL-17A was previously implicated in the crosstalk between maternal microbiome and fetal brain, promoting the autistic-like phenotype\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In the current settings, however, we found no significant variations in the IL-17A levels in maternal serum of autistic models or LGR-1-pretreated mice (Supplementary Fig.\u0026nbsp;3a), suggesting that IL-17A might not be the prime target for LGR-1 on dams. Additionally, given the possibility of direct inheritance of LGR-1 by the offspring, our metagenomic data did not find evidence of \u003cem\u003eLimosilactobacillus rhamnosus\u003c/em\u003e in some LGR-1-pretreated pups (Supplementary Fig.\u0026nbsp;3b). Furthermore, when LGR-1 was supplemented during lactation, the pup microbiome was not altered in a manner comparable to the \u003cem\u003ein utero\u003c/em\u003e treatment (Supplementary Fig.\u0026nbsp;3c-e).\u003c/p\u003e \u003cp\u003eNext, we cross-fostered pups with dams from different groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Along with the restructured microbiome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef), most pups exhibited social properties resembling their foster mothers, both in sociability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg) and social novelty (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). The data underscored the significance of maternal microbiome in shaping social performance and identified lactation as a critical period for conveying the benefits of early LGR-1 use. However, it\u0026rsquo;s worth noting that not all phenotypes were consistent with those of foster dams, as exemplified by the sociability of pups with their biological mothers remedied with LGR-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). This might suggest that maternal microbiome should not be considered as the sole contributor to the offspring behavior, and that LGR-1 does not function solely through orchestrating maternal microbiome.\u003c/p\u003e \u003cp\u003eIn summary, our findings indicate a mechanistic link between maternal microbiome and the behavioral benefits brought by LGR-1.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVaginal microbiota modulated by LGR-1 contributes to the lasting effect of the microbiome.\u003c/b\u003e We next attempted to decipher other LGR-1-related factors responsible for the altered offspring microbiome and behavior. As LGR-1 originally inhabits in the urogenital system\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and vaginal bacteria are considered the seeding microbiome of newborns\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, roles of vaginal microbiota were then investigated in the studied remediation. Firstly, maternal vaginal microbiota was sequenced prior to parturition in response to LGR-1 addition. As revealed by Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, VPA injection dampened the dominant status of lactobacillus, a tide further turned by the continuous impact of LGR-1. In the aspect of compositions, LGR-1 caused a dominant presence of \u003cem\u003eLb. rhamnosus\u003c/em\u003e in the murine vagina (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), a finding consistent with the notion of vagina-rectal communication\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further test the importance of vaginal microbiome, we interrupted the LGR-1 intake at E14 (Embryonic day 14), a week preceding delivery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). As a result, the test mice were unable to normally socialize with the resident mice in a three-chamber trial, albeit administered with LGR-1 (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.07; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In the event of social novelty, the halted treatment also abrogated the benefits brought by LGR-1 (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.879; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). By analyzing the offspring microbiome, we found that pausing LGR-1 led to a disparate bacterial community in pups\u0026rsquo; intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Therefore, the LGR-1-modifying vaginal microbiota contributes to the offspring\u0026rsquo;s intestinal microbiome, as well as the resulting social behavior.\u003c/p\u003e \u003cp\u003eBy adding LGR-1 during lactation (Supplementary Fig.\u0026nbsp;3c), the impact of vaginal microbiota on the offspring\u0026rsquo;s microbiota can be artificially bypassed. In this case, LGR-1 lost its ability to rescue the social deficits (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.014 for sociability with a preference to the inanimate object, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.871 for social novelty), in contrast with the prenatal administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and h). Along with the taxa distribution at the phylum and genus level (Supplementary Fig.\u0026nbsp;3d and e), PCA also indicates that LGR-1 led to the distinct offspring microbiota when added in lactation instead of gestation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei), during which no roles of vaginal microbiota were involved.\u003c/p\u003e \u003cp\u003eIn summary, the LGR-1-modifying vaginal microbiota plays part in the offspring\u0026rsquo;s microbiome and social benefits.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLGR-1 has impact on behavior by modulating\u003c/b\u003e \u003cb\u003eAkkermansia\u003c/b\u003e \u003cb\u003ein offspring.\u003c/b\u003e To further delineate the compositional properties of \u0026ldquo;good\u0026rdquo; microbiome modified by LGR-1, the taxa with the highest fold changes were ranked and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. In this matrix, only taxa with the \u0026ldquo;reversal mode\u0026rdquo;, which abundance was partially restored by LGR-1, were incorporated. In contrast to the LGR-1-pretreated group, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e (AKK) was highly enriched in the VPA-exposed mice. In light of its importance on the central nervous system\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, AKK was selected for further studies. Of note, the offspring abundance of AKK displayed a trend inverse to that of the maternal \u003cem\u003eLb. rhamnosus\u003c/em\u003e in the dam-pup pairs revealed by the metagenomic sequencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Moreover, AKK was found lower in the offspring of the recipients engrafted with the LGR-1-modifying microbiome (\u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.005; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), suggesting a marked generational correlation. Besides, other candidate lactobacillus strains did not generate the comparable AKK alterations (Supplementary Fig.\u0026nbsp;4a).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we investigate if AKK is associated with the autism-like behavior. Tetracycline can be used to decrease AKK in murine intestine\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Our results showed that administering tetracycline at a dose of 3 g/l indeed altered the microbiome of BTBR mice, reducing AKK levels close to zero (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Consequently, AKK elimination drove the mice to behave more socially and less repetitively than their defective conspecifics, reversing the animal performance to a varying extent (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 for sociability, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.049 for stereotypies between \u0026ldquo;BTBR\u0026thinsp;+\u0026thinsp;Tet\u0026rdquo; and \u0026ldquo;BTBR\u0026rdquo;; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and f). In contrast, the addition of AKK did not yield the recovery effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee and f, Supplementary Fig.\u0026nbsp;4b-d). Additionally, tetracycline acted in a dose-dependent manner (Supplementary Fig.\u0026nbsp;4e), likely due to its complicated impact on the overall microbiome.\u003c/p\u003e \u003cp\u003eAKK was then administered to the male pups pretreated with LGR-1 at early life (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). As a result, social deficits were partly reproduced by the postweaning effect of this microbe (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 for sociability, \u003cem\u003eP\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.313 for social novelty; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh and i), indicating that the LGR-1-AKK axis may play roles in modulating autism-like behaviors. The inability to cause lesions to sociability (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eh) might indicate a dominant role of LGR-1 over AKK in this functional aspect.\u003c/p\u003e \u003cp\u003eCollectively, the LGR-1\u0026rsquo;s social effect is related to the level of AKK in offspring.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAKK influences autism-like behavior through the immune-brain pathway.\u003c/b\u003e Finally, to interrogate how AKK influences autism-like symptoms, we attempted to dissect the gut-brain axis involved in the studied intervention. The metagenomic sequencing data showed that LGR-1 modified the abundance of a list of carbohydrate-active enzymes (Supplementary Fig.\u0026nbsp;5a) and increased the gene copies of butyrate-related enzymes in microbial genomes (Supplementary Fig.\u0026nbsp;5b), suggesting potential roles of the microbe-derived metabolites. Based on it, we first performed the metabolomic analysis targeting the short chain fatty acids. As revealed by the results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), the addition of AKK reversed the LGR-1-mediated enhancement of 2-methylbutyrate and isovaleric acid. Since these BCFAs (branched-chain fatty acids) were recently found to exert anti-inflammatory effects\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, the immune route was further examined with respect to its roles in the studied gut-brain axis. Immune infiltration, indicative of a compromised intestinal barrier, was observed in PE-stained colon tissues of BTBR mice. This lesion was mitigated by the prophylactic addition of LGR-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Notably, when AKK was further administered to the offspring mice, there appeared a marked propagation of goblet cells, resulting in a new form of aberrations. This observation could be relevant to the mucin-degrading property of AKK\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, while goblet cells are known to produce mucins to compensate for the loss.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine the immune status beyond the host-microbe interface, we determined the changes of T helper cells in spleens in response to the LGR-1/AKK treatment. According to the flow cytometric data, LGR-1 inhibited the differentiation of CD4\u003csup\u003e+\u003c/sup\u003eIL-17\u003csup\u003e+\u003c/sup\u003e cells in BTBR mice, which was then partly reversed by supplementing \u003cem\u003eAkkermansia\u003c/em\u003e strains (p\u0026thinsp;=\u0026thinsp;0.011, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d), an observation different from the changes in pregnant dams\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In addition, the differentiated Th1 cells (CD4\u003csup\u003e+\u003c/sup\u003eIFNγ\u003csup\u003e+\u003c/sup\u003e) were also decreased with the prenatal use of LGR-1, but no significant influence was detected after postnatal colonization of AKK (p\u0026thinsp;=\u0026thinsp;0.447, Supplementary Fig.\u0026nbsp;5c, d). Consistent with the gut-associated and systemic immunity, brain immunity was also sensitive to signals from gut microbiota. In the prefrontal cortex, the microglial activation in BTBR mice was partly inhibited by the prenatal addition of LGR-1, and the favorable effect was then postnatally attenuated by AKK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f), suggesting that immune route was intensively involved in the studied gut-brain crosstalk.\u003c/p\u003e \u003cp\u003eMicroglial activity is tightly associated with neuronal function. Consequently, the prefrontal E/I ratio was measured in response to treatment of LGR-1 and AKK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h; Supplementary Fig.\u0026nbsp;5e-h). In alignment with previous findings, the frequency of mIPSC was increased by prenatal administration of LGR-1, a change subsequently reversed by postnatal AKK (p\u0026thinsp;=\u0026thinsp;0.035, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh). As mEPSC remained unaltered throughout this process, the E/I balance was assumed to be re-disturbed upon AKK\u0026rsquo;s colonization.\u003c/p\u003e \u003cp\u003eTo further explore the influence of CD4\u003csup\u003e+\u003c/sup\u003eIL17\u003csup\u003e+\u003c/sup\u003e cells on the studied gut-brain communication, an antibody binding to IL-17A, namely Secukinumab, was used to abrogate the functioning of IL-17A (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). When Secukinumab was intraperitoneally injected into the mice treated with the prenatal LGR-1 and postnatal AKK, the behavioral impairment caused by AKK was significantly attenuated, as evidenced by the repetitive (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ej) and social behaviors (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). This provides a piece of evidence that IL-17A plays essential roles in conveying the gut-derived signals into the brain, in the context of LGR-1/AKK intervention.\u003c/p\u003e \u003cp\u003eTaken together, AKK has impact on autism through the immune-brain pathways.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCan autism be prevented in pregnancy? Here we report that prenatal intake of LGR-1 reduced the risk of autism-like symptoms. According to the previous findings, either autism-relevant traits\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e or gut microbiome\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e persisted across generations, which implied the significance of maternal microbiome on the autistic behaviors. This was further supported by the finding that ASD-like symptoms were induced in the offspring by applying antibiotics to the maternal microbiome\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. A strategy with opposite directions was used here to confer benefits on the autistic-like animals, discovering that a specific strain, LGR-1, is potent in improving the relevant social deficits when applied at early life. Moreover, vertical transmission of microbiome was found to play important roles in this preventative paradigm, emphasizing the generational axis of LGR-1/AKK (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To our knowledge, this represents a new instance of using a single probiotic strain to, at least in part, prevent the occurrence of autism-like behaviors. Given the obvious merits of prophylactic approach, this could provide an intriguing choice for combatting the autism-related complications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePregnancy is a critical period for the onset of autism. Viral infection in pregnancy, usually accompanied by immune activation, has been employed in mice to establish autistic models\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This may pinpoint the likeliness of preventing autism as early as pregnancy. Accordingly, this notion has been examined in several studies: resveratrol corrected the ASD-like abnormalities caused by VPA\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e; antibiotic treatment before and during pregnancy attenuated the social deficits caused by maternal immune activation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Despite these advances, this strategy is still at its infancy, scarcely involving the use of the microbe-based methods. Notably, the gut microbiome in gestation is highly dynamic and unique, rendering it susceptible to intervention with a premeditated orientation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Accordingly, the present research shows that maternal microbiome can also be targeted for the positive moderation, generating the desirable behavioral outcomes rather than negative effects. However, it remains a challenging task to define a \u0026ldquo;good maternal microbiota\u0026rdquo;: although LGR-1 can induce a collection of changes among commensals, it did not necessarily generate a singular/exclusive combination to alleviate the autism-related performance. The \u0026ldquo;\u003cem\u003ebona fide\u003c/em\u003e\u0026rdquo; determinant could be the overall balance of the maternal bacteria during childbirth, accounting for the \u0026ldquo;normal\u0026rdquo; seeding and the early-life development of offspring microbiome.\u003c/p\u003e \u003cp\u003eA variety of probiotic strains have been suggested to improve autistic symptoms, exemplified by \u003cem\u003eBacteroides fragilis\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. While these microbes show robust activities in ameliorating autism-related behavior, the current study uses a single bacterial strain preemptively to achieve the prophylactic effect. Importantly, this conferred protection varies depending on the specific strain used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and e), indicating the remarkable strain selectivity. It\u0026rsquo;s intriguing to speculate that this phenomenon might be associated with the unique properties of LGR-1, a strain initially isolated from distal urethra and then known to be probiotic in the gastrointestinal tract\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Nonetheless, the key structural determinant that empowers LGR-1 to modulate social behavior remains poorly understood, warranting future investigations. But it appears unlikely to ascribe the behavioral benefits to the inheritance of LGR-1 \u003cem\u003eper se\u003c/em\u003e, as its host species \u003cem\u003eLb. rhamnosus\u003c/em\u003e was not detected in the feces of certain offspring, and LGR-1 did not improve mice behaviors during lactation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg and h). Based on this analysis, we propose that the molecular determinant of LGR-1 is likely utilized to moderate the composition of maternal commensals, as well as to reprogram their tendency of being transmitted to the next generation.\u003c/p\u003e \u003cp\u003eThe time window is critical in determining the effect of an intervention. For instance, exposure to taurin improved the social performance of BTBR mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. But this only occurred during pregnancy and lactation, while administration at later life did not yield the posivie effect. In the present study, LGR-1 was administered exclusively during the prenatal period, achieving early-life intervention of autism-like complications. Contrarily, the main effect was elicted at the postnatal stage including lactation, which may be attributed to the ongoing microbiome-reshaping impact of the maternal microbiome, as demonstrated by cross-fostering trails (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg and h). Therefore, while the prenatal route can not be entirely ruled out, the effective period of LGR-1 is supposed to separate from its initial colonization. Given the current evidence, no definitive conclusions can be drawn as to whether other strains could exert a prophylactic effect via the similar mechanisms to LGR-1.\u003c/p\u003e \u003cp\u003eThe maternal gut is the largest source of commensal bacteria living in the GI tract of offspring\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The mechanisms driving this \u0026ldquo;vertical tranmission\u0026rdquo; are largely unclear, but it might be related to the persistence of maternal bacteria and their better adaptation to the intestinal niche compared to microbes acquired from other sources\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Considering the existence of microbiota inheritance, it provides a route to use maternal intervention to improve the offspring symptoms. This was achieved in this study, with its importance best elucidated in the maternal FMT trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d). However, it remains challenging to accurately predict the transmission profile for each taxonomic unit, due to the remarkable variations of the physiological settings. Furthermore, transmission is a well-defined process associated with the strain nature, rather than its relative abundance in a microbial community, an observation (data not shown) consistent with the previous report\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. On the other side, microbiome transmission also has relevance with vaginal microbiota, as the majority of strain transmission occurred in babies delivered vaginally (74.39%), at a higher frequency than those delivered by C-section (12.56%)\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLGR-1 retains its capacity to modulate vaginal microbiota. Indeed, LGR-1 is the first strain thought to replenish the vaginal microbiota as the exogenous probiotic\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This capability is linked to its effect on social behavior, due to the importance of vaginal microbiota on the prevalence of ASD\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This notion was further substantiated by the present finding that LGR-1 acted, in part, by modulating the composition of vaginal microbiota. According to our data, the maternal vaginal microbiota, coupled with their gut microbiome, impacted the formation of offspring microbiome, probably by modulating their seeding and early-life development, which may serve as the crucial events relating to the autism-like phenotypes. This proposition also aligns with the theory that the pioneering microbes can drive the development of subsequent microbiota, which coincide with a critical time window of neurodevelopment\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Therefore, LGR-1 should be viewed as a unique strain, with modulatory activity on both populations of microbes present in the pregnant dams, constituting its major specificity of behavioral remediation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e is widely regarded as the next-generation beneficial microbe. It\u0026rsquo;s interesting to notice that this microbe is impacted by the \u0026ldquo;first generation probiotic\u0026rdquo;, lactobacillus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c), through unknown routes in separate generations. AKK can be delivered to the progeny by vertical inheritance, a finding consistent with a previous study\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Remarkably, the two microbes showed opposite trends in abundance, indicating that LGR-1 might curb the ability of AKK to transmit and thrive in the next generation. Another intriguing observation here is the unexpected high existence of AKK in the autism-like animals, which did not adhere to the positive effect of this \u0026ldquo;beneficial microbe\u0026rdquo;. In fact, this is not a rare case, as AKK was shown to be enhanced, albeit with conflicting data, in the intestines of both animal models and some ASD patients\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, which is supposedly associated with the mucin-degrading activity of AKK. In a general sense, AKK can facilitate the turnover of mucins, but its excessive growth might lead to the injuries of the gut barrier. This hypothesis was supported by our finding that, upon AKK administration, an excess of goblet cells were observed at the gut-bacteria interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggesting a compensatory response towards damage. Another possibility is the involvement of AKK with inflammation. While AKK is traditionally considered anti-inflammatory, it also promotes the maturation of leukocytes, and participates in the chemotaxis and complement cascade\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In an instance, AKK exacerbated the salmonella-mediated gut inflammation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Based on its discrete performance, AKK\u0026rsquo;s action is not predisposed, but likely dependent on the physiological context and dosage used. Besides, AKK is unlikely to act independently to influence behavior, but help building a specific microbiome that serve its function. While the focus of this study is the generational mechanism of prophylactic effect, the detailed gut-brain routes of \u003cem\u003eAkkermansia\u003c/em\u003e, in terms of the T cell trafficking or oxytocinergic system, should be explicitly dissected in the following investigations.\u003c/p\u003e \u003cp\u003eThis study has some limitations: (I) one of the challenges with the gut-brain axis is the difficulty of establishing the cause-and-effect relationship. In this case, while LGR-1 is shown to alleviate ASD-like symptoms, considerations of other upstream factors such as diet and gene cannot be excluded; (II) while animal models are useful to demonstrate key characteristics of ASD, they may not fully reflect the complexity of human behavior and brain circuits. Therefore, clinical trials are required in future to validate the effects of LGR-1 on human subjects; (III) ASD is no longer considered the dysfunction of a specialized brain area but the wide-ranging reorganization. Gut microbiome is quite unlikely to affect all brain areas of interest in a desired manner, which means it probably cannot resolve all the ASD-related aberrations; (IV) the molecular basis for strain specificity remains unclear, which may be associated with the key determinants of LGR-1 in modulating social behavior. Further research is needed to address these difficulites.\u003c/p\u003e \u003cp\u003eCollectively, given the lack of amenable medications of ASD, gut microbiome has been increasingly appreciated to remediate autism-related deficits. In this study, manipulating the maternal microbiome using a single strain, LGR-1, is effective in attenuating autism-like behaviors in newborn animals. This strategy leverages the non-genetic yet inheritable feature of gut microbiome to maneuver generational effect. In conclusion, the microbe-based preventive approach is tested here, offering a promising avenue to address the challenges of ASD-like complications by tailoring the microbiome of pregnant mothers.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eStudy design.\u003c/b\u003e The objective of this study is to assess the prophylactic effect of LGR-1 on autism-like behaviors, and to define the underlying generational mechanisms focusing on vertical transmission of gut microbiome. Both VPA-exposed and idiopathic BTBR mice were used to establish the autism-like models. For the major paradigm, LGR-1 was fed to the pregnant mice by oral gavage at a dose of 10\u003csup\u003e9\u003c/sup\u003e/mice/d, starting from conception till parturition. The control groups were treated with saline/solvent following the same paradigm. Secukinumab was intraperitoneally injected into the mice at a dose of 10 mg/kg at 10 and 6 days prior to behavioral trials. Three-chamber test and reciprocal social interaction were used to assess the sociability of the test mice, and marble burying trial was used to assess their reciprocal behaviors. The behavioral tests were only conducted when male mice reached adulthood (7\u0026ndash;9 weeks old). Electrophysiological recordings were used to assess the E/I balance in the prefrontal cortex. Microbiome samples were collected prior to the behavioral trials and then subjected for metagenomic sequencing for assessment of vertical transmission, or 16S-rRNA sequencing for other assessments. Mice with obvious health problems were excluded from the relevant test and analysis, and no data was excluded unless the mice refused to explore in the arena, too aggressive or always tended to climb upwards along the wall or cages during the behavioral test. Sample size was determined by the availability of pregnant dams and reagents and was not predetermined, with at least two different litters used in each treatment group. The order of measurements was randomized towards treatment groups. In all experiments, data obtained in individual mice was considered as one biological replicate. A minimum of three biological replicates were used in each experiment. Behavioral experiments were replicated at least twice with independent cohort of mice, along with most microbiome sequencing and other analysis. Data collection was not halted until the experiments were completed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMice.\u003c/b\u003e C57BL/6J mice were housed under SPF (specific pathogen-free) conditions at a 12-hour light/dark cycle per day and had access to food and water \u003cem\u003ead libitum\u003c/em\u003e. These mice were originally obtained from the animal facility of Anhui Medical University and bred in-house. The VPA-treated mice were generated by administering a single dose (400 mg/kg) of sodium valproate (CSNpharm, Chicago, USA) to the pregnant C57BL/6J mice at embryonic day 12.5 (E12.5)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe BTBR T\u003csup\u003e+\u003c/sup\u003e Itpr3\u003csup\u003etf\u003c/sup\u003e/J (BTBR) inbred mice, known to display an inherent autistic-like phenotype, were purchased from The Jackson Laboratory (Bar Harbor, USA). They were housed in the cycle of 14-hour light and 10-hour dark with \u003cem\u003ead libitum\u003c/em\u003e access to food and water.\u003c/p\u003e \u003cp\u003eEach dam gave birth to an average 8\u0026thinsp;~\u0026thinsp;9 offspring. The total male offspring were randomly regrouped 3\u0026thinsp;~\u0026thinsp;6/cage at weaning, without cohousing non-littermates. For transmission studies, only one litter of offspring was reserved for each dam, and one male pup was randomly selected from it to establish the mother-pup pair, the feces of which were later used for metagenomic sequencing. At least two litters of male offspring were used for each treatment group in other experiments. Female mice were not used here due to the male prevalence of ASD, as well as the behavioral misrepresentation for females in the VPA-exposed\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e and BTBR models\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Throughout the trial, body weight and food/water consuming of mice were monitored, with any unhealthy or aberrant animals culled prior to test. All experiments were conducted following protocols approved by the Institutional Animal Care and Use Committee of Hefei University of Technology.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial strains and culture.\u003c/b\u003e \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1, GG and \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e RC-14 were obtained from our laboratory stocks. Cultures were maintained on MRS medium (Huankai, Guangzhou, China) in an anerobic chamber (HYQX-Ⅱ, Yuejin, Shanghai, China) with a gas mix of 10% hydrogen, 5% carbon dioxide and 85% nitrogen. The purity of the cultures was monitored by plating with serial dilutions. For inoculation, the lactobacillus strains were grown at 37\u003csup\u003eo\u003c/sup\u003eC for 12 h. The bacteria were collected by centrifugation at 8 000 g for 5 minutes, washed twice with sterile PBS, and then suspended at a final concentration of 10\u003csup\u003e9\u003c/sup\u003e CFU/ml for further use. For LGR-1, the culture supernatant was filtered through 0.22 \u0026micro;m membrane filter and used as parabiotic. \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e DSM 22959 was purchased from Biobw (Beijing, China) and maintained in Brain Heart Infusion medium (Sigma-Aldrich, Shanghai, China) supplemented with 0.25 g/l mucin (Sigma-Aldrich, Shanghai, China). AKK was grown in the anerobic chamber for 48 h to obtain the bacterial samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBacterial supplementation.\u003c/b\u003e The pregnant dams received \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1, GG, or \u003cem\u003eLimosilactobacillus reuteri\u003c/em\u003e RC-14 at a concentration of 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU per mouse/day, and the oral gavage was carried out solely during gestation. According to the metagenomic data (PRJNA1033296), the relative abundance of \u003cem\u003eLb. rhamnosus\u003c/em\u003e in the VPA-exposed dams was increased from 5.33 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e to 5.55 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e after LGR-1 treatment. For other paradigms, the postnatal administration of LGR-1 was conducted from weaning till the behavioral test (gavage through pups), or from birth to the first week of lactation respectively (gavage through dams). Male offspring received \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e DSM 22959 prepared under anaerobic conditions at a concentration of 1 \u0026times; 10\u003csup\u003e10\u003c/sup\u003e CFU per mouse/day through daily oral gavage during PNW4-7. All bacterial supplementation was controlled by saline gavage. The bacterial viability was randomly checked and confirmed by plate counting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDrug treatment.\u003c/b\u003e For antibiotic administration, tetracycline hydrochloride (Bio Basic, Markham, Canada) was dissolved in 1% sucrose, resulting in final concentrations of 1.5 (L), 3 (M) and 10 g/l (H) respectively. The drug solution was sterilized with a 0.22 \u0026micro;m filter, and then supplied to the BTBR mice during PNW6 and 7 in their drinking water renewed every three days. For IL-17A signaling inhibition, Secukinumab (Ambeed, Beijing China) was dissolved in 0.9% saline, and i.p. injected into the mice treated with prenatal LGR-1 and postnatal AKK at a dosage of 10 mg/kg. The injection was conducted twice, specifically on 6 and 10 days before behavioral trials.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThree-chamber trial.\u003c/b\u003e All behavioral tests were conducted on 7- to 9-week-old mice. Three-chamber trial was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Briefly, the subject mouse was first habituated for 10 minutes in an empty 60 \u0026times; 40 \u0026times; 23 cm Plexiglass area formed by three interconnected chambers. Subsequently, an age/gender matched conspecific (Mouse 1) was placed into the left chamber, and the moving tracks of the subject mouse were recorded in the left and right chamber (inanimate object; Empty) in the following 10-min session (Supplementary videos were made from a bird\u0026rsquo;s-eye view). Sociability was then assessed by measuring the time spent by test mouse interacting with Mouse 1 compared to the inanimate chamber. Social novelty was evaluated in the third 10-min session, whereas a stranger (Mouse 2) was introduced to the right chamber, and the time distribution of the subject mouse was recorded and analyzed by ANY-maze (Stoelting, Wood Dale, USA) using the same methods. The arena was cleaned up between sessions. The human observers and analyzers were blinded to the treatment group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMarble burying test.\u003c/b\u003e Marble burying test was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Briefly, the subject mouse was put into a 50 \u0026times; 30 cm Plexiglass arena with a 5 cm-thick corncob bedding, with the surface covered by 24 regularly-spaced royal blue marbles. The mouse was allowed to freely explore inside the arena for 10 min. Subsequently, the mouse was removed, and marbles with at least two-thirds of their diameter obscured by bedding were counted as buried. The ratio of buried marbles was calculated to assess the severity of repetitive behavior.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFecal microbiota transplant.\u003c/b\u003e Fresh fecal samples were collected from donor mice and homogenized on ice in sterile PBS under aseptic conditions. The resulting slurry was spun at 700 g for 1 min at 4\u003csup\u003eo\u003c/sup\u003eC. The supernatants were then collected for oral gavage in a dosage of 0.2 ml per recipient mouse. For offspring transplant, donor samples were collected from 8-week-old male mice exhibiting autistic traits or those treated with LGR-1 prenatally. The transplant was then performed on the 5-week-old mice with social deficits daily and continued for 3 weeks. For maternal fecal transplant, the pregnant dams approaching delivery were used as donors, and their fecal samples were transferred to the defective mothers throughout pregnancy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCross-fostering experiment.\u003c/b\u003e Pups were cross-fostered at birth with the conspecifics born on the same day. The whole litters were removed from their biological mothers and gently introduced to a new cage with the foster mother. Pups from the socially-deficient mothers were cross-fostered to the LGR-1-intervening mothers, and vice versa. This experiment was controlled by cross-fostering pups to another mother within the same treatment group.\u003c/p\u003e \u003cp\u003e \u003cb\u003e16S rRNA sequencing and analysis.\u003c/b\u003e 16S rRNA sequencing was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, the fresh feces from male mice and female dams were collected before behavioral assays and delivery, respectively. Vaginal samples were collected by swabbing the vaginal sidewalls four times from pregnant dams on days approaching delivery, ensuring full contact with the swabs. All samples were then homogenized, subjected to DNA extraction using the HiPure Stool DNA Kit B (Magen Biotech, Guangzhou, China). The 16S rDNA V4 region was then amplified, subjected to library construction, and sequenced in the IonS5\u003csup\u003eTM\u003c/sup\u003eXL platform (Thermo Fisher Scientific, Beijing, China).\u003c/p\u003e \u003cp\u003eData analysis was performed using the QIIME 2 (Quantitative Insights into Microbial Ecology)\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Chimeric sequences were detected using UCHIME algorithm, and reads were clustered into OTUs using the Uparse software (v7.0.1001, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://drive5.com/uparse/\u003c/span\u003e\u003cspan address=\"http://drive5.com/uparse/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with cutoff value set as 97% in similarity. Abundance data was normalized according to the sample with the fewest reads.\u003c/p\u003e \u003cp\u003eIn-house Perl scripts in QIIME 2 were used to analyze alpha-diversity (within samples) and beta-diversity (among samples). R (version 4.3.1) with the built-in function \u0026ldquo;prcomp\u0026rdquo; was used to perform Principal Component Analysis (PCA). Statistical significance of sample groupings was determined by adonis method. The graphical presentation was conducted using OriginLab 2024b. All 16S rRNA sequencing data are publicly available (PRJNA1032734, 1033214, 1033221, 1033224, 1146173 and 1033320).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetagenomic sequencing and analysis.\u003c/b\u003e Metagenomic sequencing was used to detect the microbiome transmission during LGR-1 intervention. A total amount of 1 \u0026micro;g DNA per sample was used as input material for library generation using NEBNext Ultra\u0026trade; DNA Library Prep Kit for Illumina (NEB, Ipswich, USA). Briefly, the DNA sample was fragmented by sonication to a size of 350 bp, which was further end-polished, A-tailed and ligated with the full-length adaptor for Illumina sequencing, followed by PCR amplification. The resulting PCR products were purified using the AMPure XP system (Beckman Coulter Diagnostics, Brea, USA). The libraries were analyzed for size distribution by Agilent2100 Bioanalyzer and quantified with qPCR. Amplified and barcoded libraries were then pooled in approximately equimolar ratios before being sequenced on an Illumina Hiseq platform 4000, generating pair-end reads.\u003c/p\u003e \u003cp\u003eThe raw reads (6377.41\u0026thinsp;\u0026plusmn;\u0026thinsp;36.59 Mbp, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) were examined, with low-quality and unwanted reads (0.168\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017%, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) filtered by Bowtie2.2.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bowtiebio.sourceforge.net/bowtie2/index.shtml\u003c/span\u003e\u003cspan address=\"http://bowtiebio.sourceforge.net/bowtie2/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). MEGAHIT (v1.0.4-beta) was employed to assemble the clean data. For gene prediction, MetaGeneMark (V2.10, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://topaz.gatech.edu/GeneMark/\u003c/span\u003e\u003cspan address=\"http://topaz.gatech.edu/GeneMark/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was run to filter out sequences shorter than 100 nucleotides from the predicted results with default parameters. Subsequently, non-redundant, unique initial genes (Unigene) were derived with the assistance of the CD-HIT software (V4.5.8, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.org/cd-hit\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.org/cd-hit\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaxonomy prediction was performed using DIAMOND software (V0.9.9, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/bbuchfink/diamond/\u003c/span\u003e\u003cspan address=\"https://github.com/bbuchfink/diamond/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), via blasting the Unigenes against sequences of Bacteria extracted from the NR database (Version: 2018-01-02, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The functional gene pathways were profiled by blasting Unigenes against functional databases including KEGG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.kegg.jp\u003c/span\u003e\u003cspan address=\"http://www.kegg.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and CAZy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cazy.org\u003c/span\u003e\u003cspan address=\"http://www.cazy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The best Blast Hit was used for subsequent analysis.\u003c/p\u003e \u003cp\u003eTo perform further data-mining at the species- and strain-level, StrainPhlAn (an integral part of MetaPhlAn, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/biobakery/MetaPhlAn\u003c/span\u003e\u003cspan address=\"https://github.com/biobakery/MetaPhlAn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to identify a tandem of conserved and unique markers, enabling the retrieval of taxonomic profiles and inter-sample genetic distances by detecting single nucleotide polymorphisms (SNPs) for each identified taxon. 12 taxa were then recovered with a minimum prevalence of 16 across all samples. The genetic distance for each taxon was then derived according to the RAxML algorithm. For one specified taxon, genetic distances between the tested dam-pup pairs were counted, while normalized genetic distances (nGDs) were calculated as data normalized by the median of all genetic distances. To depict the strain distribution within samples and groups, some reference strains of the subject species were incorporated into phylogenetic analysis and comparisons. The cutoff value for strain similarity is set as the lowest 15% of all genetic distances incorporated.\u003c/p\u003e \u003cp\u003eThe maximum likelihood phylogenetic tree of the top 100 abundant species was established by PhyloPhlAn (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/biobakery/phylophlan\u003c/span\u003e\u003cspan address=\"https://github.com/biobakery/phylophlan\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All relevant phylogenetic data was subjected to iTOL (interactive tree of life)\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e for graphical presentation. In terms of microbiome transmission rate, nTR (normalized transmission rate) was calculated as the ratio of inter-pair nGDs to the total (inter-pair plus intra-pair) nGDs. The metagenomic data is available at PRJNA1033296.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunohistochemistry.\u003c/b\u003e Immunohistochemistry was performed as previously described with some modifications\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were anesthetized and perfused with 10 ml 0.9% sodium chloride followed by 30 ml 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer. The separated brain tissues were post-fixed in 4% PFA at 4\u003csup\u003eo\u003c/sup\u003eC for 24 h, and then cryoprotected in 30% sucrose till tissue deposition. Coronal slices (40 \u0026micro;m thick) were obtained from frozen tissue with a sliding blade microtome and rinsed in PBS containing 0.7% of Triton X-100 (PBSTX; Aladdin Scientific) for 8 h to penetrate the plasma membrane. Slices were blocked with 10% fetal bovine serum (FBS) for 2 h and then incubated in primary antibodies overnight. Primary antibodies were visualized using second antibodies rotating in the dark for 1 h at 4\u003csup\u003eo\u003c/sup\u003eC. Subsequently, the nucleus was stained using DAPI Staining solution (1:5000; Biosharp, Hefei, China), rinsed and mounted with Antifade Mounting Medium (Beyotime Biotechnology, Shanghai, China). Fluorescent imaging and data acquisition were performed on a Nikon C2 Confocal Microscope. The primary antibody used here is Rb Anti-Iba1 (Abcam, AB_2636859) with a dilution of 1:400, and the second antibody is Cy3-goat-anti-rabbit IgG (1:200; Proteintech, AB_2890957).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMetabolomic analysis targeting SCFAs (short-chain fatty acids).\u003c/b\u003e The stock solution of individual SCFAs (Novogene, Beijing, China) were mixed and prepared in SCFA-free matrix to obtain a series of SCFA calibrators. The fecal samples of various treatment groups were first resuspended with liquid nitrogen, homogenized with methanol (80%) and centrifuged at 11000 g for 10 min to remove the protein. The supernatant was then subjected to derivatization, dilution with 80% methanol and homogenization with 5 \u0026micro;l mixed internal standard solution.\u003c/p\u003e \u003cp\u003eAn ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) system (Vanquish Flex UHPLC-TSQ Altis, Thermo, Dreieich, Germany) was used to quantify SCFAs. Separation was performed on a C18 column (2.1 \u0026times; 100 mm, 1.7 \u0026micro;m, Waters, Milford, USA). The mobile phase consisted of 10 mM ammonium acetate in water (solvent A) and acetonitrile: isopropanol (1:1). The mass spectrometer was operated in negative multiple reaction mode (MRM). Parameters were set as follows: ionspray voltage (-4500 V), sheath gas (35 psi), ion source temp (550\u0026deg;C), auxiliary gas (50 psi) and collision gas (55 psi).\u003c/p\u003e \u003cp\u003e \u003cb\u003eH\u0026amp;E staining.\u003c/b\u003e Hematoxylin and eosin (H\u0026amp;E) staining was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, the colons were dissected from the anesthetized mice, with fat removed using aseptic forceps. The tissues were then fixed in 4% paraformaldehyde solution at 4\u003csup\u003eo\u003c/sup\u003eC for 48 h, followed by soaking in liquid paraffin at 65\u003csup\u003eo\u003c/sup\u003eC for 1 h and air-dried. The slices (5 \u0026micro;m thick) were obtained using a microtome, and subjected to gradient dehydration with 75, 80, 95 and 100% ethanol. The tissue sections were stained with H\u0026amp;E and observed with a light microscope (Eclipse 80i; Nikon, Tokyo, Japan).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFlow cytometry.\u003c/b\u003e Spleens were dissected from the sacrificed mice and rinsed in Hanks' Balanced Salt Solution (HBSS; Thermo Fisher Scientific, Beijing, China). The excised and fat-free tissues were incubated with collagenase D (Thermo Fisher Scientific, Beijing, CHina), strained and filtered to prepare cell suspensions. For flow cytometry, the cells were stained with Zombie Aqua (1:1000; BioLegend), Anti-Mouse CD16/32 (TruStain FcX\u0026trade;, 1:200; BioLegend, AB_1574973), CoraLite594-conjugated-Anti-Mouse CD3 (1:200; Proteintech, 17A2, AB_3064914), and BV421-conjugated-Anti-Mouse CD4 (1:200; Proteintech, RM4-4, AB_3064915). For intracellular staining, cells were treated with PMA (50 ng/ml; Thermo Fisher Scientific), ionomycin (500 ng/ml; Thermo Fisher Scientific) and Brefeldin A solution (1Х; BioLegend) for restimulation. The cells were then permeabilized and stained with PE-conjugated-Anti-Mouse IFNγ (1:200; Proteintech, XMG1.2, AB_2883916) and PerCP-Cy5.5-conjugated IL-17A antibody (1:200; BioLegend, eBio17B7, AB_2565780) using Intracellular Fixation/Permeabilization Buffer (E-CK-A109; Elabscience). Flow cytometric analysis was performed on an FACSAria III (BD Biosciences, New Jersey, USA). All data were re-analyzed using FlowJo software (BD Biosciences, New Jersey, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrophysiological recordings.\u003c/b\u003e mEPSC recordings were performed as described previously with some modifications\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Briefly, coronal slices of mPFC were prepared (300 \u0026micro;m thick) and transferred to oxygenated artificial cerebrospinal fluid (ACSF), containing 119 mM NaCl, 2.5 mM KCl, 1 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 1.3 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 26.2 mM NaHCO\u003csub\u003e3\u003c/sub\u003e and 11 mM glucose. The incubation was performed at 34\u003csup\u003eo\u003c/sup\u003eC for 30 min and subsequently at 27\u003csup\u003eo\u003c/sup\u003eC for 1 h. Afterwards, slices were placed in a recording chamber perfused constantly with ACSF at a flow rate of 2 ml/min. After 5 minutes of equilibration, the recording was initiated by identifying layer V pyramidal neurons in mPFC based on cell size and morphology. Patch pipettes (3\u0026thinsp;~\u0026thinsp;6 MΩ) were filled with an internal solution containing 110 mM potassium gluconate, 40 mM KCl, 10 mM HEPES, 3 mM MgATP, 0.5 mM Na\u003csub\u003e2\u003c/sub\u003eGTP and 0.2 mM EGTA. ACSF was supplemented with 1 \u0026micro;M tetrodotoxin and 250 \u0026micro;M picrotoxin to record mEPSC. In terms of mIPSC, the internal solution was replaced with 140 mM CsCl, 2 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 5 mM EGTA, 10 mM HEPES, 0.36 mM Na\u003csub\u003e3\u003c/sub\u003eGTP and 4.39 mM Na\u003csub\u003e2\u003c/sub\u003eATP. ACSF was added with 1 \u0026micro;M tetrodotoxin, 10 \u0026micro;M CNQX and 50 \u0026micro;M APV to sequester and record mIPSC.\u003c/p\u003e \u003cp\u003eRecordings were low-pass filtered at 2 kHz and acquired using an Multiclamp 700B amplifier (Molecular Devices, San Jose, USA) in conjunction with pClamp 11 software (Molecular Devices, San Jose, USA). Only cells with access resistance\u0026thinsp;\u0026lt;\u0026thinsp;25 MΩ and input resistance\u0026thinsp;\u0026gt;\u0026thinsp;100 MΩ were selected for analysis. Visual inspection of detected signals was allowed to reject noise artifacts.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e Data were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from at least three biological replicates with at least two independent experiments. Unless otherwise stated, statistical analyses performed include Student\u0026rsquo;s t-test, one-way ANOVA with Tukey post hoc analysis, or two-way ANOVA with Sidak post hoc analysis. The number of samples (\u003cem\u003en\u003c/em\u003e) and \u003cem\u003eP\u003c/em\u003e values were presented in the figure legends. \u003cem\u003eP\u003c/em\u003e values were FDR-corrected when multiple comparisons (\u0026gt;\u0026thinsp;3) were performed. Analysis was performed and presented using GraphPad\u0026rsquo;s Prism (version 8.0) and OriginLab (version 2024b). Paired t-test was used to compare nGDs for the specified taxon. For the three-chamber experimental data, paired t test (two groups, Mann-Whitney U test for not-normally distributed data) or two-way ANOVA (\u0026gt;\u0026thinsp;3 groups) with post hoc Sidak comparisons were performed to analyze the same mouse\u0026rsquo;s traces in the left and right chambers. Unpaired t-test was applied in other comparisons between two groups. One-way ANOVA was performed when 3 or more treatment groups were involved in the statistics except the 3-chamber trial.\u003c/p\u003e \u003cp\u003eAll statistical analysis for 16S rRNA data was performed with QIIME 2. Alpha- and beta- diversity were analyzed with the in-house Perl scripts, while PCA was performed using the R software (version 4.3.1) with the built-in function \u0026ldquo;prcomp\u0026rdquo;.\u003c/p\u003e \u003cp\u003eFor metagenomic analysis, DIAMOND was conducted to indicate enrichment in pathways of KEGG and CAZy. StrainPhlAn was used to detect species or strains based on SNPs across most treatment samples. The genetic distances were deduced based on RAxML algorithm, which were further normalized and compared through paired t-test.\u003c/p\u003e \u003cp\u003e*\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, **\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01 and ***\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.001 were considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eH.L.W. and Y.X. conceived project and designed the study; R.Y., J.X. and Y.X. conducted most experiments and analyzed the sequencing data; C.H. performed the electrophysiological trials and the relevant analysis; F.Z. and T.W. conducted some animal trails and did the relevant analysis; R.K. aided in the animal studies; J.X. aided in the data visualization; X.G. conducted the functional test of LGR-1 and supervised the research; Y.X. and R.Y. summarized data and generated figures; Y.X. and H.L.W. wrote the manuscript. All authors reviewed and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by National Natural Science Foundation of China (no. 81673624), Fundamental Research Funds for the Central Universities (no. JZ2020HGTB0053), and Anhui Provincial Key Research and Development Plan (no. 201904e01020001). We thank Dr. V. Pedicord and Dr. S. Suyama from University of Cambridge for the constructive advice to improve this manuscript; Prof. H. Tao, Prof. G. Liu and Dr. Z. Liu from Hefei University of Technology for the utility of animal facility; Dr. N. Bi for the technological advice for the electrophysiological recordings.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eMetagenomic and 16S rRNA-seq results have been deposited in the ncbi database, assigned with accession number of PRJNA1033296 (metagenomics), PRJNA1033320, 1032734, 1033214, 1033221, 1146173 and 1033224 (16S rRNA-seq); Unprocessed data is deposited in the Mendeley database assigned with URL of \u0026ldquo;https://doi.org/10.17632/85vrrttt4y.1\u0026rdquo;.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to Yi Xu or Hui-Li Wang.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCryan JF, \u003cem\u003eet al.\u003c/em\u003e The Microbiota-Gut-Brain Axis. Physiol Rev 99, 1877\u0026ndash;2013 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaenner MJ, \u003cem\u003eet al.\u003c/em\u003e Prevalence and Characteristics of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2020. MMWR Surveill Summ 72, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao E, \u003cem\u003eet al.\u003c/em\u003e TAU ablation in excitatory neurons and postnatal TAU knockdown reduce epilepsy, SUDEP, and autism behaviors in a Dravet syndrome model. Sci Transl Med 14, eabm5527 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, \u003cem\u003eet al.\u003c/em\u003e De novo genic mutations among a Chinese autism spectrum disorder cohort. Nat Commun 7, 13316 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, \u003cem\u003eet al.\u003c/em\u003e Genetic evidence of gender difference in autism spectrum disorder supports the female-protective effect. Transl Psychiatry 10, 4 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSgritta M, \u003cem\u003eet al.\u003c/em\u003e Mechanisms Underlying Microbial-Mediated Changes in Social Behavior in Mouse Models of Autism Spectrum Disorder. Neuron 101, 246\u0026ndash;259 e246 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLou M, \u003cem\u003eet al.\u003c/em\u003e Deviated and early unsustainable stunted development of gut microbiota in children with autism spectrum disorder. Gut, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYap CX, \u003cem\u003eet al.\u003c/em\u003e Autism-related dietary preferences mediate autism-gut microbiome associations. Cell 184, 5916\u0026ndash;5931 e5917 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColey-O'Rourke EJ, Hsiao EY. Microbiome alterations in autism spectrum disorder. Nature Microbiology 8, 1615\u0026ndash;1616 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharon G, \u003cem\u003eet al.\u003c/em\u003e Human Gut Microbiota from Autism Spectrum Disorder Promote Behavioral Symptoms in Mice. Cell 177, 1600\u0026ndash;1618 e1617 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuffington SA, Di Prisco GV, Auchtung TA, Ajami NJ, Petrosino JF, Costa-Mattioli M. Microbial Reconstitution Reverses Maternal Diet-Induced Social and Synaptic Deficits in Offspring. Cell 165, 1762\u0026ndash;1775 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi GB, \u003cem\u003eet al.\u003c/em\u003e The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring. Science 351, 933\u0026ndash;939 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim S, \u003cem\u003eet al.\u003c/em\u003e Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring. Nature 549, 528\u0026ndash;532 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebovitz Y, \u003cem\u003eet al.\u003c/em\u003e Lactobacillus rescues postnatal neurobehavioral and microglial dysfunction in a model of maternal microbiome dysbiosis. Brain Behav Immun 81, 617\u0026ndash;629 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi Z, \u003cem\u003eet al.\u003c/em\u003e A Novel and Reliable Rat Model of Autism. Front Psychiatry 12, 549810 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlsayouf HA, Talo H, Biddappa ML, De Los Reyes E. Risperidone or Aripiprazole Can Resolve Autism Core Signs and Symptoms in Young Children: Case Study. Children (Basel) 8, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLord C, Elsabbagh M, Baird G, Veenstra-Vanderweele J. Autism spectrum disorder. Lancet 392, 508\u0026ndash;520 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHsiao EY, \u003cem\u003eet al.\u003c/em\u003e Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155, 1451\u0026ndash;1463 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTabouy L, \u003cem\u003eet al.\u003c/em\u003e Dysbiosis of microbiome and probiotic treatment in a genetic model of autism spectrum disorders. Brain Behav Immun 73, 310\u0026ndash;319 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, \u003cem\u003eet al.\u003c/em\u003e Gut microbiota shapes social dominance through modulating HDAC2 in the medial prefrontal cortex. Cell Rep 38, 110478 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrova MI, Reid G, Ter Haar JA. Lacticaseibacillus rhamnosus GR-1, a.k.a. Lactobacillus rhamnosus GR-1: Past and Future Perspectives. Trends Microbiol 29, 747\u0026ndash;761 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu X, \u003cem\u003eet al.\u003c/em\u003e Probiotic Lactobacillus rhamnosus GR-1 supplementation attenuates Pb-induced learning and memory deficits by reshaping the gut microbiota. Front Nutr 9, 934118 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y, \u003cem\u003eet al.\u003c/em\u003e Changes to gut amino acid transporters and microbiome associated with increased E/I ratio in Chd8(+/-) mouse model of ASD-like behavior. Nat Commun 13, 1151 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerretti P, \u003cem\u003eet al.\u003c/em\u003e Mother-to-Infant Microbial Transmission from Different Body Sites Shapes the Developing Infant Gut Microbiome. Cell Host Microbe 24, 133\u0026ndash;145 e135 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Palma G, \u003cem\u003eet al.\u003c/em\u003e Transplantation of fecal microbiota from patients with irritable bowel syndrome alters gut function and behavior in recipient mice. Sci Transl Med 9, (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrova MI, Reid G, Ter Haar JA. Lacticaseibacillus rhamnosus GR-1, a.k.a. Lactobacillus rhamnosus GR-1: Past and Future Perspectives. Trends Microbiol 29, 747\u0026ndash;761 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominguez-Bello MG, \u003cem\u003eet al.\u003c/em\u003e Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 107, 11971\u0026ndash;11975 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReid G, \u003cem\u003eet al.\u003c/em\u003e Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 35, 131\u0026ndash;134 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlacher E, \u003cem\u003eet al.\u003c/em\u003e Potential roles of gut microbiome and metabolites in modulating ALS in mice. Nature 572, 474\u0026ndash;480 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnsaldo E, \u003cem\u003eet al.\u003c/em\u003e Akkermansia muciniphila induces intestinal adaptive immune responses during homeostasis. Science 364, 1179\u0026ndash;1184 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaormina VM, Unger AL, Schiksnis MR, Torres-Gonzalez M, Kraft J. Branched-Chain Fatty Acids-An Underexplored Class of Dairy-Derived Fatty Acids. \u003cem\u003eNutrients\u003c/em\u003e 12, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDerrien M, Vaughan EE, Plugge CM, de Vos WM. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int J Syst Evol Microbiol 54, 1469\u0026ndash;1476 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeltzer A, Van de Water J. The Role of the Immune System in Autism Spectrum Disorder. Neuropsychopharmacology 42, 284\u0026ndash;298 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShin Yim Y, \u003cem\u003eet al.\u003c/em\u003e Reversing behavioural abnormalities in mice exposed to maternal inflammation. Nature 549, 482\u0026ndash;487 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuybari KB, \u003cem\u003eet al.\u003c/em\u003e Sex dependent alterations of resveratrol on social behaviors and nociceptive reactivity in VPA-induced autistic-like model in rats. Neurotoxicol Teratol 81, 106905 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstes ML, McAllister AK. Brain, Immunity, Gut: \"BIG\" Links between Pregnancy and Autism. Immunity 47, 816\u0026ndash;819 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuriel-Ohayon M, Neuman H, Koren O. Microbial Changes during Pregnancy, Birth, and Infancy. Front Microbiol 7, 1031 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, \u003cem\u003eet al.\u003c/em\u003e Vertical Transmission of Gut Microbiome and Antimicrobial Resistance Genes in Infants Exposed to Antibiotics at Birth. J Infect Dis 224, 1236\u0026ndash;1246 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYassour M, \u003cem\u003eet al.\u003c/em\u003e Strain-Level Analysis of Mother-to-Child Bacterial Transmission during the First Few Months of Life. Cell Host Microbe 24, 146\u0026ndash;154 e144 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao Y, \u003cem\u003eet al.\u003c/em\u003e Stunted microbiota and opportunistic pathogen colonization in caesarean-section birth. Nature 574, 117\u0026ndash;121 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrova MI, \u003cem\u003eet al.\u003c/em\u003e Comparative Genomic and Phenotypic Analysis of the Vaginal Probiotic Lactobacillus rhamnosus GR-1. Front Microbiol 9, 1278 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBokulich NA, \u003cem\u003eet al.\u003c/em\u003e Antibiotics, birth mode, and diet shape microbiome maturation during early life. Sci Transl Med 8, 343ra382 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurran EA, \u003cem\u003eet al.\u003c/em\u003e Research review: Birth by caesarean section and development of autism spectrum disorder and attention-deficit/hyperactivity disorder: a systematic review and meta-analysis. J Child Psychol Psychiatry 56, 500\u0026ndash;508 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharon G, Sampson TR, Geschwind DH, Mazmanian SK. The Central Nervous System and the Gut Microbiome. Cell 167, 915\u0026ndash;932 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlamoudi MU, Hosie S, Shindler AE, Wood JL, Franks AE, Hill-Yardin EL. Comparing the Gut Microbiome in Autism and Preclinical Models: A Systematic Review. Front Cell Infect Microbiol 12, 905841 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Li J, Wu F, Zheng H, Peng Q, Zhou H. Altered composition and function of intestinal microbiota in autism spectrum disorders: a systematic review. Transl Psychiatry 9, 43 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEverard A, \u003cem\u003eet al.\u003c/em\u003e Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 110, 9066\u0026ndash;9071 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelzer C, de Vos WM. Microbes inside\u0026ndash;from diversity to function: the case of Akkermansia. ISME J 6, 1449\u0026ndash;1458 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim JS, Lim MY, Choi Y, Ko G. Modeling environmental risk factors of autism in mice induces IBD-related gut microbial dysbiosis and hyperserotonemia. Mol Brain 10, 14 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKazlauskas N, Seiffe A, Campolongo M, Zappala C, Depino AM. Sex-specific effects of prenatal valproic acid exposure on sociability and neuroinflammation: Relevance for susceptibility and resilience in autism. Psychoneuroendocrinology 110, 104441 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiLiberto E, Phatarpekar S, Theodorakis K, Chadman KK. Does the stranger mouse strain matter to female BTBR mice? Behavioural Brain Research 437, 114132 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReed MD, \u003cem\u003eet al.\u003c/em\u003e IL-17a promotes sociability in mouse models of neurodevelopmental disorders. Nature 577, 249\u0026ndash;253 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaporaso JG, \u003cem\u003eet al.\u003c/em\u003e QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7, 335\u0026ndash;336 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiccarelli FD, Doerks T, von Mering C, Creevey CJ, Snel B, Bork P. Toward automatic reconstruction of a highly resolved tree of life. Science 311, 1283\u0026ndash;1287 (2006).\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":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5930312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5930312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs a prevalent neurodevelopmental disease, whether ASD (autism spectrum disorder) can be ameliorated by the early use of a single microbe remains unknown. Here we report that \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e GR-1 (LGR-1) prevented the occurrence of autism-like behaviors when administered exclusively to the pregnant mice, as evidenced by the improved behaviors and restored E/I balance in the prefrontal cortex of male pups. In parallel, the offspring microbiome was reshaped by LGR-1 treatment, mediated by the vertical transmission of maternal microbiome, with its roles validated by microbiota transplant and cross-fostering. In addition to gut commensals, the LGR-1-shaping vaginal microbiota also contributed to the establishment of \u0026ldquo;beneficial\u0026rdquo; microbiome. Regarding key taxa in offspring, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e was influenced by LGR-1 and exerted effect on the ensuing behavior, through modulating immune pathways related to IL-17-producing cell population. In conclusion, a single microbe applied \u003cem\u003ein utero\u003c/em\u003e protects offspring from autism-like behaviors via microbiome transmission, shedding light on the microbe-based avenue to mitigate the risk of ASD.\u003c/p\u003e","manuscriptTitle":"Lacticaseibacillus rhamnosus GR-1 prevents autism-like behaviors by reshaping the maternal and offspring microbiome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 09:49:26","doi":"10.21203/rs.3.rs-5930312/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-19T13:02:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-16T02:41:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-10T10:08:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-08T18:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49632739956112626861631649978626881915","date":"2025-02-26T19:44:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308727925706982164607187856882035589472","date":"2025-02-23T11:13:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296336170564943555442065425320992725338","date":"2025-02-21T09:26:59+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-21T08:35:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-15T15:11:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-06T06:05:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Biofilms and Microbiomes","date":"2025-01-30T12:31:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-biofilms-and-microbiomes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjbiofilms","sideBox":"Learn more about [npj Biofilms and Microbiomes](http://www.nature.com/npjbiofilms/)","snPcode":"41522","submissionUrl":"https://submission.springernature.com/new-submission/41522/3","title":"npj Biofilms and Microbiomes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fead0841-bf6b-4552-9178-0d42cb1cd20c","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43919992,"name":"Biological sciences/Microbiology/Communities/Microbiome"},{"id":43919993,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2025-09-29T16:00:46+00:00","versionOfRecord":{"articleIdentity":"rs-5930312","link":"https://doi.org/10.1038/s41522-025-00808-5","journal":{"identity":"npj-biofilms-and-microbiomes","isVorOnly":false,"title":"npj Biofilms and Microbiomes"},"publishedOn":"2025-09-24 15:57:39","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-02-07 09:49:26","video":"","vorDoi":"10.1038/s41522-025-00808-5","vorDoiUrl":"https://doi.org/10.1038/s41522-025-00808-5","workflowStages":[]},"version":"v1","identity":"rs-5930312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5930312","identity":"rs-5930312","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}