Altered Behaviour Associated With Autism in a Mouse Model of Fragile X Syndrome Treated With Bacteroides Fragilis BF839

doi:10.21203/rs.3.rs-76080/v1

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Altered Behaviour Associated With Autism in a Mouse Model of Fragile X Syndrome Treated With Bacteroides Fragilis BF839

https://doi.org/10.21203/rs.3.rs-76080/v1

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Background: Fragile X syndrome (FXS), tightly related to the morbidity of Autism spectrum disorder (ASD), is a common hereditary syndrome often associated with retardation of intelligence. Some key symptoms of ASD such as anxiety, cognitive impairment and social anxiety disorder are also the predominant features in FXS. Children with ASD are often performed with gastrointestinal symptoms. According to the existing research, with the treatment with Bacteroides Fragilis BF839, mice with ASD will have better performance in communication and social behaviours with less anxiety and perceptual disorder. In this article, we have observed the impact of Bacteroides Fragilis BF839, a well-established Chinese bacteria strain with the human intestine origin, on mice with FXS and their behavioural disorders accordingly.

Result: Based on the Open Field test, compared to the Fmr1KO group, mice treated with BF839 showed prolonged staying time in the center of the container. This finding suggests that BF839 can improve Fmr1KO mice's self-exploration behaviour and dented their anxiety. The Elevated Plus Maze test indicated BF839 treated mice presented more activities in entering open arms, prolonged time of staying and significantly less distance travelled at the plus-maze, along with less entering behaviours in the closed arms with less time of staying and more distance travelled. This result proved that with the treatment of BF839, Fmr1KO mice have improved ability in recognizing the surrounding environment and greater senses at detecting danger. Three-box Social Interaction test confirmed that BF839 strengthens the social novelty preference of the Fmr1KO mice, proven by their increasing duration and frequency in social interacting with the stranger mouse. The final experiment named the Pool Maze test presented the result that on the fourth day, BF839 treated mice have shown significantly shortened escape latency. Meanwhile, on Day 5, BF839 treated group performed increasing frequency in passing through the platform, which, along with the shortened escape latency, demonstrated BF839 has the function of improving Fmr1KO mice's cognitive capacity and their ability to extract information from the surrounding environment.

Conclusion: Based on the outcome of each test performed, Bacteroides Fragilis BF839 can successfully improve Autism related abnormal behaviours in mice with FXS. Bacteroides Fragilis BF839 can be a potential intervention strategy in treating FXS and ASD safely and effectively.

General Microbiology

Fragile X syndrome (FXS)

Therapeutic bacteria

Gastrointestinal therapy

Autism spectrum disorder (ASD)

Behavioural disorder

the Mouse model

Recent studies have shown that gut bacteria have profound implications in a wide range of human diseases such as neurodevelopmental disorders and other mental diseases. Gut bacteria can first act on the central nervous system then contribute to the metabolism and maintenance of the human body [1,2]. Fragile X syndrome (FXS) is one of the most common hereditary diseases associated with intelligence retardation, with the morbidity of 1/4000 in males and 1/8000 in females [3]. FXS is diagnosed by the loss of the function of Fragile X mental retardation gene 1 (Fmr1) characterized by the instability of repetitive CGG trinucleotide sequence and abnormality methylation at the upstream CpG island [4,5,6,7]. Due to the failure in expressing the Fmr1 gene, the lack of Fmr1 protein has caused some abnormal changes in physical features in patients, including enlarged testicles, large ears, a long and narrow face and a prominent jaw and forehead [4]. Patients with FXS often present autism, anxiety, cognitive disorder and social interaction challenge [8,9,10]. Strictly associated with ASD, about 5~25% of patients with ASD also have FXS, and among male patients with FXS, 90% of them are presented symptoms of ASD, and above 60% of them could be diagnosed with ASD [4,11,12]. Based on current research, children with ASD are often presented with gastrointestinal symptoms [12,14,15]. Studies have shown that the use of Bacteroides fragilis NCTC9343 can reduce anxiety-like behavior in the mouse as well as other behavioral abnormalities such as communication and perception difficulties [17]. However, compared to Bacteroides fragilis BF893 which has entered the Chinese market for 20 years, Bacteroides fragilis NCTC9343 has not yet been commercialized [18]. Bacteroides fragilis BF839, also called Totem probiotics, has been proved to be effective in improving T cells' immune function and can be beneficial in treating gastrointestinal diseases [19]. Therefore, in this study, we aimed at investigating if Bacteroides fragilis BF839 has the potential therapeutic function in intervening Fmrl1 KO mice's exploring behavior, anxiety-like behavior, cognitive capacity, social behavior and learning as well as memorizing capacity.

1.1 Subject Animals:

Under a specific pathogen-free (SPF) condition, the gene knockout (KO) homozygous (-/-) and the hybrid of the wild-type (WT) and the homozygous (+/+) FVB inbreeding mice were donated by Professor B. A. Oostra (Cellular Biology and Genetics Research Center, Erasmus University, Rotterdam, Netherlands) and bred by Experimental Animal Center at The Second Affiliated Hospital of Guangzhou Medical University [20]. After the approval of the Instiutional Animal Care and Use Committee of The Second Affiliated Hospital of Guangzhou Medical University, this experiment chose three-week-old weaned mice as subjects, divided them into 2 groups: the Fmr1KO group (the control group) and the BF839 group (the intervention group). Each group contained 15 mice, sub-packing them into 3 different cages with 5 mice in one cage. The experiment allowed the BF839 group to drink freely from Bacteroides fragilis BF839 solution for four weeks, while the Fmr1KO group was drinking autoclaved tap water for the same period. The animal behavioral experiments were carried on the subject mice when they were seven to eight weeks old and were last for two weeks from 9:00 a.m. to 4:00 p.m..

1.2 The Identification of Experiment Animal's Genotypes:

Before the experiment, all the subject mice were verified by the PCR test, aiming at the genotype of Fmr1 KO and the hybrid of WT and FVB mice to design the proper primer. The designed primer M2 (5′-AGTCATGCTATGGATATCAG-3′) and N2 (5′-TGGGCTCTATGGCTTCTGA-3′) was used for detecting WT mice's Fmr1 allele fragments (amplified size of 468 bp). Refer to the related literature for more information on identifying the genotypes of the experiment animals [20].

1.3The Origins of Bacteroides fragilis BF839:

In this study, Bacteroides fragilis BF839 (Totem probiotics) solutions were provided by DALIAN TOTEM BIOLOGICAL ENGINEERING CO., LTD. Each solution contains 25 mL, with 25 x 108 CFU/ solution viable amount of Bacteroides fragilis BF839. Each two of the solutions were provided to one cage of BF839 group mice per day, last of the duration of four weeks.

1.4 Open Field Test:

Without the lid, a 30 cm tall, 70 cm wide cube box was used as the experimental analysis box. Each mouse was placed in the center of the box one at the time, allowing 5 minutes free movement. Meanwhile, the mouse's trajectory was recorded by a camera with its distance travelled and its staying duration spent at the boundary as well as at the center of the box was tracked by SMART (Smart v 2.5.21; Panlab, Spain). The data collection period would last for 5 minutes each time. The experiment method was drawn from related literature listed in the References section [17, 21, 22, 23].

1.5 Elevated Plus-Maze Test:

The elevated maze was designed in a cruciform structure, consisting of one 50 cm tall stand and four 30 cm long, 5 cm wide arms. Two of the arms on the opposite side were blocked by 15 cm tall partition, creating a closed area. The rest two arms also are on the opposite side were unobstructed, creating an open area. All four arms were connected by a 5 x 5 cm central platform. At the beginning of the test, each mouse was placed in the centre of the platform with a position facing the open are. At each trial, the subject would be allowed to move freely for 5 minutes. A camera would record the trajectory of the subjects and the app SMART was used to record the frequency, distance travelled and duration of staying time for 5 minutes. The experiment method was drawn from related literature listed in the References section [21, 22, 23].

1.6 Three-box Social Interaction Test:

The three transparent plastic boxes were separated into different cells by two clapboards, creating a right, a middle and a left cell. Each cell would have a size of 20 x 40 x 23 cm, with a 6 x 6 cm square entrance open for subjects to enter freely. Before the test, a metal cage would be placed beside the right and left cells. The subject mouse would be put into the middle cell right beside the cell walls for 10 minutes to fit in the environment. In the first phase of the test, the subject mouse was removed from the cells. A sphere was placed at the left cage while a distinct mouse coming from the same cage as the subject mouse was placed in the right cage. The mice were chosen from the same sex on purpose. When everything is settled, the subject would be put back into the middle cell as before and would be allowed to move freely for 10 minutes. In the second phase of the test, the subject mouse would be removed from the three-box environment again and the sphere was removed from the left cell, replaced with a new mouse from the different cage as the subject mouse. The new mouse would have the same sex as the subject mouse. Then the subject mouse would enter the middle cell again to move freely for another 10 minutes. At each phase, the camera would record the trajectory of the subject mouse and the app SMART would be used to record the frequency and duration of time spent at each cage (the contacting area would be 4.0 cm around the cage) for 10 minutes. The experiment method was drawn from related literature listed in the References section [17, 22, 23, 24].

1.7 Pool Maze Test:

The round platform with a diameter of 5 cm and a height of 30 cm was fixed into a round pool with a diameter of 120 cm and a height of 50 cm. The water temperature would be around 21-22°C with the water depth was measured to be 1 cm higher than the platform. The experiment consisted of two phases: the first phase was the positioning navigation phase, following the space exploration phase. In the first phase, the subject was trained four times a day with 30 seconds breaks between each training for four days. The escape latency was defined as the period when the subject entered the pool until it successfully climbs up on the platform. The escape latency was recorded in order as the subject entered the pool from different quadrants. A camera was recording the trajectory of the subject and the app SMART was used to record the subject's escape latency. The average data of the escape latency of the 4 pieces of training would be noted as a final grade of the day. If the subject fails to figure out the location of the platform within 60 seconds, the subject would be guided to the platform and stranded for 10 seconds and its escape latency would be recorded as 60 seconds. In the space exploration phase, the platform would be removed on the fifth day, and the subject would be placed at the opposite quadrant to the original ones. A camera was recording the trajectory of the subject and the app SMART was used to record and analyze how many times did the subject mouse pass through the invisible platform. After each trial, the subject mouse would be removed from the experimental setting and be properly dried before putting back to the cage. The experiment method was drawn from related literature listed in the References section [21].

1.8 Statistical Analysis:

All data collected from this study would be analyzed by SPSS 17.0 statistics software and would be presented as (x ± s). According to the normality and the homogeneity of the variance of the data, the two-sided independent sample t-test or nonparametric test was used for comparison between two groups. The repeated-measures ANOVA was demonstrated to compare and analyze the escape latency from the positioning navigation stage groups. The data would be considered as statistically significant when *p<0.05, **p<0.01, ***p<0.001.

2.1 The Identification of Experiment Animal's Genotypes:

About 800 bp DNA segments were produced by the PCR on Fmr1KO mice; 468 bp DNA segments were produced on the wild type mice( Figure. 1). Due to the presence Neomycin segment on the fifth exon of the Fmr1 gene, the amplification of 468 bp DNA was interdicted, which claimed that there was a gene defect on FXS mice.

2.2 Open Field Test:

To evaluate the subject mouse's exploratory behaviours and anxiety in a new environment, the Open Field test was performed to observe the intervention of Bacteroides fragilis BF839 on the subjects. During this 5-minute experiment, the difference in the time spent and distance travelled at the boundary or in the centre of the field between the subjects was detected. Compared to Fmr1 KO mice, mice treated with Bacteroides fragilis BF839 showed less distance travelled at the boundary of the field (t=3.631, p<0.001), with more distance travelled in the centre of the field (t=4.657, p<0.001), demonstrated a statically significant difference (Figure. 2a). Subjects treated with Bacteroides fragilis BF839 also showed less time spent at the boundary of the field (t=5.959, p<0.001) with prolonged time spent in the centre of the field (t=6.024, p<0.001), demonstrated a statically significant difference (Figure. 2b). Figure. 3 displays the trajectory of the subjects. Based on these results, it could be claimed that Bacteroides fragilis BF839 can strengthen Fmr1KO mice's exploratory behaviours in a new environment with less anxiety.

2.3: Elevated Plus-Maze Test:

To evaluate the subjects' cognitive capacity and their crisis awareness, the Elevated Plus-Maze test was performed to observe the intervention of Bacteroides fragilis BF839 on the subjects. In this 5-minute experiment, the difference in the frequency, duration of stay and distance travelled in the open and closed area between two groups of subjects. Compared to Fmr1 KO mouse, Bacteroides fragilis BF839 treated mouse showed decreasing in frequency entering the open area (t=2.506, p<0.05) with an increasing number of times entering the closed area (t=2.499, p<0.05). The result demonstrated a statically significant difference (Figure. 4a). Also, Bacteroides fragilis BF839 treated subjects performed shorten in the duration of stay in the open area (t=2.610, p<0.05) with prolonged time staying in the closed area (t=5.845, p<0.001). The result demonstrated a statically significant difference (Figure. 4b). Bacteroides fragilis BF839 treated subjects showed less distance travelled in the open area compared to the Fmr1 KO mouse (t=2.825, p<0.01) and increasing in distance travelled in the closed area (t=3.553, p<0.01). The result demonstrated a statically significant difference (Figure. 4c). Figure. 5 displays the trajectory of the subjects in the elevated plus-maze. Based on the data analyzed, Bacteroides fragilis BF839 treated mice preferred to stay in the closed area which deemed to be less dangerous to them. This finding proved that Bacteroides fragilis BF839 can improve Fmr1 KO mice's cognitive capacity and crisis awareness.

2.4: Three-box Social Interaction Test:

To evaluate the subjects' social behaviors and social novelty preference, the Three-box Social Interaction test was performed. In the first phase of the experiment, the time (t=3.524, p<0.01) and approaches (t=1.476, p＞0.05) to the familiar mouse than to the sphere was observed in Fmr1 KO mouse with a significant difference in the contacting time. As for BF839 treated mouse, the result found that they presented less frequent approaches to the familiar mouse compared with the number of approaches to the sphere but with prolonged contacting time. None of these findings demonstrated a significant difference (p>0.05) (Figure. 6a, 6b). Comparing to the Fmr1 KO group, BF839 treated mice spent more time with both the familiar mouse and the sphere with an increasing number of approaches. Thereinto, the number of approaches (t=2.841, p<0.05) to the sphere and the time spent with it (t=2.148, p<0.05) has shown a statically significant difference (Figure. 6a, 6b). The trajectory of the first stage of the experiment was shown in Figure. 7a. The result of this experiment demonstrated that with the intervention of Bacteroides fragilis BF839, the overall activeness of Fmr1 KO mouse was generally improved, including its social and non-social behaviour with the particular increase on the non-social behaviour.

During the second phase of the Three-box Social Interaction test, there was no significant difference between the contact with the familiar mouse and the stranger mouse in the Fmr1 KO group (p>0.05). However, the number of approaches (t=5.741, p<0.001) and contacting time (t=3.310, p<0.05) was significantly higher with the stranger mouse than the familiar mouse observed in the BF839 treated mouse (Figure. 6c, 6d). Figure.7b displayed the trajectory of the subject during the second phase of the experiment. The above results demonstrated that when facing the familiar and the stranger mouse of its kind, BF839 treated mouse would rather spend more time with a higher frequency with the stranger mouse than the familiar mouse. This behaviour indicated that Bacteroides fragilis BF839 could strengthen the Fmr1 KO mouse's social novelty preference.

2.5 Pool Maze Test:

To evaluate the subjects' study, memorize and cognitive capacity, the Pool Maze test was performed to observe the intervention of Bacteroides fragilis BF839 on the subjects. During the positioning navigation (Figure. 8a), there was a statically significant difference in the escape latency (F=5.500, p<0.05) and the timing (F=6.849, p<0.001). Within the first three days, there was no difference between the two groups of subjects in the duration of escape latency (p>0.05). At Day four, Bacteroides fragilis BF839 treated mouse then showed significantly shorten escape latency than Fmr1 KO mouse, with a statically significant difference (p<0.001). Figure. 9a displays the trajectory of the subjects during the positioning navigation phase of the Pool Maze test. During the space exploring phase of the experiment (Figure. 8b), after the withdrawal of the platform, Bacteroides fragilis BF839 treated mouse has demonstrated increasing frequency passing through the platform compared to the Fmr1 KO group within one minute, with a statically significant difference (t=2.800, p<0.05). The exploring trajectory of the subjects during the space exploration phase of the experiment is displayed in Figure. 9b. Based on the information above, Bacteroides fragilis BF839 treated mouse used less time to figure out the location of the invisible platform than the Fmr1 KO group, and this difference became even more obvious over time. This finding demonstrated that Bacteroides fragilis BF839 can effectively improve the cognitive and memory capacity of the Fmr1 KO mice.

As an ideal animal model of studying FXS, Fmr1 KO mouse is targeted to destroy the Fmr1 gene to ensure it fails to express Fmr1 protein, which then leads to enlarged testicles, loss of memory, abnormal behaviours and retardation of intelligence [21, 24]. Researches show that Fmr1 KO mouse can also be an animal model to study mouse with ASD [25].

In the Open Field test, mice spent more time and travelled more in the centre area indicated that they have performed more investigating behaviours with a decrease in anxiety [25]. During our experiment, compared to Fmr1 KO mice, Bacteroides Fragilis BF839 treated mice demonstrated more activities in the center area. This result is alike researches have done by Hsiao and others [17]: after treated by Bacteroides Fragilis NCTC 9343, the mouse with MIA was showing more entering and staying behaviours in the centre area. Therefore, this experiment gives a clue that Bacteroides Fragilis BF839 is capable of enhancing Fmr1 KO mouse's self-exploring behaviour and lower its anxiety.

In the Elevated Plus-Maze test, the behaviour presenting more entrances into the open area with prolonged staying time and more distances travelled would be normally considered as lower in anxiety [22]. When comparing to the normal WT mouse, Fmr1 KO mouse demonstrated increasing activities at the open area proven by more staying time, the number of entrances and distances travelled [26]. This result may indicate the decrease in cognitive capacity and the loss of sense of danger instead of reduced anxiety. Since for healthy human beings, an environment like the open area would be considered dangerous like the edge of a cliff, we considered mouse with normal cognitive capacity and with the sense of danger would prefer safer environment as the closed area in this experiment. Based on this test, compared to the Fmr1 KO mouse, BF839 treated mice demonstrated less entering, staying time and distances travelled into the open area, more entering staying and distances travelled into the closed area. Thus, we infer that Bacteroides fragilis BF839 can improve Fmr1 KO mouse's cognitive capacity to sensing the surrounding environment and their crisis awareness.

In the Three-box Social Interaction test, it is normally agreed that if the subjects display increasing time and frequency interacting with the familiar mouse than the sphere at the first stage of the experiment, the subjects could be considered to have stronger social behaviour compared to the non-social behaviour. However, there is limited research published on how to evaluate this kind of behaviour of the Fmr1 KO mouse. Only 1 research has conducted on this topic and the result of this article showed that the Fmr1 KO mouse demonstrated a longer time and increased frequency with the familiar mouse than the sphere [22], which is consistent with our result. This finding may allude that there is a pre-existing preference in the Fmr1 KO mouse to prefer social behaviour rather than non-social behaviour. Other than this, BF 839 treated mouse would have fewer approaches but more contacting time towards the familiar mouse than the sphere with no significant difference. Based on these findings, there is insufficient evidence to safely use this indicator to determine if there is an improvement from the treatment of Bacteroides fragilis BF839. At the first stage of the experiment, compared to the Fmr1 KO mouse, the BF 839 treated mouse showed synchronous growth in contact with both familiar mouse and the sphere with no statistically significant difference in the time contacting with the familiar mouse but with the sphere. Therefore, we believed that Bacteroides fragilis BF839 could strengthen the overall activeness of the Fmr1 KO mouse including social and non-social behaviour; the increasing approaches towards the sphere then may reflect its improved in social novelty preference. According to Tabouy L and other researched indicated in another article, Lactobacillus reuteri treated Shank3 KO mouse has demonstrated weakened non-social behaviour in male mice [22], which differed from our result. This might result from the genetic background of the selected mice and the difference between each probiotics strain.

Normally, when the subject mouse demonstrates more time and approaches towards a stranger mouse than a familiar mouse at the second stage of the experiment, it would be considered that the subject mouse is presenting social novelty preference [22]. During the second stage of the experiment, BF839 treated mice presented significant differences in prolonged social contact and approach frequency towards the stranger mouse, reflecting its social novelty preferences. Plus, according to Hsiao and other researchers, with the intervention of Bacteroides fragilis NCTC9343, there was no significant improvement of MIA mouse (a mouse model with Autism)'s social behaviour and social novelty preference [17]. Thus, based on our positive result, Bacteroides fragilis BF839 did have a better ability in improving Fmr1 KO mouse's social behaviour compared to the other bacteria strains mentioned above. As well-known by the public, there is strain specificity in the efficacy of probiotics.

During the Water Maze experiment of Morris, the shortened in escape latency and an increasing number of times crossing the platform indicates the better study, memorize and cognitive ability of the subjects [27]. Compared with the normal WT mouse, the prolonged escape latency and less frequency in crossing the platform showing that the Fmr1 KO mouse has decreased in the study and memorize ability as well as cognitive impairment [27]. Based on the result, compared to Fmr1 KO mouse, BF839 treated mouse demonstrated shortened escape latency on the fourth day and increasing in the number of times crossing the platform on the fifth day. Therefore, this experiment indicated that Bacteroides fragilis BF839 can strengthen Fmr1 KO mouse's study and memorize ability as well as its cognitive function.

By far, it is still unclear the molecular mechanism of how micro-bacteria monitor the brain activities and behaviour through brain-gut axis regulation. Research has suggested that Bacteroides fragilis NCTC9343 could potentially address the alteration of the microbial dependent metabolites 4-ethylbenzene sulfate/ester (4EPS) and Indolyl-3-acryloylglycine that cause anxiety-like behaviour changes in the ASD model MIA offspring mice [17]. Plus, the study claimed that Bacteroides fragilis NCTC9343 can also make an impact on the change of serum 5-HT in hippocampus learning and memory, repair the gastrointestinal barrier defects in the offspring of MIA and reduce intestinal permeability and regulate the expression of inflammatory cytokines [17]. Clinical researchers have found that probiotics can improve the GI symptoms of ASD patients and even some non-core autism symptoms as well [28, 29, 30]. We will carry out further clinical research on the molecular mechanism of Bacteroides fragilis BF839 in the treatment of Fmr1 KO mice.

In sum, Bacteroides fragilis BF839 can strengthen Fmr1 KO mice's self-exploration behaviour in a new environment; decrease its anxiety; improve its danger sensibility; enhancing its social behaviour and improve its study and memorizing ability. Thus, Bacteroides fragilis BF839 could potentially be a new intervention to treat FSX and ASD.

Acknowledgement:

The authors would like to thank Dr. B.A. Oostra from Cellular Biology and Genetics Research Center, Erasmus University for donating the FVB Fmr1 KO mice,and also thank DALIAN TOTEM BIOLOGICAL ENGINEERING CO., LTD.for cooperating to provide Bacteroides fragilis BF839 (Totem probiotics) solutions.

Funding:

This work was supported and funded by the Guangzhou Municipal Health Technology General Project of Guangzhou Municipal Health Commission in China (NO. 20201A011081).

Consent for publication:

Not applicable

Availability of data and materials:

All data generated or analysed during this study are included in this published article.

Competing Interest:

The authors declare that they have no competing interest

Ethics Approval:

All procedures treated with mice were in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals in China. This experiment was approved by the Instiutional Animal Care and Use Committee of The Second Affiliated Hospital of Guangzhou Medical University (Number of Animal use permit:SYXK 2018-0192, Acceptance number:A2019-029).

Author information:

First Author

Chu-Hui Lin & Ting Zeng

Corresponding Author

Sheng-Qiang Chen & Yu-Hong Deng

Institution

Department of Clinical Nutrition ,The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510260, China

Chu-Hui Lin, Ting Zeng, Yu-Hong Deng

Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University; Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China

Bing-Mei Li,Sheng-Qiang Chen

Guangzhou Medical University, Guangzhou, 511436, China

Jian-Hong Lin, Feng Xiao

Authors' contributions:

Lin. C, Chen. S and Deng. Y conceived the study and designed the experiment, analyzed the feasibility and performed the experiment, wrote and refined the manuscript then audited the research article to ensure its quality. Lin. C, Zeng. T, Xiao. F and Li. B monitored the Open Field test, the Elevated Maze test, the Three-box Social Interaction test and the Pool Maze test. Lin. J fed the subject mice and ran the gene test of the subjects. Lin. C collected, organized the outcome, ran the statistical analysis of the data collected and concluded the final research.

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Altered Behaviour Associated With Autism in a Mouse Model of Fragile X Syndrome Treated With Bacteroides Fragilis BF839

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