Gut Microbiota-Mediated Modulation of Neurodevelopmental Behavior in CLCN4- Deficient Mice

Gut Microbiota-Mediated Modulation of Neurodevelopmental Behavior in CLCN4- Deficient Mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Gut Microbiota-Mediated Modulation of Neurodevelopmental Behavior in CLCN4- Deficient Mice Yura Choi, Shambhunath Bose, Ji-Hong Oh, Eun-Ji Song, Young-Do Nam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7514097/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neurodevelopmental disorders (NDDs) are associated with gut–brain axis dysfunction, and chloride ion channel 4 (CLCN4) has been implicated in their pathology. We investigated whether CLCN4 knockout (KO) alters gut microbiota and contributes to NDD-like phenotypes in mice. CLCN4-KO mice displayed behavioral abnormalities, microbial dysbiosis, and increased serum p-cresol levels, along with altered hippocampal signaling proteins (PSD95, AKT, ERK). Treatment with haloperidol (Halo) modified gut microbiota, reduced p-cresol, and improved behavior, effects accompanied by increased hippocampal protein activation in homozygous KO mice but abolished by antibiotic-induced dysbiosis. Prevotellaceae_UCG-001 abundance correlated positively with hippocampal protein activation and negatively with hyperactivity, and Halo treatment significantly increased this population. Fecal microbiota transplantation (FMT) from wild-type mice restored gut microbial balance, memory, and protein phosphorylation in KO mice. These findings indicate that CLCN4 deficiency contributes to NDD-like behaviors via microbiota-mediated mechanisms and highlight Halo and FMT as promising microbiota-targeted strategies. Health sciences/Diseases Biological sciences/Microbiology Biological sciences/Neuroscience CLCN4 knockout Neurodevelopmental disorders Gut microbiota Gut–brain axis Fecal microbiota transplantation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Background Neurodevelopmental disorders (NDDs), also referred to as Raynaud–Claes syndrome, are a group of conditions characterized by deficits in nervous system development, affecting memory, language, motor function, and the ability to learn, socialize, and maintain self-control 1 . These deficits often manifest as behavioral and mental health problems. Initially, NDD was identified as a rare X-linked disorder associated with global developmental delay or intellectual disability, language delay, autism spectrum disorder (ASD), anxiety, hyperactivity, epilepsy, brain abnormalities, bipolar disorder, and facial dysmorphism 2 – 4 . Behavioral problems in NDD are commonly linked to social–emotional difficulties influenced by familial and early educational backgrounds. These problems are categorized into internalizing symptoms, which include emotional disturbances such as depression and anxiety, and externalizing symptoms, which involve behavioral and hyperactivity-related issues 5 . Ion channels in axons regulate the movement of ions, such as Na + , K + , Ca 2+ , and Cl − across the axolemma 6 , 7 , playing a crucial role in neuronal functions, including action potential initiation and propagation as well as neurotransmitter release 6 , 8 . Chloride ion channels (CLCs) constitute a family of proteins essential for various physiological processes 9 . Chloride ion channel 4 ( CLCN4 ), a voltage-dependent 2Cl − / H + exchanger encoded by the CLCN4 gene, is a member of this family 10 . In most mammals, including humans and wild Mediterranean mice ( Mus spretus ), the CLCN4 gene is X-linked, whereas in the inbred C57BL/6 laboratory mouse strain ( Mus musculus ), it is autosomal, located on chromosome 7, and contains truncated introns 11 , 12 . Although CLC4 is expressed in multiple tissues, its predominant expression is in the nervous system 3 , 13 , particularly in the hippocampus and cerebellum 10 . Additionally, CLC4 is implicated in intracellular pH regulation 14 , 15 , cell volume maintenance 9 and ion homeostasis 16 , intracellular trafficking 17 , and neuronal differentiation 18 . The dysfunction of some CLC proteins, including CCL4, manifests as lysosomal storage diseases and severe neurological disorders, including leukodystrophy and neurodegeneration 10 . More than 60 different CLCN4 variants have been recorded in the Human Gene Mutation Database, with missense variants being the most common 2 . Pathogenic variants of CLCN4 impair Cl − /H + exchange activity, disrupt homeostasis and intracellular vesicular transport, alter protein function, and affect neuronal differentiation 19 . These variants have been linked to the development of X-linked NDD, presenting with symptoms such as global developmental delay, intellectual disability, epilepsy, behavioral disorders, mental health disorders, and dysmorphic features, predominantly in males 10 , 20 , 21 . Recent studies showed that CLCN4 knockout (KO) in C57BL/6 mice manifests neurodevelopmental endophenotypes consistent with ASD in humans 22 . The gut microbiota, a complex community of microorganisms, has gained attention for its role in health and disease 23 , 24 . Dysbiosis, or microbial imbalance, has been associated with the pathogenesis of various disorders, including inflammatory bowel disease, diabetes, obesity, and cardiovascular disease 25 . Notably, NDDs, including autism and attention deficit hyperactivity disorder (ADHD), have also been linked to alterations in gut microbiota composition 26 . The gut–brain axis (GBA), a bidirectional communication network between the central and enteric nervous systems, facilitates connections between the emotional and cognitive centers of the brain and peripheral intestinal functions 27 , 28 . This connection operates through neural, endocrine, immune, and humoral pathways, allowing gut microbes to influence brain development, cognition, and behavior 28 – 31 . Studies in both mice 32 , 33 and humans 34 have demonstrated the significant role of commensal bacteria in modulating social, emotional, and anxiety-related behaviors. In a maternal immune activation mouse model on a C57BL/6N background with ASD-like features, gut microbiota alterations regulated behavioral and physiological abnormalities associated with NDD 35 . Additionally, elevated systemic levels of gut microbial metabolites have been implicated in anxiety-like behavior, reinforcing the molecular connection between gut microbiota and GBA in ASD and other NDDs. Among these metabolites, p-cresol (4-methylphenol), a product of bacterial fermentation of dietary tyrosine and phenylalanine in the colon 36 , undergoes extensive conjugation to form p-cresyl sulfate (p-CS) and p-cresyl glucuronide (p-CG). Children with autism have higher p-cresol, p-CS, and p-CG levels in urine samples than healthy controls 37 indicating the involvement of these metabolites in ASD. This study hypothesized that CLCN4 KO in C57BL/6 mice alters gut microbiota composition, leading to NDD-like phenotypes. To investigate this hypothesis and elucidate potential mechanisms, behavioral tests, gut microbial analysis, fecal microbial transplantation (FMT), and analyses of expression levels of postsynaptic density-95 (PSD95), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) proteins—key regulators in NDD pathogenesis 38 – 40 —were conducted. Additionally, the effect of haloperidol (Halo)—an antipsychotic that alleviates behavioral symptoms and improves clinical outcomes in ASD 41 – 43 —was examined in relation to both behavior and gut microbiota in CLCN4 KO (CLCN4-KO) mice. Serum p-cresol levels were also measured to explore their potential role as a mediator linking gut microbiota alterations to NDD pathogenesis. The findings of this study have significant implications. By identifying the intricate relationships among CLC4, NDD, and the gut microbiota, this study aims to provide comprehensive insights that could direct future research and therapeutic strategies. Therefore, this study lays the foundation for developing novel therapeutic strategies, expanding treatment options for NDD, and advancing precision medicine approaches in NDDs. 2. Methods 2.1. Generation of CLCN4-KO mice CLCN4-KO mice were purchased from Toolgen Inc. (Seoul, Republic of Korea). This mouse model, which has a deletion in exon 5 of CLC4N on chromosome 7, was generated using clustered regularly interspaced short palindromic repeats/Cas9 genome-editing technology on a C57BL/6N genetic background (Fig. 1 ). Male wild-type (WT) C57BL/6N and female homozygous (Ho) CLCN4-KO mice were mated to produce heterozygous (He) CLCN4-KO mice. CLC4 gene deficiency in KO animals was confirmed through PCR, as described in section 2.2 . Male WT mice were used as controls for the entire study. All animals were housed under controlled temperatures (22 ± 1 ℃) and relative humidity (40–60%) with a 12 h light/12 h dark cycle (light on at 9:00 a.m.) and allowed ad libitum access to a standard normal chow diet (Soyagreentec, Hwaseong-Si, Gyeonggi-do, South Korea) and water. All animal study procedures, including animal care and handling, were performed according to international guidelines (Guide for the Care and Use of Laboratory Animals, Institute of laboratory Animal Resources, Commission on Life Sciences, National Research Council, USA; National Academy Press: Washington D.C., 1996). The aim, outline, protocols, and ethical aspects of this study were approved by the Institutional Animal Care and Use Committee of Dongguk University (approval number: IACUC-2022-047-1). To avoid potential confounding effects of anesthesia on brain physiology, animals were euthanized by cervical dislocation. 2.2. Confirmation of CLCN4 gene deletion A PCR analysis using genomic DNA extracted from the ear tissue of the KO mice was performed to confirm the deletion of CLCN4 in the mice. Extraction was performed using tissue lysis buffer supplemented with proteinase K (Sigma-Aldrich, St. Louis, MO, USA). PCR was performed in a mixture containing Perfect Premix (Bioneer, Daejeon, Republic of Korea), 50 ng of extracted DNA, 1 µL of the forward primer (5′-CAT GTC ATG GGT GTG TCC TC-3′), 1 µL of the reverse primer (5′-TAC TTC ACC CAC GGC TTA CC-3′), and nuclease-free water (Bioneer). Touchdown PCR conditions were as follows: initial denaturation at 95 ℃ for 3 min; 10 cycles at 95 ℃ for 30 s, 72 ℃ for 30 s (-1 ℃/cycle), and 72 ℃ for 45 s; 25 cycles at 95 ℃ for 30 s, 62 ℃ for 30 s, and 72 ℃ 45 s; and final elongation at 72 ℃ for 5 min. After amplification, the final PCR products were electrophoresed on 1% agarose gel. The sizes of the PCR bands were 554 base pairs (bp) for WT, 554 and 422 bp for He, and 422 bp for Ho mice (Fig. 2 ). 2.3. Administration of antibiotics and drugs The animals were treated with Halo according to the schedule to evaluate the effect of this drug on mice (Fig. 4 A and 5 A). Briefly, Halo was dissolved in sterile water and administered by oral gavage at a dose of 1 mg/kg body weight/day for 4 weeks. The dose of this drug was selected based on previous studies 44 . The mice were treated with an antibiotic cocktail to induce gut dysbiosis as previously described 45 , 46 . However, the method of antibiotic administration was modified in the present study because prolonged exposure to antibiotics adversely affects animal health. Briefly, the mice were orally administered 100 µL of an antibiotics cocktail containing vancomycin (500 mg/L, MBcell, Seoul, Republic of Korea), neomycin (500 mg/L, MBcell), ampicillin (500 mg/L, MBcell), metronidazole (500 mg/L, MBcell), and gentamycin (500 mg/L, MBcell) every day for 5 days (Fig. 5 A and Fig. 6 A). 2.4. FMT study Recipient animals received antibiotic treatment for gut dysbiosis before fecal transplantation to perform FMT. Fresh feces from the WT, He, and Ho groups were collected from the anus of the animals and placed in sterile conical tubes. The fecal samples from a particular group were pooled, weighed, and mixed with sterile phosphate-buffered saline at a dilution ratio of 1 mg/10 µL. The mixture was shaken vigorously and centrifuged at 900 × g for 3 min. The supernatant was collected and orally gavaged to the recipient mice (10 µL/g bodyweight) every day for 5 days after completing antibiotic treatment. Fecal samples were freshly prepared on each treatment day. The animal groups used in the experiment and the fecal transplantation regimens are listed in Table 1 . Table 1 S ymbols used for donor and recipient mice before and after fecal microbiota transplantation Donor Recipient Group name 1 WT WT WT-wt 2 He WT WT-he 3 Ho WT WT-ho 4 WT He He-wt 5 He He He-he 6 WT Ho Ho-wt 7 Ho Ho Ho-ho 2.5. Behavioral tests Behavioral tests were performed in mice at five weeks of age to assess the effect of CLCN4 deletion and the responses of CLCN4-KO mice to various treatment types. All experiments were conducted after the animals were allowed to adapt for 30 min in the experimental room. 2.5.1. Open field test The open field test (OFT) is widely used to measure the quality and quantity of exploration and locomotor activity, as well as anxiety-like behavior in rodent models 47 . The OFT was performed using a chamber with dimensions of 45 cm (length) × 45 cm (width) × 45 cm (height). The chamber was composed of white, high-density, and nonporous plastic materials. After the desired treatment schedule, each mouse was placed in the center of the chamber and allowed to move freely for 5 min to allow for adaptation. Subsequently, animal behavior and movements were recorded using a video camera (Samsung, Seoul, Republic of Korea) for 5 min. The total distance traveled and time spent in the central zone of the chamber were automatically recorded and analyzed using an ANY-maze video tracking system (version 5.14; Kim & Friends, Inc., Geumcheon-gu, Republic of Korea). 2.5.2. Passive avoidance test Spatial learning and memory function tests were conducted using the passive avoidance test (PAT) as previously described 48 with some modifications. The test was performed using a two-compartment apparatus consisting of an illuminated dark chamber. Briefly, the mice were placed in the illuminated chamber and allowed to explore for 30 s on the training day. The door was then opened to allow the animals to enter the dark chamber. Three seconds after the mouse entered the dark chamber with all four paws, the door was closed, and the animal was exposed to a foot shock (50 V, 3 s duration). Subsequently, each mouse was removed from the apparatus and transferred to its home cage. On the test day (the day after training), the mice were placed again in the light chamber. After 5 s, the door was opened, and the latency of the animal entering the dark chamber was recorded. 2.5.3. Novel object recognition test The novel object recognition test (NORT) is widely used to evaluate the cognitive ability of rodents. This test was performed using an NORT device identical to the OFT chamber. Each mouse was allowed to habituate to an empty arena for 5 min before the familiarization session. During the familiarization phase, two identical objects were placed 5 cm away from the wall of the chamber and the mouse was allowed to explore each object freely for 5 min. Then, one familiar object was replaced by a new object, and the duration of interest in the new object was recorded for up to 5 min. The object discrimination index percentage was calculated to measure recognition memory in each mouse. 2.6. Western blotting After the desired treatments, the mice were anesthetized, and their brains were surgically removed. Hippocampal tissues were carefully dissected, washed with phosphate-buffered saline, and homogenized on ice in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA) using a Vibra-Cell™ ultrasonic liquid processor (Sonics & Materials Inc., Newtown, CT, USA). The tissue homogenates were centrifuged at 14,000 rpm for 30 min at 4°C, and the supernatants were collected and stored at − 80°C. The protein content of each supernatant was measured using the Bradford assay. An aliquot of 30 µg of protein was denatured at 100 ℃ in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% β-mercaptoethanol. The protein was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel under a constant voltage of 100 V for 90 min and transferred to a 0.45 µm polyvinylidene fluoride membrane (Amersham™, GE Healthcare, Munich, Germany). Membranes were blocked with 5% skim milk (Becton Biosciences, Franklin Lakes, NJ, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min. The membranes were then washed thrice each for 10 min with TBST and incubated overnight with anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-ERK (Thr202/Tyr204), anti-ERK, anti-phospho-PSD95 (Ser295), anti-PSD95 (Cell Signaling Technology, Beverly, MA, USA), and alpha-tubulin antibodies (AbFrontier, Geumcheon, Seoul, South Korea) at 4 ℃ in TBST supplemented with 5% bovine serum albumin. After washing twice with TBST, the membranes were incubated for 90 min with appropriate horseradish peroxidase-conjugated anti-IgG secondary antibodies. Immunoreactive protein bands were detected using a Bio-Rad ChemiDoc XRS imaging system (BioRad, Hercules, CA, USA) with a Super Signal West Pico ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA). Band densities were determined using ImageJ software, which is a public-domain Java image processing program inspired by NIH Image for Macintosh ( https://imagej.net/ij/ ). 2.7. Measurement of serum p-cresol levels Before euthanizing the animals, blood samples were collected from the heart using a 1 mL syringe. The blood was allowed to clot at room temperature and then centrifuged at 1000 × g for 20 min at 4 ℃. The serum was separated and stored at -80 ℃. Serum p-cresol levels were measured using a commercial enzyme-linked immunosorbent assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions. 2.8. 16s rRNA gene sequencing Bacterial genomic DNA was extracted from stool samples using a QIAamp Fast DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Amplification of the V1-V2 region of bacterial 16S rRNA gene sequences was performed using a C1000 Touch Thermal Cycler with a 96-deep-well reaction module (BioRad) under the following conditions: initial denaturation at 94°C for 2 min; 26 amplification cycles (each one with denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min) and an additional extension cycle for 10 min at 72°C. The primer sets used for this reaction contained an eight-base sample-specific barcode to tag the PCR products. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). Finally, purified PCR amplicons (100 ng) tagged with sample-specific barcode sequences were pooled, and sequencing reactions were performed at the Korea Food Research Institute on an Ion Torrent Personal Genome Machine system (Thermo Fisher Scientific) according to the manufacturer’s instructions. High-quality reads were selected for further bioinformatics analyses, and all selected reads from the samples were clustered into operational taxonomic units based on 97% sequence similarity (SILVA database: http://www.arb-silva.de ). Representative sequences were then selected using the Quantitative Insights into Microbial Ecology software package 49 . High-quality raw data were filtered by eliminating sequences lacking the V1-V2 primers or barcode sequence, containing a short read length (< 300 bp) or low-quality reads (average quality score < 20). The raw sequencing reads are publicly available at the NCBI under Project ID PRJNA1247208. 2.9. Analysis of sequenced data A linear discriminant analysis effect size (LEfSe) evaluation was performed using a web-based program ( http://huttenhower.sph.harvard.edu/galaxy ) to identify taxa with differential relative abundance among the experimental groups. The threshold for the logarithmic linear discriminant analysis score was fixed at > 2.0. 2.10. Co-occurrence network analysis Pearson’s correlation among the vital gut microbiota, behavioral indices, hippocampal levels of PSD95, AKT, and ERK, as well as serum p-cresol levels, were analyzed using the SciPy library of the Python programming language 50 with statistical significance set at p < 0.05. The correlation network was visualized using Cytoscape 3.9.1 51 . 2.11. Mutual information analysis Mutual information (MI) analysis is a generic side-channel distinguishing technique. The MI of two random variables is a measure of the relationship and dependence between variables. As MI can determine linear and nonlinear relationships between variables, it can be used to examine the dependence between any number of variables (n-dimensions) 52 . MI analysis was performed using the mutual_info_regression module from the scikit-learn library to investigate the relationship between microbial composition and other variables 53 . The MI values were scaled between 0 and 1, where 0 indicates that the two variables are independent, and the closer they are to 1, the higher the dependency. 2.12. Statistical analysis Data are expressed as the mean ± standard error of the mean (SEM) unless otherwise indicated. Statistical analysis was performed using GraphPad Prism 8 (GraphPad software, La Jolla, CA, USA), and the results were tested for significance using the Student’s t-test. Statistical significance was set at p < 0.05. A heatmap was constructed to demonstrate the relationship among microbial taxa with significant differential abundances across the study groups, as assessed by LEfSe, animal behavioral parameters, vital cell signaling kinases, and serum p-cresol levels using Spearman’s correlation test. For this analysis, only statistically significant correlations (p < 0.05) were considered. The color scale indicates the value of the correlation coefficient, whereas red indicates a positive correlation and green indicates a negative correlation. 3. Results 3.1. Confirmation of CLCN4 genotype The recombination of CLC4 in mice was confirmed by genetic analysis using PCR. The sizes of the resultant PCR bands were 554 bp for WT, 554 and 422 bp for He, and 422 bp for Ho mice (Fig. 2 ). 3.2. CLCN4 deletion causes behavioral disorders and alters gut microbiota in mice The animals were subjected to several tests starting from 5 weeks of age to investigate whether CCLN4 KO could alter behavioral disorders in mice (Fig. 3 A). The OFT (Fig. 3 B), PAT (Fig. 3 C), and NORT (Fig. 3 D) were performed to compare the various behavioral characteristics of the animals among the WT, He, and Ho groups. In the OFT, mice in the He and Ho groups showed significantly higher total distance traveled (locomotor activity) and total time spent in the central area of the open field (anxiety-like behavior) than those in the WT group (Fig. 3 B). Spatial learning and memory in CLCN4-KO mice were investigated using PAT. After the electric shock, the animals in the WT group demonstrated a significantly higher retention latency when entering the dark box in the test session than in the training session. In contrast, mice in the He and Ho groups did not exhibit significant differences in this parameter between the test and training sessions (Fig. 3 C). The time that a mouse spent interacting with a novel or familiar object was assessed using NORT, a commonly used behavioral assay in animals, to evaluate cognitive ability. The mice in both He and Ho groups had significantly lower cognitive function than the animals in the WT group (Fig. 3 D). Furthermore, the serum p-cresol level was significantly higher in the He and Ho groups than in WT mice (Fig. 5 E). Next, 16S rRNA gene sequencing of stool samples was performed to investigate whether the gut microbiota differed among the WT, He, and Ho groups. Principal coordinate analysis (PCoA), a commonly used data analysis platform used to elucidate the β-diversity of the microbial community representing differences in the overall microbial taxonomic profile among the samples, was performed. Analysis of the unweighted UniFrac distance matrix generated from the processed sequence data revealed some overlap yet distinguishable clustering patterns of the gut microbes among the WT, He, and Ho groups (Fig. 3 E, left panel). Furthermore, the He and Ho groups showed significant differences in principal coordinate 1 (PCo1), and the He group demonstrated significant differences in principal coordinate 2 (PCo2) compared to the WT group (Fig. 3 E, right panel). Additionally, a significantly lower Chao1 value, a qualitative measure of α-diversity that estimates species richness within a microbial community, was observed in the He and Ho groups than in the WT group (Fig. 3 F). Differences in gut microbiota among the three experimental groups were determined using LEfSe taxonomic rank profiles and the outcome of unweighted UniFrac analysis at the genus level (Fig. 3 G and 3 H). The abundance of Prevotella _UCG-001 was significantly lower, and the Gastranaerophilales and Osillibacter populations were significantly higher in the He and Ho groups than in the WT group. The abundance of Alistipes and Muribaculaceae was significantly higher in the He and Ho groups than in the WT group, respectively. 3.3. Halo treatment improves behavioral disorders and dysbiosis in CLCN4-KO mice The potential of Halo, a first-generation (typical) antipsychotic drug used to improve behavioral abnormalities, was assessed to validate the laboratory-generated CLCN4-KO mouse models. CLCN4-KO mice were administered either a vehicle or Halo for four weeks (Fig. 4 A). Following the treatment period, behavioral assessments were conducted using the OFT, PAT, and NORT. Aberrant locomotor activity, anxiety-like behavior, impaired spatial learning and memory function, and cognitive deficits were observed in the He and Ho groups when treated with a vehicle (Fig. 4 B– 4 D). However, such impairments in locomotor activity as well as spatial learning and memory function of animals in both groups were ameliorated in response to Halo treatment (Fig. 4 B, left panel, and 4C). Halo treatment also improved anxiety-like behavior and cognitive impairment in the He group but not in the Ho group (Fig. 4 B, right panel, and 4D). These antipsychotic effects of Halo further support the validity of the CLCN4-KO mouse model. Next, the effect of Halo on the gut microbial population was investigated to determine whether gut bacteria and the antipsychotic effect of Halo are related. Halo treatment significantly attenuated the serum p-cresol levels in the He and Ho mice (Fig. 5 E). The clustering profile of gut microbes in the WT and Ho animals, as revealed by PCoA, demonstrated close and overlapping distributional patterns between the pre- and post-Halo treatments (Fig. 4 E, left panel). Furthermore, both the WT and Ho groups exhibited a significant change in PCo2 but not in PCo1 in response to Halo exposure (Fig. 4 E, middle and right panels). In contrast, He animals showed nearly distinguishable clustering patterns of gut microbes, accompanied by a significant difference in PCo1, but not in PCo2, between the pre- and post-Halo treatments (Fig. 4 E, middle and right panels). No significant change was observed in the α-diversity index of Chao1 in WT animals in response to Halo treatment (Fig. 4 F). Aligning with previous results (Fig. 3 F), a significantly lower Chao1 value was observed in the vehicle-treated He and Ho mice than in the vehicle-treated WT mice, which remained unaltered in response to Halo treatment (Fig. 4 F). In addition, significant differences were observed in the relative abundances of Gastranaerophilales, Prevotellaceae_ UCG - 001, Alistipes, Osillibacter , and Muribaculaceae in both the vehicle-treated He and Ho groups compared to the vehicle-treated WT group (Fig. 4 G and 4 H), aligning with the previous findings (Fig. 3 G and 3 H). However, an apparent trend in the changes in the abundance profiles of these bacterial taxa towards that of WT was evident in both the He and Ho groups in response to Halo treatment. These findings were further supported by LEfSe analysis, which revealed the enrichment of Prevotellaceae _UCG-001 and Ruminococcus within the Halo-treated He (He-H) and Ho (Ho-H) groups, respectively (see Additional file 1). 3.4. Antibiotic treatment inhibits the beneficial effect of Halo in CLC4 KO mice CLCN4-KO mice were administered a cocktail of antibiotics for three consecutive days and then treated with Halo through oral gavage daily for four weeks to further examine the gut microbial influence on Halo-mediated improvement in behavioral disorders. The motor activity, cognition, and memory of the animals were tested. The beneficial effects of Halo on OFT parameters were significantly impaired in the He and Ho groups after treatment with antibiotics (Fig. 5 B). A similar suppressive effect of antibiotics on cognitive function was observed in the He-H group (Fig. 5 C). However, spatial learning and memory function as well as the serum p-cresol level in both the He-H and Ho-H groups remained unaltered in response to antibiotic treatment (Fig. 5 D and 5 E, respectively). Next, the PSD95, AKT, and ERK phosphorylation levels were examined in the hippocampus of all experimental animal groups to assess whether such cell-signaling proteins, which are involved in neuronal communication, plasticity, and function, are involved in mediating the effect of antibiotic treatment on the antipsychotic effect of Halo. The phosphorylation levels of all three proteins in the Ho group and PSD95 in the He group were significantly lower than those in the WT group (Fig. 5 F). However, exposure to Halo significantly increased the phosphorylation levels of all three proteins in Ho mice, but not in He animals. Treatment of both the He-H and Ho-H groups with antibiotics significantly increased the phosphorylation of all three proteins in the He-H group, as well as AKT and ERK in the Ho-H group. 3.5. Amelioration of behavioral disorders in CLCN4-KO mice by FMT FMT was performed to further evaluate the involvement of gut microbes in the behavior of CLCN4-KO mice. Solutions of fecal matter from WT, He, and Ho mice were prepared and administered orally for five days after the induction of intestinal microbial destabilization by antibiotic treatment (Fig. 6 A and Table 1 ). After the desired FMT, the OFT, PAT, and NORT parameters were compared among various animal groups as follows: WT-wt vs. WT-he, WT-wt vs. WT-ho, He-he vs. He-wt, and Ho-ho vs. Ho-wt. The OFT parameter “total distance traveled” and the NORT parameter “object discrimination” did not vary significantly among the inter-group comparisons (Fig. 6 B and 6 D). Similarly, another OFT parameter, “time spent in the center zone,” did not differ among all comparisons, except for its significantly higher value in the WT-he group than in the Wt-wt group (Fig. 6 C). A significantly higher value of the PAT parameter “latency time to enter the dark box” was observed in the test than in the training period in the He and Ho mice after receiving FMT from the WT group (Fig. 6 E, middle and right panels, respectively). A similar increase was also noted in WT animals after receiving FMT from the WT, He, or Ho groups (Fig. 6 E, left panel). Next, a detailed analysis of the vital gut microbial population in all experimental groups was performed before and after fecal transplantation to elucidate the major contributors to the FMT-mediated improvement of behavioral disorders in CLCN4-KO mice. PCoA results showed that the diversity of the gut microbiota in recipient mice was markedly altered in response to FMT (Fig. 7 A– 7 C). Furthermore, the following significant changes in the gut microbial population in response to FMT were observed: an increase in Alistipes and a decline in Prevotellaceae_ UCG - 001 in both the WT-he and WT-ho groups; a decrease in Oscillibacter in the WT-he group and an increase in Muribaculaceae in the WT-ho group; and a decline in Alistipes , Muribaculaceae , Oscillibacter , Gastranaerophilales , and Prevotellaceae_ UCG - 001 in both the HE-wt and HO-wt groups (Fig. 8 A– 8 D). Next, PSD95, AKT, and ERK phosphorylation levels in the hippocampus of all FMT donor/recipient animal groups were measured to investigate whether these vital cells signaling proteins play a role in mediating the effect of FMT on animal behavioral indices. Significantly higher pERK levels were observed in the WT-he group than in the WT-wt group (Fig. 9 ). Both pPSD95 and pAKT levels were significantly lower in the WT-ho group than in the WT-wt group. In contrast, significantly higher levels of pAKT and pERK were observed in the Ho-wt groups than in the He-he and Ho-ho groups. 3.6. Gut microbiomes are strongly associated with behavioral disorders in CLCN4-KO mice MI, heatmap, and co-occurrence network analyses were performed to determine possible correlations among the vital gut microbiota, behavioral parameters, hippocampal levels of key signaling proteins (PSD95, AKT, and ERK), and serum p-cresol levels. MI analysis conducted on Prevotellaceae_ UCG - 001, Oscillibacter , Alistipes , Muribaculaceae , and Gastranaerophilales revealed that anxiety, hyperactivity, and serum p-cresol levels were the most frequent parameters strongly associated with these microbes (80% occurrence, indicated by red text) (see Additional file 2). Other correlated parameters included PSD95 (60% overall occurrence), NORT, ERK, and AKT (each with an overall 40% occurrence). However, heatmap and co-occurrence network analyses (Fig. 10 ) revealed that Prevotellaceae_ UCG - 001 was positively correlated with hippocampal PSD95, AKT, and ERK levels and negatively correlated with hyperactivity. Oscillibacter was positively correlated with anxiety and serum p-cresol levels. In contrast, Gastranaerophilales showed a negative correlation with ERK expression. 4. Discussion This study evaluated the effect of gut microbiota on the behavioral abnormalities of CLCN4-KO mice with a C57BL/6 genetic background and investigated the potential molecular mechanisms underlying these effects. Humans and mice share common genetic features, with over 90% of their genomes partitioned into corresponding regions of conserved synteny 54 , while the protein-coding regions exhibit approximately 85% sequence similarity 55 . Numerous KO and knock-in mouse models are widely used for studying human diseases 56 . Experimental manipulation of the mouse genome, particularly gene KO, provides a powerful approach for generating animal models of human genetic disorders 57 . Mutations in the X-linked CLCN4 gene, both inherited and de novo , have been associated with behavioral disorders 58 . In contrast, CLCN4-KO mice with a C57BL/6 background exhibit neurodevelopmental endophenotypes, including impaired social interaction and increased stereotypic behavior 22 . Various animal behavioral models are available for evaluating the cognitive and locomotor abilities of rodents. These models assess features such as anxiety, autonomic functions, learning, memory, and locomotor activity 59 . The OFT is one of the most widely used psychological assessment platforms 60 , measuring locomotor activity based on distance traveled in the periphery of an open arena in an enclosed specialized box within a defined time. Time spent in the center of the open field is a more selective indicator of anxiety-like behavior 61 , 62 . In this study, both He and Ho groups exhibited significantly higher total distances traveled and total time spent in the center area of the open field than the WT group. Learning and memory were assessed using the PAT, a fear-based task in which animals were trained to avoid entering a dark compartment associated with an aversive stimulus (a mild electric shock). WT mice exhibited significantly higher retention latency in the test than those in the training group after the electric shock, indicating normal spatial learning and memory function. In contrast, the He and Ho groups showed no significant differences between test and training retention latencies, suggesting impaired spatial learning and memory. Restricted and repetitive behaviors are characteristic features of ASD 63 . To evaluate these behaviors, the NORT was performed, which measured the time spent interacting with a novel or familiar object. Both He and Ho mice exhibited significantly lower cognitive abilities than WT mice. These findings confirm that the deletion of CLCN4 induces behavioral symptoms in mouse models. This finding was further supported by the observation that treatment with Halo, a first-generation (typical) antipsychotic drug used to manage behavioral symptoms associated with autism 41 , 42 , markedly reversed the above-mentioned adverse effects of CLCN4 KO in the He and Ho mice, except for anxiety-like behavior and cognitive deficits in the Ho group. Gut microbiota plays a crucial role in brain function, influencing neurogenesis, myelination, microglial maturation, development and maintenance of the blood–brain barrier integrity, development of the hypothalamic–pituitary–adrenal (HPA) axis, and HPA axis stress response development and function 32 , 33 . Disruptions in these processes can contribute to NDD 64 . Gut microbiota influences behavioral and physiological abnormalities associated with NDD 35 , and gut dysbiosis in early life has been linked to an increased risk of conditions such as autism and ADHD 26 . Furthermore, the gut microbiota form part of the unconscious regulatory system that modulates cognitive function and fundamental behavioral patterns, including social interaction and stress management 65 . The bi-directional crosstalk between the gut microbiota and GBA—referred to as the gut microbiome-brain axis—is implicated in regulating complex characteristics, including social, emotional, and anxiety-like responses. Previous studies on ASD have reported gut microbial variations in β-diversity, although no consistent microbial signature has been identified across studies 66 . In this investigation, some overlapping yet distinct clustering patterns of gut microbiota were observed among the WT, He, and Ho groups, indicating significant differences in microbial composition. Significant differences in PCo1 were detected between the WT group and both He and Ho groups, as well as in PCo2 between the WT and He groups, suggesting differential diversity of gut microbes among these groups. Further analysis revealed that the gut microbiota composition in the He and Ho groups deviated from that of WT mice, as indicated by significantly lower Chao1 indices—an α-diversity measure reflecting species richness—compared to the WT group. Similar reductions in Chao1 and Shannon indices have been reported in patients with ASD compared to non-related neurotypical controls 67 , aligning with findings from ASD mouse models 68 . Consistent with the observed β-diversity differences, significant changes in the relative abundances of key gut microbial genera were detected following CLCN4 KO. Specifically, higher abundances of Gastranaerophilales, Alistipes, Oscillibacter , and Muribaculaceae , along with a lower population of Prevotellaceae_ UCG-001 were observed in He, Ho, or both groups than in WT mice. These differential microbial compositions were further supported by LEfSe analysis, which assessed the effect of Halo on the taxonomic rank profiling of gut microbes. Notably, Gastranaerophilales was enriched in the vehicle-treated (control) Ho group (see Additional file 1D). These findings are consistent with those of previous studies 69 – 74 . Alterations in the gut microbial profile affect neurological function and behavior via the gut–microbiome–brain axis, mediated by neurotransmitters, immune activation, and neuroactive bacterial metabolites 33 , 75 . The negative effect of microbial metabolites, such as p-cresol, on neural function is attributed to multiple mechanisms, including membrane depolarization, augmented susceptibility to seizures 76 , reduced Na + -K + ATPase activity 77 and impaired synthesis of dopamine from norepinephrine caused by dopamine-ß-hydroxylase inhibition 78 . Exposure to p-cresol induces autistic-like behaviors in mice by remodeling the gut microbiota 69 . Several phylogenetically diverse gut microbial strains can produce p-cresol, and the levels of this metabolite and its conjugates, p-CS and p-CG, are associated with Muribaculaceae 69 , 79 , Alistipes [69, 79, 80, 81], and Oscillibacter 69 , 80 , 81 . Consistent with these findings, CLCN4-KO mice demonstrated higher abundances of Muribaculaceae, Alistipes , and Oscillibacter than the WT mice, along with significantly elevated p-cresol levels. These observations suggest the involvement of these gut microbiota in the etiology of NDD. Furthermore, genome-based metabolic modeling in a previous study of patients with Parkinson’s disease (PD) identified Gastranaerophilales as a key bacterium responsible for indole production, which can enter the bloodstream 82 . Indole is closely associated with various neurological and neuropsychiatric disorders 83 and induces anxiety-like behavior and depressive disorders in rats 84 . In agreement with these reports, the higher population of Gastranaerophilales observed in both the He and Ho groups than in the WT group suggests that this microbial taxon may contribute to NDD onset and progression in this mouse model. The potential involvement of gut microbiota in the beneficial effects of Halo in CLCN4-KO mice was further explored. Exposing He and Ho mice to Halo significantly reduced serum p-cresol levels. Furthermore, a clear distinction in the clustering patterns of gut microbial communities was observed before and after Halo treatment in the He group, along with significant differences in PCo1 in the He group and PCo2 in the Ho group. These results indicate that Halo influences the gut microbiota in both He and Ho animals, albeit in different manners, highlighting a potential interaction between this antipsychotic drug and the gut microbiota of the CLCN4-KO mouse models. However, Chao1 diversity indices in both the He and Ho groups remained unchanged after Halo treatment, indicating that Halo does not affect the α-diversity of the gut microbial population in CLCN4-KO mice. This observation aligns with those of a previous clinical report showing no significant changes in the α-diversity (Chao1 and Shannon indexes) of the gut microbiota in patients with schizophrenia after 6 weeks of treatment with the antipsychotic drug olanzapine 85 . However, a trend toward normalization of microbial abundance profiles was observed in response to Halo treatment, particularly in the genera Gastranaerophilales, Alistipes, Osillibacter, Muribaculaceae , and Prevotellaceae_ UCG-001, shifting toward levels observed in WT mice. This finding supports the hypothesis that antipsychotics may restore and normalize gut microbial diversity to a state more comparable to that of healthy controls, potentially offering therapeutic benefits for individuals with NDD 86 . LEfSe analysis further revealed marked alterations in the microbial communities within both He and Ho groups after Halo treatment, albeit in a distinct manner, demonstrating the enrichment of Prevotellaceae_ UCG-001 in the He-H group and Ruminococcus in the Ho-H group (see Additional file 1D). Olanzapine treatment significantly increases the abundance of Prevotellaceae UCG-001 in the gut environment of rats 87 . In contrast, Ruminococcus can regulate dopaminergic signaling 88 which plays a fundamental role in neurodevelopment 89 . The abundance of many Ruminococcus spp., including Ruminococcus sp. AT10 and Ruminococcus sp. DJF was higher in patients with typical antipsychotic-treated schizophrenia (SCZ) than in atypical patients with SCZ treated with antipsychotic drugs 90 . Modification of the gut microbiota by several types of host-targeting non-antibiotic drugs, including first-generation antipsychotics such as Halo, may influence the pharmacokinetics and dynamics of these medicines and modulate their therapeutic efficacy by metabolizing drug compounds 91 – 94 . To further investigate the association between gut microbiota and the therapeutic effects of Halo, mice were treated with a broad-spectrum antibiotic cocktail. Antibiotic-induced intestinal dysbacteriosis triggers behavioral alterations and neuronal activation in various brain regions of mice 95 . Therefore, antibiotic-mediated disruption of gut microbial communities presents a useful approach for assessing the influence of gut microbiota on cognition, emotion, and behavior 96 . In this study, exposing the Ho-H and He-H groups to antibiotics resulted in a marked reversal of behavioral improvements, returning the animals to a disease-like state, with the exception of spatial learning and memory function, as assessed by PAT. These findings provide further support for the role of gut microbial communities on the therapeutic efficacy of Halo in improving NDD. The PSD95, ERK, and AKT signaling pathways were analyzed in the hippocampus of mice to further investigate the molecular mechanisms underlying gut microbial involvement in the antipsychotic activity and the protective effect of Halo against NDD. PSD95—a key member of membrane-associated guanylate kinase family 1—plays a crucial role in glutamatergic signaling, synaptic plasticity, and dendritic spine morphogenesis during neurodevelopment 97 – 99 . Current evidence suggests a relationship between PSD95 dysfunction and NDD, including ASD 100 . The phosphorylation–dephosphorylation status of the ser-295 residue of PSD95 is a key factor in controlling synaptic strength. Specifically, phosphorylation of this residue facilitates the synaptic accumulation of PSD95 and intensifies excitatory postsynaptic currents, whereas dephosphorylation triggers long-term depression 101 . In contrast, ERK phosphorylation plays an essential role in facilitating neuronal communication and plasticity 102 . PI3K/AKT signaling is essential for brain development, maintenance, repair, and plasticity during adulthood 103 . ERK phosphorylation levels are significantly lower in patients with autism than in neurotypical controls 104 . Similarly, dysregulated PI3K/AKT signaling is associated with ASD 39 , 40 . Consistent with these findings, in this study, the phosphorylation level of PSD95 in both the He and Ho groups and that of ERK and AKT in the Ho group were lower than those in the WT group. Treatment of the Ho group with Halo, but not the He group, significantly increased the phosphorylation of all three signaling proteins. These differences in phosphorylation levels and the responses of pPSD95, pAKT, and pERK to Halo between the He and Ho groups may be attributed to their distinct genetic backgrounds. However, with the exception of PSD95 in the Ho group, the phosphorylation levels of all three signaling proteins were significantly attenuated in both He and Ho animals upon co-treatment with antibiotics. These results suggest that the PSD95, ERK, and AKT signaling pathways play a crucial role in the gut microbial contribution to the effects of Halo on NDD. The association between gut microbes and NDD was further confirmed through experiments involving FMT, a technique commonly used to alter gut microbial composition by administering a solution of fecal matter from a donor into the intestinal tract of a recipient to achieve beneficial health effects 105 . Accumulating evidence indicates the transmission of depressive and anxiety-like symptoms and behaviors through FMT from psychologically ill donors to healthy recipients. The inverse has also been observed, with improvements in depressive and anxiety-like symptoms following FMT from healthy controls 106 . FMT findings and overall behavioral assessment of the animals (Fig. 6 and Fig. 3 B– 3 D) indicated that FMT from the WT mice largely contributed to the behavioral transition of the spatial learning and memory function. However, no significant effect was observed on locomotor activity, anxiety-like behavior, or cognitive function. In mice, FMT from aged donors impaired spatial learning and memory in young adult recipients. In contrast, anxiety, explorative behavior, and locomotor activity remained unaltered 107 , and aging-related symptoms improved in older WT animals after receiving FMT from young WT donors 108 . Furthermore, apart from a significantly higher value of “time spent in the center zone in the WT-he group compared to the Wt-wt group, no significant differences in behavioral indices were observed between the WT-wt group and the WT-he or WT-ho groups. These results suggest that the gut microbiota of WT mice may play a dominant role in shaping spatial learning and memory function in both the He and Ho groups, driving them toward a "WT-type" profile while potentially resisting the influence of transplanted microbes from CLCN4-KO animals. A comprehensive analysis of the gut microbiota was conducted to further elucidate the molecular mechanisms underlying the FMT-mediated improvement of behavioral disorders in CLCN4-KO mice. The phosphorylation levels of PSD95, AKT, and ERK signaling proteins in the hippocampus of all studied groups were measured before and after fecal transplantation. The results demonstrated declines in Alistipes , Muribaculaceae , Oscillibacter , and Gastranaerophilales in both the He-wt and Ho-wt groups after FMT. Given the observed gut microbial profile (Fig. 3 H) and the potential associations of Alistipes , Muribaculaceae , and Oscillibacter with the serum p-cresol level, as well as the relationship of Gastranaerophilales with indole production, it is plausible that the fecal preparation from WT mice contains gut microbes capable of suppressing the growth of these four bacterial taxa in the gut environment of the He and Ho groups. This suppression may contribute to the improvement of neurodevelopmental disorder (NDD) parameters in CLCN4-KO WT animals. The wild mouse gut microbiota improves disease resistance 109 . Clinical trials in patients with ASD have demonstrated the beneficial effects of FMT from healthy donors on neurological symptoms 110 . Additionally, multiple studies have reported improvements in depressive and anxiety-like symptoms and behaviors following the transplantation of healthy microbiota 106 . The dominance of gut microbes in WT mice over those of the CLCN4-KO animals was further reflected in the response of the WT animals to FMT from CLCN4-KO WT mice. Specifically, no significant changes were observed in the abundance of Gastranaerophilales and Muribaculaceae , whereas a significant decline in the Oscillibacter population was noted after FMT from the He group. Furthermore, no significant alterations in the abundance of Gastranaerophilales and Oscillibacter were observed following FMT from the He group. Analysis of the phosphorylation levels of key hippocampal signaling molecules revealed the following features: no significant differences between the WT-wt and WT-he groups for pPSD95 and pAKT, or between the WT-wt and WT-ho groups for pERK; significantly higher levels of pPSD95 and pAKT in the WT-wt compared to WT-ho groups; increased pAKT levels in the He-wt compared to He-he groups; and elevated pERK levels in the WT-he compared to WT-wt groups, as well as in the Ho-wt compared to Ho-ho groups. These results suggest that the gut microbiota of WT mice plays a crucial role in shaping the gut microbiota, long-term memory, and learning functions, as well as the phosphorylation status of PSD95, AKT, and ERK signaling proteins in the recipient mice, shifting their profiles toward a WT-like pattern and indicating the dominance of WT gut microbiota over that of the He and Ho groups. MI, heatmap, and co-occurrence network analyses were conducted to confirm the association between key gut microbiota, behavioral parameters, serum p-cresol levels, and neuronal signaling proteins. Prevotellaceae_ UCG - 001, Oscillibacter , Alistipes , Muribaculaceae , and Gastranaerophilales were strongly and significantly associated with anxiety, hyperactivity, and serum p-cresol levels. Furthermore, Prevotellaceae_ UCG - 001, which exhibited lower abundance in both the He and Ho groups than in the WT group, was positively correlated with the hippocampal levels of PSD95, AKT, and ERK but negatively correlated with hyperactivity. A lower abundance of Prevotellaceae is associated with autism 74 . Given the observed increase in Prevotellaceae_ UCG - 001 in CCLN4-KO after Halo treatment, this bacterial taxon likely contributes to the beneficial effects of Halo in improving NDD. Osillibacter , which exhibited higher abundances in the He and Ho groups than in the WT group, demonstrated a positive correlation with p-cresol levels and anxiety. These findings align with those of previous studies reporting a higher abundance of Osillibacter in patients with ASD than in the control volunteers 73 , in high anxiety mice compared to low anxiety mice 111 , and in patients with PD with moderate depression compared to patients with PD without depression 112 Gastranaerophilales , which displayed higher abundances in the He and Ho groups than in the WT group, was negatively correlated with the hippocampal ERK level. Signaling through ERK/MAPK proteins is essential for the proper development of the nervous system from neural progenitor cells originating from the embryonic mesoderm, highlighting these proteins as potential novel therapeutic targets in several NDDs 113 . 5. Conclusions This study used the CLCN4-KO mouse model and applied Halo as an antipsychotic agent, with or without an antibody cocktail, to elucidate the involvement of gut microbiota, particularly Gastranaerophilales, Alistipes, Oscillibacter , and Muribaculaceae in the etiology of NDD. The microbial metabolite p-cresol and the key hippocampal neuronal signaling proteins PSD95, AKT, and ERK played significant roles in this process. These findings indicate that Halo, which exhibited its characteristic antipsychotic effects in the He and Ho groups, was negatively correlated with the abundance of these bacteria and serum p-cresol levels in both groups and positively associated with the activation status of PSD9, AKT, and ERK in the Ho group. Many of these effects were inhibited by the application of an antibody cocktail, suggesting that gut microbiota influences the beneficial effect of Halo in CLCN4-KO mice. Additionally, Prevotellaceae_ UCG - 001, which exhibited a lower abundance in both the He and Ho groups than in the WT group, was positively correlated with the hippocampal levels of activated PSD95, AKT, and ERK and negatively correlated with hyperactivity. Treatment with Halo significantly increased the Prevotellaceae_ UCG - 001 population in both He and Ho groups, suggesting that this bacterial taxon may contribute to the beneficial effects of Halo in improving NDD. The FMT study revealed that the gut microbiota of WT mice plays a dominant role in shaping the gut microbiota, long-term memory, and learning functions, as well as the phosphorylation status of hippocampal PSD95, AKT, and ERK in recipient mice towards the WT phenotype. Considering these results and the applicability of KO and knock-mouse models in studying human diseases 56 , FMT from healthy patients fortified with Prevotellaceae_ UCG - 001 may be an effective treatment strategy for patients with NDD. However, extensive studies and clinical trials are required to validate this approach. Abbreviations ADHD attention deficit hyperactivity disorder ASD autism spectrum disorder CLC chloride ion channel CLC4 chloride ion channel 4 FMT fecal microbiota transplantation GBA gut–brain axis Halo haloperidol He heterozygous He-H Halo-treated He,Ho,homozygous Ho-H Halo-treated Ho HPA hypothalamic–pituitary–adrenal KO knockout LEfSe linear discriminant analysis effect size MI mutual information NDD neurodevelopmental disorders NORT novel object recognition test OFT open field test PAT passive avoidance test PCoA principal coordinate analysis PCo1 principal coordinate 1 PCo2 principal coordinate 2 p-CG p-cresyl glucuronide p-CS p-cresyl sulfate PD Parkinson’s disease SEM standard error of the mean TBST Tris-buffered saline containing 0.1% Tween 20 WT wild-type Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare no competing interests. Funding The authors are grateful for funding support from a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (RS-2020-KH087713) to Prof. Kim, and by the Main Research Program (E0170601–09) of the Korea Food Research Institute funded by the Ministry of Science and ICT to Dr. Nam Author Contribution YC, SJ and HK jointly conceived and designed the study. YC performed animal behavior analysis, FMT, Western blotting, real-time PCR and analyzed all data. YC and JO performed network analysis. YC, EJS and YDN performed 16s rRNA analysis. YC and BS wrote the manuscript. Data Availability The raw 16S rRNA sequencing reads from this study are available in the NCBI under the BioProject ( https://www.ncbi.nlm.nih.gov/bioproject/ ) accession number PRJNA1247208. References Straub L, Bateman BT, Hernandez-Diaz S, York C, Lester B, Wisner KL, et al. 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Gut microbiome partially mediates and coordinates the effects of genetics on anxiety-like behavior in Collaborative Cross mice. Scientific reports. 2021;11(1):270. Guo T, Chen L. Gut microbiota and inflammation in Parkinson’s disease: Pathogenetic and therapeutic insights. European Journal of Inflammation. 2022;20:1721727X221083763. Iroegbu JD, Ijomone OK, Femi-Akinlosotu OM, Ijomone OM. ERK/MAPK signalling in the developing brain: Perturbations and consequences. Neuroscience & Biobehavioral Reviews. 2021;131:792–805. Additional Declarations No competing interests reported. Supplementary Files supplefigures.zip Additional information Additional file 1. LEfSe-based comparison of gut microbial abundance and haloperidol-induced changes in WT, He, and Ho groups. (A) Comparison among the vehicle-treated WT (WT_V), He (He_V), and Ho (Ho_V) groups. (B) Comparison between the He_V and Halo-treated He (He-H) groups. (C) Comparison between the Ho_V and Halo-treated Ho (Ho-H) groups. (D) Comparison among the WT_V, He_V, He-H, Ho_V, and Ho-H groups. Additional file 2. Mutual information (MI) data of the gut microbiota are plotted as bar graphs. (A) Prevotellaceae _UCG-001. (B) Oscillibacter . (C) Alistipes . (D) Muribaculaceae , (E) Gastranaerophilales . The vertical line indicates the threshold at 0.01, whereas the labels in red denote variables whose MI with the corresponding microbiota composition exceeds the threshold. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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18:17:13","extension":"tif","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33314636,"visible":true,"origin":"","legend":"","description":"","filename":"figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/142d478704e386dd4da55343.tif"},{"id":93167343,"identity":"8d6198b7-1ca1-4a1b-904f-fecc410e2ae3","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"tif","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13536300,"visible":true,"origin":"","legend":"","description":"","filename":"figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/d00311949bd80624e3e48b92.tif"},{"id":93167358,"identity":"7bfc1540-c283-4e10-9cfd-e10ecd6311b8","added_by":"auto","created_at":"2025-10-09 18:17:13","extension":"tif","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6138220,"visible":true,"origin":"","legend":"","description":"","filename":"figure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/a0f7765fa793c84653500e3e.tif"},{"id":93168148,"identity":"48b92ce6-7064-494c-b0e6-bae5856b794a","added_by":"auto","created_at":"2025-10-09 18:25:13","extension":"tif","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19075632,"visible":true,"origin":"","legend":"","description":"","filename":"figure8.tif","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/fced0363377ddb6f61e19b1f.tif"},{"id":93167357,"identity":"1bc764e4-26be-4126-8981-85506c16dcb9","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"tif","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":26581352,"visible":true,"origin":"","legend":"","description":"","filename":"figure9.tif","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/0240fa94eeaf80a860fccad0.tif"},{"id":93167342,"identity":"81ac8123-a1e5-4c95-87c1-66f9a10e79c8","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"xml","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":246610,"visible":true,"origin":"","legend":"","description":"","filename":"04b571c8b90f47f88fd059665c41adc91structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/e90977e7053477b3d770d539.xml"},{"id":93168143,"identity":"bae1926d-1b25-471d-a115-a9d3604a9bf4","added_by":"auto","created_at":"2025-10-09 18:25:12","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":261681,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/67b3ac35f1908d8f9067679d.html"},{"id":93167323,"identity":"6eb5c8c2-ded2-4dc3-a305-78eaefae52c9","added_by":"auto","created_at":"2025-10-09 18:17:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1847012,"visible":true,"origin":"","legend":"\u003cp\u003eGenerating a CLCN4-knockout mouse model using clustered regularly interspaced short palindromic repeats/Cas9 gene-editing\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/cff0df1eea74f7fe569177bd.png"},{"id":93167325,"identity":"8cee6fe1-1180-45a0-9d7b-7724a93eaca5","added_by":"auto","created_at":"2025-10-09 18:17:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2317615,"visible":true,"origin":"","legend":"\u003cp\u003ePCR genotyping results for CLCN4-wild-type (CLCN4-WT), -heterozygous (hetero, He), and -homozygous (homo, Ho)-knockout (CLCN4-KO) mice. PCR products from the mouse ear tissue samples were resolved using agarose gel electrophoresis. The sizes of the PCR bands were 554 bp for WT mice, 554 and 422 bp for He mice, and 422 bp for Ho mice.\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/638c06415488679dd814c90a.png"},{"id":93167327,"identity":"224f5dc7-fab9-41ed-bdd8-6a07666c1df0","added_by":"auto","created_at":"2025-10-09 18:17:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12102101,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Schematic illustration of the overall experimental design for assessing the behavioral patterns in the WT, He, and Ho groups. (B) Total distance traveled (m) (left panel) and time spent in the central zone (s) of the chamber (right panel) by the WT, He, and Ho mice in the open field test (OFT). (C) Latency time of the WT, He, and Ho groups to enter the dark box (s) in the passive avoidance test (PAT). (D) Object discrimination index percentage of the WT, He, and Ho groups in the novel object recognition test (NORT). (E) Principal coordinate analysis (PCoA) of unweighted UniFrac distances reveals overlapping yet distinguishable clustering patterns of gut microbes among the WT, He, and Ho groups (left panel), indicating apparent differences in the gut microbiota among the groups. The differences in principal coordinate 1 (PCo1) and 2 (PCo2) among these three animal groups are shown in the middle and right panels, respectively. (F) Difference in the Chao1 value among the WT, He, and Ho mice. (G) Overview of the differential abundance of gut microbes at the genus level among the WT, He, and Ho groups, as evaluated by linear discriminant analysis effect size (LEfSe). (H) Relative abundances of \u003cem\u003eGastranaerophilales\u003c/em\u003e, \u003cem\u003ePrevotellaceae\u003c/em\u003e_UCG-001, \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eOscillibacter\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e in the WT, He, and Ho groups based on unweighted UniFrac analysis results. Results are expressed as the mean ± standard error of the mean (SEM). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 are the levels of significance in the difference of a parameter between the two experimental groups, as determined by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Onlinefigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/c1cc317f6e9cdbbe8cecb771.png"},{"id":93167329,"identity":"8719ae9f-ec11-4b14-93f5-f850a37cd34e","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12285295,"visible":true,"origin":"","legend":"\u003cp\u003eAmelioration of neurodevelopmental disorder (NDD)-related behavioral abnormalities in CLCN4-KO mice after haloperidol (Halo) treatment. (A) Schematic representation of the overall experimental procedure to evaluate the effect of Halo on the He and Ho mice. (B) Total distance traveled (m) (left panel) and time spent in the central zone (s) of the chamber (right panel) by the WT, He, He-H, Ho, and Ho-H groups in the OFT. (C) Latency time of the mice to enter the dark box (s) in the PAT. (D) Object discrimination index percentage of the mice in the NORT. (E) PCoA of unweighted UniFrac distances reveals nearly distinguishable clustering patterns of gut microbes among the WT, WT + Halo, He, He-H, Ho, and Ho-H groups (left panel), indicating apparent differences in the gut microbiota among these groups. The differences in PCo1 and PCo2 among these groups are shown in the middle and right panels, respectively. (F) The differences in Chao1 values between the WT and WT + Halo groups (left panel) and among the WT, He, He-H, Ho, and Ho-H groups (right panel). (G) Overview of the abundance of gut microbes in the WT, He, and Ho groups before and after Halo treatments, as evaluated by LEfSe. (H) Relative abundances of \u003cem\u003eGastranaerophilales\u003c/em\u003e, \u003cem\u003ePrevotellaceae\u003c/em\u003e_UCG-001, \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eOscillibacter\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e in the WT, He, He-H, Ho, and Ho-H groups, based on the unweighted UniFrac analysis results. Results are expressed as the mean ± SEM. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 are the levels of significance in the difference of a parameter between the two experimental groups, as determined by the Student’s test.\u003c/p\u003e","description":"","filename":"Onlinefigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/19a7e5316544cd9b0e53c703.png"},{"id":93167331,"identity":"20691530-965a-484b-9db4-ff7cdb2e4189","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":15507034,"visible":true,"origin":"","legend":"\u003cp\u003ePartial suppression of the Halo-mediated improvement in behavioral patterns of the CLCN4-KO mice by antibiotics. (A) Schematic representation of the overall experimental design to evaluate the effect of antibiotic-mediated gut dysbiosis on the He and Ho groups treated with or without Halo. (B) Total distance traveled (m) (left panel) and time spent in the central zone (s) of the chamber (right panel) by the WT, He, He-H, He + antibiotic + Halo, Ho, Ho-H, and Ho + antibiotic + Halo groups in the OFT. (C) Latency time of the animals of these groups to enter the dark box (s) in the PAT. (D) Object discrimination index percentage of the mice in these groups in the NORT. (E) The concentration of serum p-cresol in the study groups. (F) Phosphorylation levels of the vital cell signaling proteins PSD95, AKT, and ERK in the hippocampus of the mice of the WT, He, He-H, He + antibiotic + Halo groups (upper panel) and WT, Ho, Ho-H, Ho + antibiotic + Halo groups (lower panel). Results are expressed as the mean ± SEM. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 are the levels of significance in the difference of a parameter between the two experimental groups, as determined by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Onlinefigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/1a5d834f86b516f377c95345.png"},{"id":93167328,"identity":"f7ccca1b-06f3-4eda-8f10-bfe86b0f2e70","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5670615,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Effect of fecal microbial transplantation on behavioral patterns in the WT, He, and Ho groups. (B) Total distance traveled (m) by the mice in the WT-wt, WT-he, WT-ho groups (left panel), and He-he, He-wt, Ho-ho, Ho-wt groups (right panel) in the OFT. (C) Time spent in the central zone (s) of the chamber by the mice in the WT-wt, WT-he, WT-ho groups (left panel), and He-he, He-wt, Ho-ho, and Ho-wt groups (right panel) in the OFT. (D) Latency time of the mice in the WT-wt, WT-he, WT-ho groups (left panel); He-he, He-wt groups (middle panel); and Ho-ho, Ho-wt groups (right panel) in the PAT. (E) Object discrimination index percentage of the mice in the WT-wt, WT-he, WT-ho groups (left panel), and He-he, He-wt, Ho-ho, Ho-wt groups (right panel) in the NORT. Results are expressed as the mean ± SEM. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, and **** p \u0026lt; 0.0001 are the levels of significance in the difference of a parameter between the two experimental groups, as determined by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Onlinefigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/037e89d3f8f78304f94008d6.png"},{"id":93168142,"identity":"d0f3723a-373c-4a58-a7cc-b233afb47d42","added_by":"auto","created_at":"2025-10-09 18:25:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3123466,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCoA profile of each study group before and after FMT. (A) The pre- (left panel) and post-FMT (right panel) PCoA of the Wt-wt, He-wt, and Ho-wt groups. (B) The pre- (left panel) and post-FMT (right panel) PCoA of the He-he and WT-he groups. (C) The pre- (left panel) and post-FMT (right panel) PCoA of the Ho-ho and WT-ho groups.\u003c/p\u003e","description":"","filename":"Onlinefigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/5804935391a4e8205c79b9a7.png"},{"id":93167336,"identity":"0fb0dc32-cb5a-4cce-ba79-24dac35c275a","added_by":"auto","created_at":"2025-10-09 18:17:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":10605254,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in the relative abundances of \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, \u003cem\u003eOscillibacter\u003c/em\u003e, \u003cem\u003eGastranaerophilales\u003c/em\u003e, and \u003cem\u003ePrevotellaceae\u003c/em\u003e_UCG-001 in mice. Differences between the pre- and post-FMT in the (A) WT-he, (B) WT-ho, (C) He-wt, and (D) Ho-wt groups. Results are expressed as the mean ± SEM. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, and **** p \u0026lt; 0.0001 are the levels of significance in the difference in the relative abundance between the two experimental groups, as determined by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Onlinefigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/e8c9ffa105ec6715e2b1bdff.png"},{"id":93168147,"identity":"8a73cfeb-016e-41dd-a918-bddb3e47c15a","added_by":"auto","created_at":"2025-10-09 18:25:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":15230764,"visible":true,"origin":"","legend":"\u003cp\u003ePhosphorylation levels of PSD95, AKT, and ERK in the hippocampus of mice. (A) WT-wt, WT-he, WT-ho groups (upper panel); He-he, He-wt groups (middle panel); and Ho-ho, Ho-wt groups (lower panel). Results are expressed as the mean ± SEM. * p \u0026lt; 0.05 and ** p \u0026lt; 0.01 are the levels of significance in the difference of a parameter between the two experimental groups, as determined by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Onlinefigure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/894362f5c1d8d11e1b1aaf94.png"},{"id":93168145,"identity":"933b71a2-eb76-47cf-845b-f5fcf8d3aefb","added_by":"auto","created_at":"2025-10-09 18:25:12","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":64552,"visible":true,"origin":"","legend":"\u003cp\u003eCo-occurrence network and heatmap analysis of gut microbiota, behavior, signaling proteins, and p-cresol levels. (A) In the co-occurrence network analysis (left panel), the nodes represent the relative abundances of microbiota (circles, filled with peach color); PSD95, AKT, and ERK genes (triangles, filled with gray color); serum biomarkers (diamonds, filled with red color); and behavioral indices (rectangles, filled with pink color). The links between nodes denote significant negative (broken blue color lines) and positive correlations (solid red color lines). The degree of the node (number of links) is mapped to the node size. (B) A heatmap was constructed (right panel) to visualize the relationship among the microbial taxa with significantly differential abundances across the study groups, as assessed by LEfSe; animal behavioral parameters hyperactivity, anxiety, and NORT; hippocampal levels of key signaling proteins PSD95, AKT, and ERK; and serum p-cresol levels using the Spearman’s correlation test. Only significant correlations with p \u0026lt; 0.05 were considered. The color scale indicates the value of the correlation coefficient, whereas the red color indicates a positive correlation, and the green color shows a negative correlation.\u003c/p\u003e","description":"","filename":"Onlinefigure10.png","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/5e201a31b9adcf41ccd98078.png"},{"id":93795220,"identity":"fb49526f-75ea-4237-b979-e52538de0692","added_by":"auto","created_at":"2025-10-17 15:47:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10026151,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/362dae8e-042a-4488-a959-48f48e937c74.pdf"},{"id":93168138,"identity":"b3326d60-90bd-49db-b416-c97042efca2b","added_by":"auto","created_at":"2025-10-09 18:25:12","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5603441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1.\u003c/strong\u003e LEfSe-based comparison of gut microbial abundance and haloperidol-induced changes in WT, He, and Ho groups. (A) Comparison among the vehicle-treated WT (WT_V), He (He_V), and Ho (Ho_V) groups. (B) Comparison between the He_V and Halo-treated He (He-H) groups. (C) Comparison between the Ho_V and Halo-treated Ho (Ho-H) groups. (D) Comparison among the WT_V, He_V, He-H, Ho_V, and Ho-H groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2.\u003c/strong\u003e Mutual information (MI) data of the gut microbiota are plotted as bar graphs. (A) \u003cem\u003ePrevotellaceae\u003c/em\u003e_UCG-001. (B) \u003cem\u003eOscillibacter\u003c/em\u003e. (C) \u003cem\u003eAlistipes\u003c/em\u003e. (D) \u003cem\u003eMuribaculaceae\u003c/em\u003e, (E) \u003cem\u003eGastranaerophilales\u003c/em\u003e. The vertical line indicates the threshold at 0.01, whereas the labels in red denote variables whose MI with the corresponding microbiota composition exceeds the threshold.\u003c/p\u003e","description":"","filename":"supplefigures.zip","url":"https://assets-eu.researchsquare.com/files/rs-7514097/v1/640e6a9913a070399c393b4c.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gut Microbiota-Mediated Modulation of Neurodevelopmental Behavior in CLCN4- Deficient Mice","fulltext":[{"header":"1. Background","content":"\u003cp\u003eNeurodevelopmental disorders (NDDs), also referred to as Raynaud\u0026ndash;Claes syndrome, are a group of conditions characterized by deficits in nervous system development, affecting memory, language, motor function, and the ability to learn, socialize, and maintain self-control \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These deficits often manifest as behavioral and mental health problems. Initially, NDD was identified as a rare X-linked disorder associated with global developmental delay or intellectual disability, language delay, autism spectrum disorder (ASD), anxiety, hyperactivity, epilepsy, brain abnormalities, bipolar disorder, and facial dysmorphism \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. Behavioral problems in NDD are commonly linked to social\u0026ndash;emotional difficulties influenced by familial and early educational backgrounds. These problems are categorized into internalizing symptoms, which include emotional disturbances such as depression and anxiety, and externalizing symptoms, which involve behavioral and hyperactivity-related issues \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIon channels in axons regulate the movement of ions, such as Na\u003csup\u003e+\u003c/sup\u003e, K\u003csup\u003e+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e across the axolemma \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, playing a crucial role in neuronal functions, including action potential initiation and propagation as well as neurotransmitter release \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Chloride ion channels (CLCs) constitute a family of proteins essential for various physiological processes \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Chloride ion channel 4 (\u003cem\u003eCLCN4\u003c/em\u003e), a voltage-dependent 2Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e/ H\u003csup\u003e+\u003c/sup\u003e exchanger encoded by the \u003cem\u003eCLCN4\u003c/em\u003e gene, is a member of this family \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In most mammals, including humans and wild Mediterranean mice (\u003cem\u003eMus spretus\u003c/em\u003e), the \u003cem\u003eCLCN4\u003c/em\u003e gene is X-linked, whereas in the inbred C57BL/6 laboratory mouse strain (\u003cem\u003eMus musculus\u003c/em\u003e), it is autosomal, located on chromosome 7, and contains truncated introns \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Although CLC4 is expressed in multiple tissues, its predominant expression is in the nervous system \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, particularly in the hippocampus and cerebellum \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Additionally, CLC4 is implicated in intracellular pH regulation \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, cell volume maintenance \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and ion homeostasis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, intracellular trafficking \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and neuronal differentiation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe dysfunction of some CLC proteins, including CCL4, manifests as lysosomal storage diseases and severe neurological disorders, including leukodystrophy and neurodegeneration \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. More than 60 different \u003cem\u003eCLCN4\u003c/em\u003e variants have been recorded in the Human Gene Mutation Database, with missense variants being the most common \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Pathogenic variants of \u003cem\u003eCLCN4\u003c/em\u003e impair Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e exchange activity, disrupt homeostasis and intracellular vesicular transport, alter protein function, and affect neuronal differentiation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These variants have been linked to the development of X-linked NDD, presenting with symptoms such as global developmental delay, intellectual disability, epilepsy, behavioral disorders, mental health disorders, and dysmorphic features, predominantly in males \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Recent studies showed that \u003cem\u003eCLCN4\u003c/em\u003e knockout (KO) in C57BL/6 mice manifests neurodevelopmental endophenotypes consistent with ASD in humans \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe gut microbiota, a complex community of microorganisms, has gained attention for its role in health and disease \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Dysbiosis, or microbial imbalance, has been associated with the pathogenesis of various disorders, including inflammatory bowel disease, diabetes, obesity, and cardiovascular disease \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Notably, NDDs, including autism and attention deficit hyperactivity disorder (ADHD), have also been linked to alterations in gut microbiota composition \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The gut\u0026ndash;brain axis (GBA), a bidirectional communication network between the central and enteric nervous systems, facilitates connections between the emotional and cognitive centers of the brain and peripheral intestinal functions \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This connection operates through neural, endocrine, immune, and humoral pathways, allowing gut microbes to influence brain development, cognition, and behavior \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Studies in both mice \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and humans \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e have demonstrated the significant role of commensal bacteria in modulating social, emotional, and anxiety-related behaviors. In a maternal immune activation mouse model on a C57BL/6N background with ASD-like features, gut microbiota alterations regulated behavioral and physiological abnormalities associated with NDD \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, elevated systemic levels of gut microbial metabolites have been implicated in anxiety-like behavior, reinforcing the molecular connection between gut microbiota and GBA in ASD and other NDDs. Among these metabolites, p-cresol (4-methylphenol), a product of bacterial fermentation of dietary tyrosine and phenylalanine in the colon \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, undergoes extensive conjugation to form p-cresyl sulfate (p-CS) and p-cresyl glucuronide (p-CG). Children with autism have higher p-cresol, p-CS, and p-CG levels in urine samples than healthy controls \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e indicating the involvement of these metabolites in ASD.\u003c/p\u003e\u003cp\u003eThis study hypothesized that \u003cem\u003eCLCN4\u003c/em\u003e KO in C57BL/6 mice alters gut microbiota composition, leading to NDD-like phenotypes. To investigate this hypothesis and elucidate potential mechanisms, behavioral tests, gut microbial analysis, fecal microbial transplantation (FMT), and analyses of expression levels of postsynaptic density-95 (PSD95), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK) proteins\u0026mdash;key regulators in NDD pathogenesis \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e\u0026mdash;were conducted. Additionally, the effect of haloperidol (Halo)\u0026mdash;an antipsychotic that alleviates behavioral symptoms and improves clinical outcomes in ASD \u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e\u0026mdash;was examined in relation to both behavior and gut microbiota in \u003cem\u003eCLCN4\u003c/em\u003e KO (CLCN4-KO) mice. Serum p-cresol levels were also measured to explore their potential role as a mediator linking gut microbiota alterations to NDD pathogenesis.\u003c/p\u003e\u003cp\u003eThe findings of this study have significant implications. By identifying the intricate relationships among CLC4, NDD, and the gut microbiota, this study aims to provide comprehensive insights that could direct future research and therapeutic strategies. Therefore, this study lays the foundation for developing novel therapeutic strategies, expanding treatment options for NDD, and advancing precision medicine approaches in NDDs.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.1. Generation of CLCN4-KO mice\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eCLCN4-KO mice were purchased from Toolgen Inc. (Seoul, Republic of Korea). This mouse model, which has a deletion in exon 5 of \u003cem\u003eCLC4N\u003c/em\u003e on chromosome 7, was generated using clustered regularly interspaced short palindromic repeats/Cas9 genome-editing technology on a C57BL/6N genetic background (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Male wild-type (WT) C57BL/6N and female homozygous (Ho) CLCN4-KO mice were mated to produce heterozygous (He) CLCN4-KO mice. \u003cem\u003eCLC4\u003c/em\u003e gene deficiency in KO animals was confirmed through PCR, as described in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e. Male WT mice were used as controls for the entire study.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAll animals were housed under controlled temperatures (22\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃) and relative humidity (40\u0026ndash;60%) with a 12 h light/12 h dark cycle (light on at 9:00 a.m.) and allowed \u003cem\u003ead libitum\u003c/em\u003e access to a standard normal chow diet (Soyagreentec, Hwaseong-Si, Gyeonggi-do, South Korea) and water. All animal study procedures, including animal care and handling, were performed according to international guidelines (Guide for the Care and Use of Laboratory Animals, Institute of laboratory Animal Resources, Commission on Life Sciences, National Research Council, USA; National Academy Press: Washington D.C., 1996). The aim, outline, protocols, and ethical aspects of this study were approved by the Institutional Animal Care and Use Committee of Dongguk University (approval number: IACUC-2022-047-1). To avoid potential confounding effects of anesthesia on brain physiology, animals were euthanized by cervical dislocation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Confirmation of \u003cem\u003eCLCN4\u003c/em\u003e gene deletion\u003c/h2\u003e\u003cp\u003eA PCR analysis using genomic DNA extracted from the ear tissue of the KO mice was performed to confirm the deletion of \u003cem\u003eCLCN4\u003c/em\u003e in the mice. Extraction was performed using tissue lysis buffer supplemented with proteinase K (Sigma-Aldrich, St. Louis, MO, USA). PCR was performed in a mixture containing Perfect Premix (Bioneer, Daejeon, Republic of Korea), 50 ng of extracted DNA, 1 \u0026micro;L of the forward primer (5\u0026prime;-CAT GTC ATG GGT GTG TCC TC-3\u0026prime;), 1 \u0026micro;L of the reverse primer (5\u0026prime;-TAC TTC ACC CAC GGC TTA CC-3\u0026prime;), and nuclease-free water (Bioneer). Touchdown PCR conditions were as follows: initial denaturation at 95 ℃ for 3 min; 10 cycles at 95 ℃ for 30 s, 72 ℃ for 30 s (-1 ℃/cycle), and 72 ℃ for 45 s; 25 cycles at 95 ℃ for 30 s, 62 ℃ for 30 s, and 72 ℃ 45 s; and final elongation at 72 ℃ for 5 min. After amplification, the final PCR products were electrophoresed on 1% agarose gel. The sizes of the PCR bands were 554 base pairs (bp) for WT, 554 and 422 bp for He, and 422 bp for Ho mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Administration of antibiotics and drugs\u003c/h2\u003e\u003cp\u003eThe animals were treated with Halo according to the schedule to evaluate the effect of this drug on mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Briefly, Halo was dissolved in sterile water and administered by oral gavage at a dose of 1 mg/kg body weight/day for 4 weeks. The dose of this drug was selected based on previous studies \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe mice were treated with an antibiotic cocktail to induce gut dysbiosis as previously described \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. However, the method of antibiotic administration was modified in the present study because prolonged exposure to antibiotics adversely affects animal health. Briefly, the mice were orally administered 100 \u0026micro;L of an antibiotics cocktail containing vancomycin (500 mg/L, MBcell, Seoul, Republic of Korea), neomycin (500 mg/L, MBcell), ampicillin (500 mg/L, MBcell), metronidazole (500 mg/L, MBcell), and gentamycin (500 mg/L, MBcell) every day for 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. FMT study\u003c/h2\u003e\u003cp\u003eRecipient animals received antibiotic treatment for gut dysbiosis before fecal transplantation to perform FMT. Fresh feces from the WT, He, and Ho groups were collected from the anus of the animals and placed in sterile conical tubes. The fecal samples from a particular group were pooled, weighed, and mixed with sterile phosphate-buffered saline at a dilution ratio of 1 mg/10 \u0026micro;L. The mixture was shaken vigorously and centrifuged at 900 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 min. The supernatant was collected and orally gavaged to the recipient mice (10 \u0026micro;L/g bodyweight) every day for 5 days after completing antibiotic treatment. Fecal samples were freshly prepared on each treatment day. The animal groups used in the experiment and the fecal transplantation regimens are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eS\u003c/b\u003eymbols used for donor and recipient mice before and after fecal microbiota transplantation\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDonor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRecipient\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGroup name\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWT-wt\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWT-he\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWT-ho\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHe-wt\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHe-he\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHo-wt\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHo-ho\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Behavioral tests\u003c/h2\u003e\u003cp\u003eBehavioral tests were performed in mice at five weeks of age to assess the effect of \u003cem\u003eCLCN4\u003c/em\u003e deletion and the responses of CLCN4-KO mice to various treatment types. All experiments were conducted after the animals were allowed to adapt for 30 min in the experimental room.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1. Open field test\u003c/h2\u003e\u003cp\u003eThe open field test (OFT) is widely used to measure the quality and quantity of exploration and locomotor activity, as well as anxiety-like behavior in rodent models \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. The OFT was performed using a chamber with dimensions of 45 cm (length) \u0026times; 45 cm (width) \u0026times; 45 cm (height). The chamber was composed of white, high-density, and nonporous plastic materials. After the desired treatment schedule, each mouse was placed in the center of the chamber and allowed to move freely for 5 min to allow for adaptation. Subsequently, animal behavior and movements were recorded using a video camera (Samsung, Seoul, Republic of Korea) for 5 min. The total distance traveled and time spent in the central zone of the chamber were automatically recorded and analyzed using an ANY-maze video tracking system (version 5.14; Kim \u0026amp; Friends, Inc., Geumcheon-gu, Republic of Korea).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2. Passive avoidance test\u003c/h2\u003e\u003cp\u003eSpatial learning and memory function tests were conducted using the passive avoidance test (PAT) as previously described \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e with some modifications. The test was performed using a two-compartment apparatus consisting of an illuminated dark chamber. Briefly, the mice were placed in the illuminated chamber and allowed to explore for 30 s on the training day. The door was then opened to allow the animals to enter the dark chamber. Three seconds after the mouse entered the dark chamber with all four paws, the door was closed, and the animal was exposed to a foot shock (50 V, 3 s duration). Subsequently, each mouse was removed from the apparatus and transferred to its home cage. On the test day (the day after training), the mice were placed again in the light chamber. After 5 s, the door was opened, and the latency of the animal entering the dark chamber was recorded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3. Novel object recognition test\u003c/h2\u003e\u003cp\u003eThe novel object recognition test (NORT) is widely used to evaluate the cognitive ability of rodents. This test was performed using an NORT device identical to the OFT chamber. Each mouse was allowed to habituate to an empty arena for 5 min before the familiarization session. During the familiarization phase, two identical objects were placed 5 cm away from the wall of the chamber and the mouse was allowed to explore each object freely for 5 min. Then, one familiar object was replaced by a new object, and the duration of interest in the new object was recorded for up to 5 min. The object discrimination index percentage was calculated to measure recognition memory in each mouse.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Western blotting\u003c/h2\u003e\u003cp\u003eAfter the desired treatments, the mice were anesthetized, and their brains were surgically removed. Hippocampal tissues were carefully dissected, washed with phosphate-buffered saline, and homogenized on ice in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA) using a Vibra-Cell\u0026trade; ultrasonic liquid processor (Sonics \u0026amp; Materials Inc., Newtown, CT, USA). The tissue homogenates were centrifuged at 14,000 rpm for 30 min at 4\u0026deg;C, and the supernatants were collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. The protein content of each supernatant was measured using the Bradford assay. An aliquot of 30 \u0026micro;g of protein was denatured at 100 ℃ in Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) containing 5% β-mercaptoethanol. The protein was electrophoresed in sodium dodecyl sulfate-polyacrylamide gel under a constant voltage of 100 V for 90 min and transferred to a 0.45 \u0026micro;m polyvinylidene fluoride membrane (Amersham\u0026trade;, GE Healthcare, Munich, Germany). Membranes were blocked with 5% skim milk (Becton Biosciences, Franklin Lakes, NJ, USA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min. The membranes were then washed thrice each for 10 min with TBST and incubated overnight with anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-ERK (Thr202/Tyr204), anti-ERK, anti-phospho-PSD95 (Ser295), anti-PSD95 (Cell Signaling Technology, Beverly, MA, USA), and alpha-tubulin antibodies (AbFrontier, Geumcheon, Seoul, South Korea) at 4 ℃ in TBST supplemented with 5% bovine serum albumin. After washing twice with TBST, the membranes were incubated for 90 min with appropriate horseradish peroxidase-conjugated anti-IgG secondary antibodies. Immunoreactive protein bands were detected using a Bio-Rad ChemiDoc XRS imaging system (BioRad, Hercules, CA, USA) with a Super Signal West Pico ECL reagent (Thermo Fisher Scientific, Waltham, MA, USA). Band densities were determined using ImageJ software, which is a public-domain Java image processing program inspired by NIH Image for Macintosh (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Measurement of serum p-cresol levels\u003c/h2\u003e\u003cp\u003eBefore euthanizing the animals, blood samples were collected from the heart using a 1 mL syringe. The blood was allowed to clot at room temperature and then centrifuged at 1000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min at 4 ℃. The serum was separated and stored at -80 ℃. Serum p-cresol levels were measured using a commercial enzyme-linked immunosorbent assay kit (MyBioSource, San Diego, CA, USA) according to the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8. 16s rRNA gene sequencing\u003c/h2\u003e\u003cp\u003eBacterial genomic DNA was extracted from stool samples using a QIAamp Fast DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer\u0026rsquo;s instructions. Amplification of the V1-V2 region of bacterial 16S rRNA gene sequences was performed using a C1000 Touch Thermal Cycler with a 96-deep-well reaction module (BioRad) under the following conditions: initial denaturation at 94\u0026deg;C for 2 min; 26 amplification cycles (each one with denaturation at 94\u0026deg;C for 30 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 1 min) and an additional extension cycle for 10 min at 72\u0026deg;C. The primer sets used for this reaction contained an eight-base sample-specific barcode to tag the PCR products. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen). Finally, purified PCR amplicons (100 ng) tagged with sample-specific barcode sequences were pooled, and sequencing reactions were performed at the Korea Food Research Institute on an Ion Torrent Personal Genome Machine system (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. High-quality reads were selected for further bioinformatics analyses, and all selected reads from the samples were clustered into operational taxonomic units based on 97% sequence similarity (SILVA database: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arb-silva.de\u003c/span\u003e\u003cspan address=\"http://www.arb-silva.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Representative sequences were then selected using the Quantitative Insights into Microbial Ecology software package \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. High-quality raw data were filtered by eliminating sequences lacking the V1-V2 primers or barcode sequence, containing a short read length (\u0026lt;\u0026thinsp;300 bp) or low-quality reads (average quality score\u0026thinsp;\u0026lt;\u0026thinsp;20). The raw sequencing reads are publicly available at the NCBI under Project ID PRJNA1247208.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Analysis of sequenced data\u003c/h2\u003e\u003cp\u003eA linear discriminant analysis effect size (LEfSe) evaluation was performed using a web-based program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/galaxy\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/galaxy\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to identify taxa with differential relative abundance among the experimental groups. The threshold for the logarithmic linear discriminant analysis score was fixed at \u0026gt;\u0026thinsp;2.0.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Co-occurrence network analysis\u003c/h2\u003e\u003cp\u003ePearson\u0026rsquo;s correlation among the vital gut microbiota, behavioral indices, hippocampal levels of PSD95, AKT, and ERK, as well as serum p-cresol levels, were analyzed using the SciPy library of the Python programming language \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e with statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The correlation network was visualized using Cytoscape 3.9.1 \u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.11. Mutual information analysis\u003c/h2\u003e\u003cp\u003eMutual information (MI) analysis is a generic side-channel distinguishing technique. The MI of two random variables is a measure of the relationship and dependence between variables. As MI can determine linear and nonlinear relationships between variables, it can be used to examine the dependence between any number of variables (n-dimensions) \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. MI analysis was performed using the mutual_info_regression module from the scikit-learn library to investigate the relationship between microbial composition and other variables \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The MI values were scaled between 0 and 1, where 0 indicates that the two variables are independent, and the closer they are to 1, the higher the dependency.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Statistical analysis\u003c/h2\u003e\u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) unless otherwise indicated. Statistical analysis was performed using GraphPad Prism 8 (GraphPad software, La Jolla, CA, USA), and the results were tested for significance using the Student\u0026rsquo;s t-test. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. A heatmap was constructed to demonstrate the relationship among microbial taxa with significant differential abundances across the study groups, as assessed by LEfSe, animal behavioral parameters, vital cell signaling kinases, and serum p-cresol levels using Spearman\u0026rsquo;s correlation test. For this analysis, only statistically significant correlations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were considered. The color scale indicates the value of the correlation coefficient, whereas red indicates a positive correlation and green indicates a negative correlation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Confirmation of \u003cem\u003eCLCN4\u003c/em\u003e genotype\u003c/h2\u003e\u003cp\u003eThe recombination of CLC4 in mice was confirmed by genetic analysis using PCR. The sizes of the resultant PCR bands were 554 bp for WT, 554 and 422 bp for He, and 422 bp for Ho mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.2. \u003cem\u003eCLCN4\u003c/em\u003e deletion causes behavioral disorders and alters gut microbiota in mice\u003c/h2\u003e\u003cp\u003eThe animals were subjected to several tests starting from 5 weeks of age to investigate whether \u003cem\u003eCCLN4\u003c/em\u003e KO could alter behavioral disorders in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The OFT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), PAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), and NORT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) were performed to compare the various behavioral characteristics of the animals among the WT, He, and Ho groups. In the OFT, mice in the He and Ho groups showed significantly higher total distance traveled (locomotor activity) and total time spent in the central area of the open field (anxiety-like behavior) than those in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Spatial learning and memory in CLCN4-KO mice were investigated using PAT. After the electric shock, the animals in the WT group demonstrated a significantly higher retention latency when entering the dark box in the test session than in the training session. In contrast, mice in the He and Ho groups did not exhibit significant differences in this parameter between the test and training sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The time that a mouse spent interacting with a novel or familiar object was assessed using NORT, a commonly used behavioral assay in animals, to evaluate cognitive ability. The mice in both He and Ho groups had significantly lower cognitive function than the animals in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, the serum p-cresol level was significantly higher in the He and Ho groups than in WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, 16S rRNA gene sequencing of stool samples was performed to investigate whether the gut microbiota differed among the WT, He, and Ho groups. Principal coordinate analysis (PCoA), a commonly used data analysis platform used to elucidate the β-diversity of the microbial community representing differences in the overall microbial taxonomic profile among the samples, was performed. Analysis of the unweighted UniFrac distance matrix generated from the processed sequence data revealed some overlap yet distinguishable clustering patterns of the gut microbes among the WT, He, and Ho groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, left panel). Furthermore, the He and Ho groups showed significant differences in principal coordinate 1 (PCo1), and the He group demonstrated significant differences in principal coordinate 2 (PCo2) compared to the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, right panel). Additionally, a significantly lower Chao1 value, a qualitative measure of α-diversity that estimates species richness within a microbial community, was observed in the He and Ho groups than in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Differences in gut microbiota among the three experimental groups were determined using LEfSe taxonomic rank profiles and the outcome of unweighted UniFrac analysis at the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). The abundance of \u003cem\u003ePrevotella\u003c/em\u003e_UCG-001 was significantly lower, and the \u003cem\u003eGastranaerophilales\u003c/em\u003e and \u003cem\u003eOsillibacter\u003c/em\u003e populations were significantly higher in the He and Ho groups than in the WT group. The abundance of \u003cem\u003eAlistipes\u003c/em\u003e and \u003cem\u003eMuribaculaceae\u003c/em\u003e was significantly higher in the He and Ho groups than in the WT group, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Halo treatment improves behavioral disorders and dysbiosis in CLCN4-KO mice\u003c/h2\u003e\u003cp\u003eThe potential of Halo, a first-generation (typical) antipsychotic drug used to improve behavioral abnormalities, was assessed to validate the laboratory-generated CLCN4-KO mouse models. CLCN4-KO mice were administered either a vehicle or Halo for four weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following the treatment period, behavioral assessments were conducted using the OFT, PAT, and NORT. Aberrant locomotor activity, anxiety-like behavior, impaired spatial learning and memory function, and cognitive deficits were observed in the He and Ho groups when treated with a vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). However, such impairments in locomotor activity as well as spatial learning and memory function of animals in both groups were ameliorated in response to Halo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, left panel, and 4C). Halo treatment also improved anxiety-like behavior and cognitive impairment in the He group but not in the Ho group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, right panel, and 4D). These antipsychotic effects of Halo further support the validity of the CLCN4-KO mouse model.\u003c/p\u003e\u003cp\u003eNext, the effect of Halo on the gut microbial population was investigated to determine whether gut bacteria and the antipsychotic effect of Halo are related. Halo treatment significantly attenuated the serum p-cresol levels in the He and Ho mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The clustering profile of gut microbes in the WT and Ho animals, as revealed by PCoA, demonstrated close and overlapping distributional patterns between the pre- and post-Halo treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, left panel). Furthermore, both the WT and Ho groups exhibited a significant change in PCo2 but not in PCo1 in response to Halo exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, middle and right panels). In contrast, He animals showed nearly distinguishable clustering patterns of gut microbes, accompanied by a significant difference in PCo1, but not in PCo2, between the pre- and post-Halo treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, middle and right panels). No significant change was observed in the α-diversity index of Chao1 in WT animals in response to Halo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Aligning with previous results (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), a significantly lower Chao1 value was observed in the vehicle-treated He and Ho mice than in the vehicle-treated WT mice, which remained unaltered in response to Halo treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). In addition, significant differences were observed in the relative abundances of \u003cem\u003eGastranaerophilales, Prevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001, \u003cem\u003eAlistipes, Osillibacter\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e in both the vehicle-treated He and Ho groups compared to the vehicle-treated WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), aligning with the previous findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). However, an apparent trend in the changes in the abundance profiles of these bacterial taxa towards that of WT was evident in both the He and Ho groups in response to Halo treatment. These findings were further supported by LEfSe analysis, which revealed the enrichment of \u003cem\u003ePrevotellaceae\u003c/em\u003e_UCG-001 and \u003cem\u003eRuminococcus\u003c/em\u003e within the Halo-treated He (He-H) and Ho (Ho-H) groups, respectively (see Additional file 1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Antibiotic treatment inhibits the beneficial effect of Halo in CLC4 KO mice\u003c/h2\u003e\u003cp\u003eCLCN4-KO mice were administered a cocktail of antibiotics for three consecutive days and then treated with Halo through oral gavage daily for four weeks to further examine the gut microbial influence on Halo-mediated improvement in behavioral disorders. The motor activity, cognition, and memory of the animals were tested. The beneficial effects of Halo on OFT parameters were significantly impaired in the He and Ho groups after treatment with antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A similar suppressive effect of antibiotics on cognitive function was observed in the He-H group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). However, spatial learning and memory function as well as the serum p-cresol level in both the He-H and Ho-H groups remained unaltered in response to antibiotic treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, respectively).\u003c/p\u003e\u003cp\u003eNext, the PSD95, AKT, and ERK phosphorylation levels were examined in the hippocampus of all experimental animal groups to assess whether such cell-signaling proteins, which are involved in neuronal communication, plasticity, and function, are involved in mediating the effect of antibiotic treatment on the antipsychotic effect of Halo. The phosphorylation levels of all three proteins in the Ho group and PSD95 in the He group were significantly lower than those in the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). However, exposure to Halo significantly increased the phosphorylation levels of all three proteins in Ho mice, but not in He animals. Treatment of both the He-H and Ho-H groups with antibiotics significantly increased the phosphorylation of all three proteins in the He-H group, as well as AKT and ERK in the Ho-H group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.5. Amelioration of behavioral disorders in CLCN4-KO mice by FMT\u003c/h2\u003e\u003cp\u003eFMT was performed to further evaluate the involvement of gut microbes in the behavior of CLCN4-KO mice. Solutions of fecal matter from WT, He, and Ho mice were prepared and administered orally for five days after the induction of intestinal microbial destabilization by antibiotic treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAfter the desired FMT, the OFT, PAT, and NORT parameters were compared among various animal groups as follows: WT-wt vs. WT-he, WT-wt vs. WT-ho, He-he vs. He-wt, and Ho-ho vs. Ho-wt. The OFT parameter \u0026ldquo;total distance traveled\u0026rdquo; and the NORT parameter \u0026ldquo;object discrimination\u0026rdquo; did not vary significantly among the inter-group comparisons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similarly, another OFT parameter, \u0026ldquo;time spent in the center zone,\u0026rdquo; did not differ among all comparisons, except for its significantly higher value in the WT-he group than in the Wt-wt group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). A significantly higher value of the PAT parameter \u0026ldquo;latency time to enter the dark box\u0026rdquo; was observed in the test than in the training period in the He and Ho mice after receiving FMT from the WT group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, middle and right panels, respectively). A similar increase was also noted in WT animals after receiving FMT from the WT, He, or Ho groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, left panel).\u003c/p\u003e\u003cp\u003eNext, a detailed analysis of the vital gut microbial population in all experimental groups was performed before and after fecal transplantation to elucidate the major contributors to the FMT-mediated improvement of behavioral disorders in CLCN4-KO mice. PCoA results showed that the diversity of the gut microbiota in recipient mice was markedly altered in response to FMT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Furthermore, the following significant changes in the gut microbial population in response to FMT were observed: an increase in \u003cem\u003eAlistipes\u003c/em\u003e and a decline in \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 in both the WT-he and WT-ho groups; a decrease in \u003cem\u003eOscillibacter\u003c/em\u003e in the WT-he group and an increase in \u003cem\u003eMuribaculaceae\u003c/em\u003e in the WT-ho group; and a decline in \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, \u003cem\u003eOscillibacter\u003c/em\u003e, \u003cem\u003eGastranaerophilales\u003c/em\u003e, and \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 in both the HE-wt and HO-wt groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, PSD95, AKT, and ERK phosphorylation levels in the hippocampus of all FMT donor/recipient animal groups were measured to investigate whether these vital cells signaling proteins play a role in mediating the effect of FMT on animal behavioral indices. Significantly higher pERK levels were observed in the WT-he group than in the WT-wt group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Both pPSD95 and pAKT levels were significantly lower in the WT-ho group than in the WT-wt group. In contrast, significantly higher levels of pAKT and pERK were observed in the Ho-wt groups than in the He-he and Ho-ho groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Gut microbiomes are strongly associated with behavioral disorders in CLCN4-KO mice\u003c/h2\u003e\u003cp\u003eMI, heatmap, and co-occurrence network analyses were performed to determine possible correlations among the vital gut microbiota, behavioral parameters, hippocampal levels of key signaling proteins (PSD95, AKT, and ERK), and serum p-cresol levels. MI analysis conducted on \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001, \u003cem\u003eOscillibacter\u003c/em\u003e, \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, and \u003cem\u003eGastranaerophilales\u003c/em\u003e revealed that anxiety, hyperactivity, and serum p-cresol levels were the most frequent parameters strongly associated with these microbes (80% occurrence, indicated by red text) (see Additional file 2). Other correlated parameters included PSD95 (60% overall occurrence), NORT, ERK, and AKT (each with an overall 40% occurrence). However, heatmap and co-occurrence network analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e) revealed that \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 was positively correlated with hippocampal PSD95, AKT, and ERK levels and negatively correlated with hyperactivity. \u003cem\u003eOscillibacter\u003c/em\u003e was positively correlated with anxiety and serum p-cresol levels. In contrast, \u003cem\u003eGastranaerophilales\u003c/em\u003e showed a negative correlation with ERK expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study evaluated the effect of gut microbiota on the behavioral abnormalities of CLCN4-KO mice with a C57BL/6 genetic background and investigated the potential molecular mechanisms underlying these effects. Humans and mice share common genetic features, with over 90% of their genomes partitioned into corresponding regions of conserved synteny \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, while the protein-coding regions exhibit approximately 85% sequence similarity \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Numerous KO and knock-in mouse models are widely used for studying human diseases \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Experimental manipulation of the mouse genome, particularly gene KO, provides a powerful approach for generating animal models of human genetic disorders \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Mutations in the X-linked \u003cem\u003eCLCN4\u003c/em\u003e gene, both inherited and \u003cem\u003ede novo\u003c/em\u003e, have been associated with behavioral disorders \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In contrast, CLCN4-KO mice with a C57BL/6 background exhibit neurodevelopmental endophenotypes, including impaired social interaction and increased stereotypic behavior \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eVarious animal behavioral models are available for evaluating the cognitive and locomotor abilities of rodents. These models assess features such as anxiety, autonomic functions, learning, memory, and locomotor activity \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The OFT is one of the most widely used psychological assessment platforms \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, measuring locomotor activity based on distance traveled in the periphery of an open arena in an enclosed specialized box within a defined time. Time spent in the center of the open field is a more selective indicator of anxiety-like behavior \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. In this study, both He and Ho groups exhibited significantly higher total distances traveled and total time spent in the center area of the open field than the WT group. Learning and memory were assessed using the PAT, a fear-based task in which animals were trained to avoid entering a dark compartment associated with an aversive stimulus (a mild electric shock). WT mice exhibited significantly higher retention latency in the test than those in the training group after the electric shock, indicating normal spatial learning and memory function. In contrast, the He and Ho groups showed no significant differences between test and training retention latencies, suggesting impaired spatial learning and memory. Restricted and repetitive behaviors are characteristic features of ASD \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. To evaluate these behaviors, the NORT was performed, which measured the time spent interacting with a novel or familiar object. Both He and Ho mice exhibited significantly lower cognitive abilities than WT mice. These findings confirm that the deletion of \u003cem\u003eCLCN4\u003c/em\u003e induces behavioral symptoms in mouse models. This finding was further supported by the observation that treatment with Halo, a first-generation (typical) antipsychotic drug used to manage behavioral symptoms associated with autism \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, markedly reversed the above-mentioned adverse effects of \u003cem\u003eCLCN4\u003c/em\u003e KO in the He and Ho mice, except for anxiety-like behavior and cognitive deficits in the Ho group.\u003c/p\u003e\u003cp\u003eGut microbiota plays a crucial role in brain function, influencing neurogenesis, myelination, microglial maturation, development and maintenance of the blood\u0026ndash;brain barrier integrity, development of the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis, and HPA axis stress response development and function \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Disruptions in these processes can contribute to NDD \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Gut microbiota influences behavioral and physiological abnormalities associated with NDD \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, and gut dysbiosis in early life has been linked to an increased risk of conditions such as autism and ADHD \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, the gut microbiota form part of the unconscious regulatory system that modulates cognitive function and fundamental behavioral patterns, including social interaction and stress management \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. The bi-directional crosstalk between the gut microbiota and GBA\u0026mdash;referred to as the gut microbiome-brain axis\u0026mdash;is implicated in regulating complex characteristics, including social, emotional, and anxiety-like responses.\u003c/p\u003e\u003cp\u003ePrevious studies on ASD have reported gut microbial variations in β-diversity, although no consistent microbial signature has been identified across studies \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In this investigation, some overlapping yet distinct clustering patterns of gut microbiota were observed among the WT, He, and Ho groups, indicating significant differences in microbial composition. Significant differences in PCo1 were detected between the WT group and both He and Ho groups, as well as in PCo2 between the WT and He groups, suggesting differential diversity of gut microbes among these groups. Further analysis revealed that the gut microbiota composition in the He and Ho groups deviated from that of WT mice, as indicated by significantly lower Chao1 indices\u0026mdash;an α-diversity measure reflecting species richness\u0026mdash;compared to the WT group. Similar reductions in Chao1 and Shannon indices have been reported in patients with ASD compared to non-related neurotypical controls \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, aligning with findings from ASD mouse models \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Consistent with the observed β-diversity differences, significant changes in the relative abundances of key gut microbial genera were detected following \u003cem\u003eCLCN4\u003c/em\u003e KO. Specifically, higher abundances of \u003cem\u003eGastranaerophilales, Alistipes, Oscillibacter\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e, along with a lower population of \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG-001 were observed in He, Ho, or both groups than in WT mice. These differential microbial compositions were further supported by LEfSe analysis, which assessed the effect of Halo on the taxonomic rank profiling of gut microbes. Notably, \u003cem\u003eGastranaerophilales\u003c/em\u003e was enriched in the vehicle-treated (control) Ho group (see Additional file 1D). These findings are consistent with those of previous studies \u003csup\u003e\u003cspan additionalcitationids=\"CR70 CR71 CR72 CR73\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAlterations in the gut microbial profile affect neurological function and behavior via the gut\u0026ndash;microbiome\u0026ndash;brain axis, mediated by neurotransmitters, immune activation, and neuroactive bacterial metabolites \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. The negative effect of microbial metabolites, such as p-cresol, on neural function is attributed to multiple mechanisms, including membrane depolarization, augmented susceptibility to seizures \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, reduced Na\u003csup\u003e+\u003c/sup\u003e-K\u003csup\u003e+\u003c/sup\u003e ATPase activity \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e and impaired synthesis of dopamine from norepinephrine caused by dopamine-\u0026szlig;-hydroxylase inhibition \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Exposure to p-cresol induces autistic-like behaviors in mice by remodeling the gut microbiota \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Several phylogenetically diverse gut microbial strains can produce p-cresol, and the levels of this metabolite and its conjugates, p-CS and p-CG, are associated with \u003cem\u003eMuribaculaceae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eAlistipes\u003c/em\u003e [69, 79, 80, 81], and \u003cem\u003eOscillibacter\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, CLCN4-KO mice demonstrated higher abundances of \u003cem\u003eMuribaculaceae, Alistipes\u003c/em\u003e, and \u003cem\u003eOscillibacter\u003c/em\u003e than the WT mice, along with significantly elevated p-cresol levels. These observations suggest the involvement of these gut microbiota in the etiology of NDD. Furthermore, genome-based metabolic modeling in a previous study of patients with Parkinson\u0026rsquo;s disease (PD) identified \u003cem\u003eGastranaerophilales\u003c/em\u003e as a key bacterium responsible for indole production, which can enter the bloodstream \u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Indole is closely associated with various neurological and neuropsychiatric disorders \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e and induces anxiety-like behavior and depressive disorders in rats \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. In agreement with these reports, the higher population of \u003cem\u003eGastranaerophilales\u003c/em\u003e observed in both the He and Ho groups than in the WT group suggests that this microbial taxon may contribute to NDD onset and progression in this mouse model.\u003c/p\u003e\u003cp\u003eThe potential involvement of gut microbiota in the beneficial effects of Halo in CLCN4-KO mice was further explored. Exposing He and Ho mice to Halo significantly reduced serum p-cresol levels. Furthermore, a clear distinction in the clustering patterns of gut microbial communities was observed before and after Halo treatment in the He group, along with significant differences in PCo1 in the He group and PCo2 in the Ho group. These results indicate that Halo influences the gut microbiota in both He and Ho animals, albeit in different manners, highlighting a potential interaction between this antipsychotic drug and the gut microbiota of the CLCN4-KO mouse models. However, Chao1 diversity indices in both the He and Ho groups remained unchanged after Halo treatment, indicating that Halo does not affect the α-diversity of the gut microbial population in CLCN4-KO mice. This observation aligns with those of a previous clinical report showing no significant changes in the α-diversity (Chao1 and Shannon indexes) of the gut microbiota in patients with schizophrenia after 6 weeks of treatment with the antipsychotic drug olanzapine \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. However, a trend toward normalization of microbial abundance profiles was observed in response to Halo treatment, particularly in the genera \u003cem\u003eGastranaerophilales, Alistipes, Osillibacter, Muribaculaceae\u003c/em\u003e, and \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG-001, shifting toward levels observed in WT mice. This finding supports the hypothesis that antipsychotics may restore and normalize gut microbial diversity to a state more comparable to that of healthy controls, potentially offering therapeutic benefits for individuals with NDD \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. LEfSe analysis further revealed marked alterations in the microbial communities within both He and Ho groups after Halo treatment, albeit in a distinct manner, demonstrating the enrichment of \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG-001 in the He-H group and \u003cem\u003eRuminococcus\u003c/em\u003e in the Ho-H group (see Additional file 1D). Olanzapine treatment significantly increases the abundance of \u003cem\u003ePrevotellaceae\u003c/em\u003e UCG-001 in the gut environment of rats \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eRuminococcus\u003c/em\u003e can regulate dopaminergic signaling \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e which plays a fundamental role in neurodevelopment \u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e. The abundance of many \u003cem\u003eRuminococcus\u003c/em\u003e spp., including \u003cem\u003eRuminococcus\u003c/em\u003e sp. \u003cem\u003eAT10\u003c/em\u003e and \u003cem\u003eRuminococcus\u003c/em\u003e sp. \u003cem\u003eDJF\u003c/em\u003e was higher in patients with typical antipsychotic-treated schizophrenia (SCZ) than in atypical patients with SCZ treated with antipsychotic drugs \u003csup\u003e\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e\u003c/sup\u003e. Modification of the gut microbiota by several types of host-targeting non-antibiotic drugs, including first-generation antipsychotics such as Halo, may influence the pharmacokinetics and dynamics of these medicines and modulate their therapeutic efficacy by metabolizing drug compounds \u003csup\u003e\u003cspan additionalcitationids=\"CR92 CR93\" citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eTo further investigate the association between gut microbiota and the therapeutic effects of Halo, mice were treated with a broad-spectrum antibiotic cocktail. Antibiotic-induced intestinal dysbacteriosis triggers behavioral alterations and neuronal activation in various brain regions of mice \u003csup\u003e\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. Therefore, antibiotic-mediated disruption of gut microbial communities presents a useful approach for assessing the influence of gut microbiota on cognition, emotion, and behavior \u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. In this study, exposing the Ho-H and He-H groups to antibiotics resulted in a marked reversal of behavioral improvements, returning the animals to a disease-like state, with the exception of spatial learning and memory function, as assessed by PAT. These findings provide further support for the role of gut microbial communities on the therapeutic efficacy of Halo in improving NDD.\u003c/p\u003e\u003cp\u003eThe PSD95, ERK, and AKT signaling pathways were analyzed in the hippocampus of mice to further investigate the molecular mechanisms underlying gut microbial involvement in the antipsychotic activity and the protective effect of Halo against NDD. PSD95\u0026mdash;a key member of membrane-associated guanylate kinase family 1\u0026mdash;plays a crucial role in glutamatergic signaling, synaptic plasticity, and dendritic spine morphogenesis during neurodevelopment \u003csup\u003e\u003cspan additionalcitationids=\"CR98\" citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u003c/sup\u003e. Current evidence suggests a relationship between PSD95 dysfunction and NDD, including ASD \u003csup\u003e\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e\u003c/sup\u003e. The phosphorylation\u0026ndash;dephosphorylation status of the ser-295 residue of PSD95 is a key factor in controlling synaptic strength. Specifically, phosphorylation of this residue facilitates the synaptic accumulation of PSD95 and intensifies excitatory postsynaptic currents, whereas dephosphorylation triggers long-term depression \u003csup\u003e\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e\u003c/sup\u003e. In contrast, ERK phosphorylation plays an essential role in facilitating neuronal communication and plasticity \u003csup\u003e\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e\u003c/sup\u003e. PI3K/AKT signaling is essential for brain development, maintenance, repair, and plasticity during adulthood \u003csup\u003e\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u003c/sup\u003e. ERK phosphorylation levels are significantly lower in patients with autism than in neurotypical controls \u003csup\u003e\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e\u003c/sup\u003e. Similarly, dysregulated PI3K/AKT signaling is associated with ASD \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, in this study, the phosphorylation level of PSD95 in both the He and Ho groups and that of ERK and AKT in the Ho group were lower than those in the WT group. Treatment of the Ho group with Halo, but not the He group, significantly increased the phosphorylation of all three signaling proteins. These differences in phosphorylation levels and the responses of pPSD95, pAKT, and pERK to Halo between the He and Ho groups may be attributed to their distinct genetic backgrounds. However, with the exception of PSD95 in the Ho group, the phosphorylation levels of all three signaling proteins were significantly attenuated in both He and Ho animals upon co-treatment with antibiotics. These results suggest that the PSD95, ERK, and AKT signaling pathways play a crucial role in the gut microbial contribution to the effects of Halo on NDD.\u003c/p\u003e\u003cp\u003eThe association between gut microbes and NDD was further confirmed through experiments involving FMT, a technique commonly used to alter gut microbial composition by administering a solution of fecal matter from a donor into the intestinal tract of a recipient to achieve beneficial health effects \u003csup\u003e\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u003c/sup\u003e. Accumulating evidence indicates the transmission of depressive and anxiety-like symptoms and behaviors through FMT from psychologically ill donors to healthy recipients. The inverse has also been observed, with improvements in depressive and anxiety-like symptoms following FMT from healthy controls \u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. FMT findings and overall behavioral assessment of the animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) indicated that FMT from the WT mice largely contributed to the behavioral transition of the spatial learning and memory function. However, no significant effect was observed on locomotor activity, anxiety-like behavior, or cognitive function. In mice, FMT from aged donors impaired spatial learning and memory in young adult recipients. In contrast, anxiety, explorative behavior, and locomotor activity remained unaltered \u003csup\u003e\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u003c/sup\u003e, and aging-related symptoms improved in older WT animals after receiving FMT from young WT donors \u003csup\u003e\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e\u003c/sup\u003e. Furthermore, apart from a significantly higher value of \u0026ldquo;time spent in the center zone in the WT-he group compared to the Wt-wt group, no significant differences in behavioral indices were observed between the WT-wt group and the WT-he or WT-ho groups. These results suggest that the gut microbiota of WT mice may play a dominant role in shaping spatial learning and memory function in both the He and Ho groups, driving them toward a \"WT-type\" profile while potentially resisting the influence of transplanted microbes from CLCN4-KO animals.\u003c/p\u003e\u003cp\u003eA comprehensive analysis of the gut microbiota was conducted to further elucidate the molecular mechanisms underlying the FMT-mediated improvement of behavioral disorders in CLCN4-KO mice. The phosphorylation levels of PSD95, AKT, and ERK signaling proteins in the hippocampus of all studied groups were measured before and after fecal transplantation. The results demonstrated declines in \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, \u003cem\u003eOscillibacter\u003c/em\u003e, and \u003cem\u003eGastranaerophilales\u003c/em\u003e in both the He-wt and Ho-wt groups after FMT. Given the observed gut microbial profile (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) and the potential associations of \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, and \u003cem\u003eOscillibacter\u003c/em\u003e with the serum p-cresol level, as well as the relationship of \u003cem\u003eGastranaerophilales\u003c/em\u003e with indole production, it is plausible that the fecal preparation from WT mice contains gut microbes capable of suppressing the growth of these four bacterial taxa in the gut environment of the He and Ho groups. This suppression may contribute to the improvement of neurodevelopmental disorder (NDD) parameters in CLCN4-KO WT animals. The wild mouse gut microbiota improves disease resistance \u003csup\u003e\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e\u003c/sup\u003e. Clinical trials in patients with ASD have demonstrated the beneficial effects of FMT from healthy donors on neurological symptoms \u003csup\u003e\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u003c/sup\u003e. Additionally, multiple studies have reported improvements in depressive and anxiety-like symptoms and behaviors following the transplantation of healthy microbiota \u003csup\u003e\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e\u003c/sup\u003e. The dominance of gut microbes in WT mice over those of the CLCN4-KO animals was further reflected in the response of the WT animals to FMT from CLCN4-KO WT mice. Specifically, no significant changes were observed in the abundance of \u003cem\u003eGastranaerophilales\u003c/em\u003e and \u003cem\u003eMuribaculaceae\u003c/em\u003e, whereas a significant decline in the\u003cem\u003eOscillibacter\u003c/em\u003e population was noted after FMT from the He group. Furthermore, no significant alterations in the abundance of \u003cem\u003eGastranaerophilales\u003c/em\u003e and \u003cem\u003eOscillibacter\u003c/em\u003e were observed following FMT from the He group. Analysis of the phosphorylation levels of key hippocampal signaling molecules revealed the following features: no significant differences between the WT-wt and WT-he groups for pPSD95 and pAKT, or between the WT-wt and WT-ho groups for pERK; significantly higher levels of pPSD95 and pAKT in the WT-wt compared to WT-ho groups; increased pAKT levels in the He-wt compared to He-he groups; and elevated pERK levels in the WT-he compared to WT-wt groups, as well as in the Ho-wt compared to Ho-ho groups. These results suggest that the gut microbiota of WT mice plays a crucial role in shaping the gut microbiota, long-term memory, and learning functions, as well as the phosphorylation status of PSD95, AKT, and ERK signaling proteins in the recipient mice, shifting their profiles toward a WT-like pattern and indicating the dominance of WT gut microbiota over that of the He and Ho groups.\u003c/p\u003e\u003cp\u003eMI, heatmap, and co-occurrence network analyses were conducted to confirm the association between key gut microbiota, behavioral parameters, serum p-cresol levels, and neuronal signaling proteins. \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001, \u003cem\u003eOscillibacter\u003c/em\u003e, \u003cem\u003eAlistipes\u003c/em\u003e, \u003cem\u003eMuribaculaceae\u003c/em\u003e, and \u003cem\u003eGastranaerophilales\u003c/em\u003e were strongly and significantly associated with anxiety, hyperactivity, and serum p-cresol levels. Furthermore, \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001, which exhibited lower abundance in both the He and Ho groups than in the WT group, was positively correlated with the hippocampal levels of PSD95, AKT, and ERK but negatively correlated with hyperactivity. A lower abundance of \u003cem\u003ePrevotellaceae\u003c/em\u003e is associated with autism\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. Given the observed increase in \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 in CCLN4-KO after Halo treatment, this bacterial taxon likely contributes to the beneficial effects of Halo in improving NDD. \u003cem\u003eOsillibacter\u003c/em\u003e, which exhibited higher abundances in the He and Ho groups than in the WT group, demonstrated a positive correlation with p-cresol levels and anxiety. These findings align with those of previous studies reporting a higher abundance of \u003cem\u003eOsillibacter\u003c/em\u003e in patients with ASD than in the control volunteers \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, in high anxiety mice compared to low anxiety mice \u003csup\u003e\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e\u003c/sup\u003e, and in patients with PD with moderate depression compared to patients with PD without depression \u003csup\u003e\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eGastranaerophilales\u003c/em\u003e, which displayed higher abundances in the He and Ho groups than in the WT group, was negatively correlated with the hippocampal ERK level. Signaling through ERK/MAPK proteins is essential for the proper development of the nervous system from neural progenitor cells originating from the embryonic mesoderm, highlighting these proteins as potential novel therapeutic targets in several NDDs \u003csup\u003e\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study used the CLCN4-KO mouse model and applied Halo as an antipsychotic agent, with or without an antibody cocktail, to elucidate the involvement of gut microbiota, particularly \u003cem\u003eGastranaerophilales, Alistipes, Oscillibacter\u003c/em\u003e, and \u003cem\u003eMuribaculaceae\u003c/em\u003e in the etiology of NDD. The microbial metabolite p-cresol and the key hippocampal neuronal signaling proteins PSD95, AKT, and ERK played significant roles in this process. These findings indicate that Halo, which exhibited its characteristic antipsychotic effects in the He and Ho groups, was negatively correlated with the abundance of these bacteria and serum p-cresol levels in both groups and positively associated with the activation status of PSD9, AKT, and ERK in the Ho group. Many of these effects were inhibited by the application of an antibody cocktail, suggesting that gut microbiota influences the beneficial effect of Halo in CLCN4-KO mice. Additionally, \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001, which exhibited a lower abundance in both the He and Ho groups than in the WT group, was positively correlated with the hippocampal levels of activated PSD95, AKT, and ERK and negatively correlated with hyperactivity. Treatment with Halo significantly increased the \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 population in both He and Ho groups, suggesting that this bacterial taxon may contribute to the beneficial effects of Halo in improving NDD. The FMT study revealed that the gut microbiota of WT mice plays a dominant role in shaping the gut microbiota, long-term memory, and learning functions, as well as the phosphorylation status of hippocampal PSD95, AKT, and ERK in recipient mice towards the WT phenotype. Considering these results and the applicability of KO and knock-mouse models in studying human diseases \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, FMT from healthy patients fortified with \u003cem\u003ePrevotellaceae_\u003c/em\u003eUCG\u003cem\u003e-\u003c/em\u003e001 may be an effective treatment strategy for patients with NDD. However, extensive studies and clinical trials are required to validate this approach.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eADHD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eattention deficit hyperactivity disorder\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eASD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eautism spectrum disorder\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003echloride ion channel\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCLC4\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003echloride ion channel 4\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFMT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efecal microbiota transplantation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGBA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003egut\u0026ndash;brain axis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHalo\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehaloperidol\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHe\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eheterozygous\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHe-H\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHalo-treated He,Ho,homozygous\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHo-H\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHalo-treated Ho\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHPA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ehypothalamic\u0026ndash;pituitary\u0026ndash;adrenal\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eknockout\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eLEfSe\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003elinear discriminant analysis effect size\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emutual information\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNDD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eneurodevelopmental disorders\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNORT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enovel object recognition test\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOFT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eopen field test\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePAT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epassive avoidance test\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCoA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprincipal coordinate analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCo1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprincipal coordinate 1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePCo2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eprincipal coordinate 2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ep-CG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ep-cresyl glucuronide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ep-CS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ep-cresyl sulfate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eParkinson\u0026rsquo;s disease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003estandard error of the mean\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTBST\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTris-buffered saline containing 0.1% Tween 20\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ewild-type\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe authors are grateful for funding support from a grant of the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp;Welfare, Republic of Korea (RS-2020-KH087713) to Prof. Kim, and by the Main Research Program (E0170601\u0026ndash;09) of the Korea Food Research Institute funded by the Ministry of Science and ICT to Dr. Nam\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYC, SJ and HK jointly conceived and designed the study. YC performed animal behavior analysis, FMT, Western blotting, real-time PCR and analyzed all data. YC and JO performed network analysis. YC, EJS and YDN performed 16s rRNA analysis. YC and BS wrote the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw 16S rRNA sequencing reads from this study are available in the NCBI under the BioProject ( https://www.ncbi.nlm.nih.gov/bioproject/ ) accession number PRJNA1247208.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStraub L, Bateman BT, Hernandez-Diaz S, York C, Lester B, Wisner KL, et al. Neurodevelopmental disorders among publicly or privately insured children in the United States. JAMA psychiatry. 2022;79(3):232\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi S, Zhang W, Liang P, Zhu M, Zheng B, Zhou W, et al. Novel variants in the CLCN4 gene associated with syndromic X-linked intellectual disability. 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Neuroscience \u0026amp; Biobehavioral Reviews. 2021;131:792\u0026ndash;805.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CLCN4 knockout, Neurodevelopmental disorders, Gut microbiota, Gut–brain axis, Fecal microbiota transplantation","lastPublishedDoi":"10.21203/rs.3.rs-7514097/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7514097/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeurodevelopmental disorders (NDDs) are associated with gut\u0026ndash;brain axis dysfunction, and chloride ion channel 4 (CLCN4) has been implicated in their pathology. We investigated whether CLCN4 knockout (KO) alters gut microbiota and contributes to NDD-like phenotypes in mice. CLCN4-KO mice displayed behavioral abnormalities, microbial dysbiosis, and increased serum p-cresol levels, along with altered hippocampal signaling proteins (PSD95, AKT, ERK). Treatment with haloperidol (Halo) modified gut microbiota, reduced p-cresol, and improved behavior, effects accompanied by increased hippocampal protein activation in homozygous KO mice but abolished by antibiotic-induced dysbiosis. Prevotellaceae_UCG-001 abundance correlated positively with hippocampal protein activation and negatively with hyperactivity, and Halo treatment significantly increased this population. Fecal microbiota transplantation (FMT) from wild-type mice restored gut microbial balance, memory, and protein phosphorylation in KO mice. These findings indicate that CLCN4 deficiency contributes to NDD-like behaviors via microbiota-mediated mechanisms and highlight Halo and FMT as promising microbiota-targeted strategies.\u003c/p\u003e","manuscriptTitle":"Gut Microbiota-Mediated Modulation of Neurodevelopmental Behavior in CLCN4- Deficient Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 18:17:06","doi":"10.21203/rs.3.rs-7514097/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ed57d054-3b47-443a-85c8-13593bf9aa21","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55802233,"name":"Health sciences/Diseases"},{"id":55802234,"name":"Biological sciences/Microbiology"},{"id":55802235,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2025-10-17T15:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 18:17:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7514097","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7514097","identity":"rs-7514097","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}