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Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy
Journal of Clinical Neuroscience xxx (xxxx) xxx
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Experimental study
Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy Chao-Wen Luo a,1, Xiao-Yan Deng a,1, Jin-Ling Cheng a, Dan-Xian Xiao a, Chu-Yu Zhang a, Jia-Xin Feng a, Sheng-Qiang Chen b,⇑, Nan Hu b a b
KingMed school of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510182 China The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260 China
a r t i c l e
i n f o
Article history: Received 15 July 2019 Accepted 8 February 2020 Available online xxxx Keywords: Hyperbaric oxygen therapy Fragile X Syndrome Fmr1 Anxiety Social behaviors
a b s t r a c t Fragile X syndrome (FXS) is a common mental retardation syndrome. Anxiety and abnormal social behaviors are prominent features of FXS in humans. To better understand the effects of hyperbaric oxygen therapy (HBOT) on these behaviors, we analyzed anxiety-related and social behaviors in Fmr1 knockout mice treated by HBOT. In the open field test, HBOT group mice preferred the periphery to central areas and tended to run or walk along the wall. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group. In the elevated plus maze test, the percentage of distance traveled was significantly increased in the open arm and significantly decreased in the closed arm for HBOT group mice compared with control group mice. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm and exhibited fewer anxiety-related behaviors. In the three-chambered social approach test, the HBOT group mice made more approaches to the wire cup containing an acquaintance mouse than control group mice in the sociability test and made more approaches to the wire cup containing a stranger mouse than control group mice in the social novelty preference test. The results suggested that HBOT group mice showed increased levels of social interaction and decreased ‘‘social anxiety” than the control group to partner mice in this test. Our findings indicated that HBOT resulted in altered anxiety and social behavior in Fmr1 knockout mice and could possibly be used as a treatment for FXS. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability, caused by the loss of fragile X mental retardation (Fmr1) gene function [1,2], primarily due to abnormal methylation of expanded CGG trinucleotide repeats in the 50 untranslated region of the gene [3,4].The protein product of the Fmr1 gene (FMRP), highly expressed in the brain, is an RNAbinding protein related to the intracellular transport and translation of mRNAs, many of which are significant for synaptic function [3,5]. Individuals with FXS present with specific physical and behavioral phenotypes. Physically, male FXS patients have mild facial anomalies including a long narrow face, protruding ears and prominent jaw, hyper-extensible joints, flat feet and, in postpubescent males, macro-orchidism. Behaviorally, male FXS ⇑ Corresponding author at: Institute of Neuroscience, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China. E-mail address: [email protected] (S.-Q. Chen). 1 Equal contributors.
patients have features including cognitive impairment, attention deficits, hyperactivity, sensory hypersensitivity and hyperarousal, anxiety and social isolation and autistic-like behaviors, including gaze avoidance, and perseverative language and hand stereotypies [1,6–7]. Additionally, individuals with FXS are also commonly diagnosed with autism, and Fragile X syndrome is the most commonly known cause of autism [8–10]. The mechanism by which this occurs is unclear. Although clearly two different syndromes, there are also many commonalities between the two disorders [9]. The most common autistic features in Fragile X boys are repetitive motor behaviors, sensitivity to sensory stimuli of all modalities, and social anxiety. Abnormal social behaviors, especially shyness and social withdrawal, can be a prominent feature of FXS [8–10]. Several authors have speculated on a possible link between Fmr1 and autism. Hyperbaric oxygen therapy (HBOT) is the use of greater than atmospheric pressure oxygen as a pharmacological treatment for basic disease processes and their associated diseases [11,12]. HBOT has a variety of physiological effects depending on the chronicity of
https://doi.org/10.1016/j.jocn.2020.02.004 0967-5868/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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the underlying pathology [13]. In previous reports, HBOT has been found to potentially improve abnormal social behaviors in autistic individuals [14–16], but no one has yet investigated these processes in fragile X syndrome. Fmr1 knockout mice, which have undetectable levels of Fmr1 mRNA and FMRP, exhibit several of the behavioral and physical characteristics of the human syndrome [17,18]. In addition, it’s important to point out that the anxiety responses observed in FXS patients are mainly observed in social settings. Especially shyness and social withdrawal, abnormal social behaviors can be a prominent feature of FXS. Social anxiety is frequently observed in high-functioning males with solely mild or moderate deficits in FMRP and is often the presenting problem in females with FXS [8,19]. The present study was attempted to develop an animal model of aspects of the human case series to experimentally examine the efficacy of HBOT in a mouse model of FXS, and to provide information about anxiety-related and social behaviors improvement leading by HBOT in human patients. Therefore, we designed a series of experiments to further characterize anxiety-related responses and social interactions in Fmr1 KO mice treated with hyperbaric oxygen therapy. Our findings indicated that HBOT group mice displayed (a) hyperactivity with thigmotaxis in the open field test (b) a decrease in anxiety-related response in the elevated plus maze test and (c) more active social behaviors than the control group to partner mice in the three-chambered social test. 2. Materials and methods 2.1. Subjects Animal behavioral testing was performed on a colony of Fmr1 KO mice bred at Guangzhou Medical University from stock provided by S. Q. Chen, which originated from 129/OLA ES cells from B. Oostra. The genetic background and breeding strategy used follow the guidelines detailed in Crusio WE, Goldowitz D, Holmes A, Wolfer D (2009) Standards for the publication of mouse mutant studies. Mice (two to five per cage) were housed in a room with a 12-hours light/dark cycle ——lights on at 6:00 a.m., off at 18:00p.m.—— with access to food and water ad libitum. In general, behavioral testing was performed between 9:00 a.m. and15:00p. m.. All experiments were carried out in accordance with the guidelines laid down by the National Institutes of Health in the USA regarding the care and use of animals for experimental procedures and with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Correct targeting and integration were confirmed by genotyping using polymerase chain reaction. There were 11 male mice in HBOT group and 14 male mice in control group. The HBOT group was treated with hyperbaric oxygenation for a total of 21 days over two courses while the altitude control group did receive any treatment. After a total of 52 day, 25 mice in two groups were tested with a series of behavioral tests. The control group was tested at the same ages as the HBOT treated group. 2.2. Hyperbaric oxygen therapy HBOT was conducted as previously described [14,16] and similar to treatments used clinically in human patients in The Second Affiliated Hospital of Guangzhou Medical University. HBOT was delivered in a NG90IIIB mono-place chamber with 100% oxygen commencing 30 days after born. All mice receiving hyperbaric oxygen treatment were placed in the home cage of the animals. The cages were slid into the hyperbaric chamber on a gurney. During treatment sessions, the pressure in the chamber was slowly increased to 0.20 MPa(2.0 ATA) over the course of 20 min, followed
by a period of 40 min at the target pressure, and finally by a period of 15 min in which the pressure was gradually decreased back to ambient pressure. Oxygen flow rate was 5 L/min. Total treatment time was 80 min. Mice were carefully observed throughout the dives for behavioral changes or signs of distress. The group received a total of two HBOT courses (one session/day, 10 days a course, 1 day in between, then the second course). 2.3. Visual placing responses Poor visual acuity may influence the results of most behavioral tests. Before the detailed behavioral assays were conducted, as a result, visual acuity was evaluated using visual placing responses [20]. When suspended by the tail and lowered toward the table, all the Fmr1 KO mice raised their heads and reached out their forelimbs for the surface of the table, indicating no difference in visual acuity between the groups. Therefore, we used the Fmr1 KO mice for the following behavioral assays in consecutive order. 2.4. Open field test The open field test was conducted as described previously [17,20]. Each test mouse was placed in the center of an open field arena (70 70 cm each) illuminated at 600 lx and allowed to explore for 5 min. Each parameter within 5 min was evaluated with a computerized video-tracking system (Smart v 2.5.21; Panlab, Spain). 2.5. Elevated plus maze test The elevated plus maze test was conducted as previously described [17,20,21]. The elevated plus maze was elevated to a height of 50 cm above the floor and consisted of two enclosed arms surrounded by 15 cm high walls and two open arms. These arms (30 5 cm each) extended from a central area (5 5 cm). Each mouse was placed in the central area facing one of the open arms and allowed to explore for 5 min. Each parameter was analyzed using a computerized video-tracking system (Smart v 2.5.21). 2.6. Three-chambered social approach test To investigate sociability and social novelty preference, the three-chambered social approach test was conducted as described previously [18,22]. Sociability was defined by how the test mice made social contact with a familiar mouse versus an inanimate object. Social novelty preference was defined as how the test mice engaged in social contact with a novel stranger versus a familiar mouse. The three-chamber apparatus was a transparent plastic box with two transparent acrylic partitions with a rectangular opening (6 6 cm). The acrylic partitions divided the box into three chambers (left, center, and right; 20 40 23 cm each), which were illuminated at 120 lx. Each side chamber contained a cuboid wire cage (7.2 7.7 17 cm each) in the corner to hold a stimulus mouse. The wire cage consisted of wire bars, allowing minimal contact between the mice and to prevent fighting. A white bottle was placed on the top of the wire cage to prevent the test mouse from climbing to the top of the cage. All stimulus mice were of the same background, age, sex, and weight, and were acclimated to the wire cages for 10 min before beginning the test. The test consisted of three phases: acclimation, sociability, and social novelty preference. During the acclimation phase, a test mouse was placed in the center chamber and allowed to freely investigate and habituate to all three chambers and wire cages for 10 min. A familiar littermate was then placed in one of the wire cages. A wire cage containing a small ball was placed in the other side of threechamber as a non-social and inanimate object. During the second
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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phase, the test mouse was placed in the center chamber and allowed to freely investigate all three chambers for 10 min. The second phase was the sociability test. Subsequently, a novel unfamiliar mouse introduced as a novel stranger mouse was replaced for the small ball within another wire cage in the other side of the three-chamber apparatus. During the third phase, the test mouse was placed in the center chamber and allowed to freely investigate all three chambers for 10 min. The third phase was the social novelty preference test. Movement of the test mouse was recorded with a video camera and movement tracks were analyzed using Smart v 2.5.21. Parameters, including the number of events, duration, and mean time per event per test, were measured. 2.7. Statistics Statistical analyses were performed using the Statistical Package for Social Sciences version 13.0 (SPSS Inc., Chicago, IL USA). Unless noted otherwise, two-tailed P values were determined. Data are presented as mean ± SEM. One-way ANOVA was used to assess for differences between the HBOT and the control group. A probability value of p < 0.05 was considered statistically significant.
Fig. 1. The open field test.
3. Results 3.1. Open field test To evaluate spontaneous motor activity, exploratory behaviors, and emotional responses in a novel environment, we performed the open field test (Fig. 1). During the 5 min test session, both in the central (Fig. 3b, F1, 23 = 9.49, p < 0.01) and boundary areas (Fig. 3b, F1, 23 = 21.83, p < 0.01), the velocity traveled was increased in HBOT group mice compared with the control group mice. Distance traveled in the boundary (Fig. 3a, F1,23 = 21.53, p < 0.01) was also significantly increased in HBOT group mice. In addition, although time mice spent in areas did not significantly differ between groups, there were trends towards increased time spent in the boundary area (Fig. 3c, F1,23 = 0.98, p ＞ 0.05) and reduced time in the central area (Fig. 3c, F1,23 = 1.51, p ＞ 0.05) in HBOT compared with control group mice. Distance traveled in the center (Fig. 3a, F1,23 = 1.17, p ＞ 0.05) was increased in HBOT group mice, but did not significantly differ between groups. In a novel, brightly lit arena, mice preferred the periphery to the central areas and tend to run or walk along the wall, a behavior called thigmotaxis. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group mice Fig. 2. 3.2. The elevated plus maze test To investigate anxiety-related behaviors, we performed the elevated plus maze test (Fig. 4). We observed that the number of entrances was significantly increased in the open arm (Fig. 6a, F1,21 = 15.93, p < 0.01) in HBOT compared with control group mice. In addition, the distance traveled was significantly increased in the open arm (Fig. 6b, F1,21 = 22.91, p < 0.01) and significantly decreased in the closed arm (Fig. 6b, F1,21 = 4.58, p < 0.05) in HBOT group mice compared with control group mice. We also found that HBOT group mice spent significantly more time in the open arm than control group mice (Fig. 6c, F1,21 = 6.70, p < 0.05). However, the number of entrances in the closed arm and the time mice spent in the closed arm did not differ significantly between groups, but there were trend towards reduced the number of entrances in the closed arm (Fig. 6a, F1,21 = 2.78, p＞0.05) and reduced time in the closed arm (Fig. 6c, F1,21 = 3.87, p＞0.05) in the HBOT group
Fig. 2. The track of the HBOT group (left) and control group mice (right) in the open field test.
mice compared with control group mice. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm with fewer anxiety-related behaviors Fig. 5. 3.3. Social interaction test To examine social behaviors, including sociability and social novelty preference, we first performed the three-chambered social approach test in the Fmr1 knockout mice treated by hyperbaric oxygen therapy (Fig. 7). HBOT group mice exhibited a significantly higher number of approaches to the wire cup containing an acquaintance mouse (Fig. 8a, F1,23 = 9.89, p < 0.01) and the wire cup containing the object (Fig. 8a, F1,23 = 6.76, p < 0.05) than control group mice. For the duration of social interaction initiated by the subjects, HBOT group mice tended to spend more time around the wire cup containing an acquaintance mouse than control group mice (Fig. 8b, F1,23 = 3.95, p ＞ 0.05), although this result was not statistically significant. In addition, HBOT group mice spent significantly more time around the wire cup containing the object (Fig. 8b, F1,23 = 4.62, p < 0.05) than the control group mice. In the social novelty preference test, HBOT group mice also exhibited significantly more approaches to the wire cup containing a stranger mouse (Fig. 8a, F1,23 = 16.73, p < 0.01) and the wire cup containing an acquaintance mouse (Fig. 8a, F1,23 = 13.76, p < 0.01) than control group mice, but there were no significant differences between two groups for the duration of social interaction initiated by the subjects.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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Fig. 3. (a) Distance traveled in areas (cm). (b) Velocity traveled in areas (cm/s). (c) Time in areas (sec). Bars indicate means standard error of the mean for each parameter. HBOT group, n = 11; control group, n = 14; *p < 0.05,**p < 0.01. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group.
Fig. 5. The track of HBOT group (left) and control group mice (right) in the elevated plus maze test.
Fig. 4. The elevated plus maze test.
4. Discussion FXS is the most common inherited cause of intellectual disability that affects all major ethnic groups and races. The actual worldwide prevalence is estimated to range between 1:5000–7000 men and 1:4000–6000 women [1,7]. Thus, it is crucial to find the safe and effective treatment for FXS due to the rapidly growing patient population and the consequent huge burden on affected individuals, their families and care givers, and society as a whole. Fmr1
knockout mice, which have undetectable levels of Fmr1 mRNA and FMRP, exhibit several of the physical and behavioral characteristics of the human syndrome [18,23]. These mice are very useful in studies to elucidate mechanisms by which loss of FMR1 gene function alters behaviors and to test potential therapies to ameliorate these responses [23]. In additions, the X-linked nature of the disease results in considerable variation in the severity of symptoms when comparing males and females in human patients [19,24]. The symptoms in male patients tend to be more severe than in female patients with fragile X. 10% of males diagnosed with FXS are able to live independently compared with 44% of females [19,24]. As a result, the present study was attempted to develop an animal model of aspects of the human case series to experimen-
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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Fig. 6. (a) Number of entrances into arm. (b) Distance traveled in arm (cm). (c) Time in arm (sec). Bars indicate means standard error of the mean for each parameter. HBOT group, n = 9; control group, n = 14; *p < 0.05, **p < 0.01. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm and exhibited fewer anxiety-related behaviors.
Fig. 7. The three-chambered social approach test.
tally examine the efficacy of HBOT in the Fmr1 KO male mouse model of FXS, and to provide information about anxiety-related and social behaviors improvement leading by HBOT in human patients. To evaluate exploratory behaviors, spontaneous motor activity, and emotional responses in a novel environment, we performed the open field test. In the open field test, the Fmr1 knockout mice treated with hyperbaric oxygen therapy showed, contrary to
expectations, hyperactivity with thigmotaxis, suggesting a potential anxiogenic state[17,2315–16]. However, we find that distance traveled in the center with fewer anxiety-related behaviors was increased in HBOT group mice, although did not significantly differ between groups. The contradictory of the results suggests the need to conduct additional tests of anxiety to assess anxiety-like behavior in the mice. The Fmr1 KO mice are hyperactive, so increased locomotor activity could confound assessment of anxiety in these tests [25]. To further assess anxiety-related responses in Fmr1 KO mice treated with hyperbaric oxygen therapy, we performed the elevated plus maze test. In the present study, we find it interesting that the Fmr1 knockout mice treated with hyperbaric oxygen therapy enhanced motor activity in the open arm, suggesting a potential decrease in anxiety-like behavior. The anxiety-related behaviors of Fmr1 KO mice are complicated and may even appear to be contradictory. In some previous studies, Fmr1 KO mice showed decreased anxiety-like behavior both in the open-field and in the light/dark tests but increased anxiety-related responses in the mirrored chamber [17,23,25]. Therefore, it is likely to be an important factor that type of test chosen to evaluate anxietyrelated responses in FXS mouse models [23,26]. Given that both the open-field and the elevated plus maze test are tests for exploratory based anxiety. Currently, the responses of Fmr1 KO mice in non-exploratory tests of anxiety, such as light enhanced startle or stress induced hyperthermia, are being studied in our laboratory, which will be meaningful to further understand the relationship between anxiety responses and exploration in these mice.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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From these data, we can draw the following conclusions. The present results suggest that HBOT changed anxiety and social behaviors in the Fmr1 knockout mouse and could possibly be used as a treatment for FXS. There are several possible explanations for this pattern of results. HBOT has been used in several cerebral hypoperfusion conditions and can compensate for decreased blood flow by increasing the oxygen content of plasma and body tissues [12,13]. There is evidence that HBOT can reduce oxidative stress through the upregulation of antioxidant enzymes [12,13,27]. HBOT has been reported to possess strong anti-inflammatory properties and has been shown to improve immune function [12,13]. HBOT can also increase the function and production of mitochondria and improve neurotransmitter abnormalities [13,27]. In addition, enzymes can be upregulated with HBOT to help with detoxification problems [13]. Further, dysbiosis, commonly found in autistic children, can be improved by HBOT [13]. Finally, HBOT has been shown to mobilize stem cells from bone marrow to systemic circulation. Previous research showed that stem cells can enter the brain and form new neurons, astrocytes, and microglia in humans [13,28]. It is expected that amelioration of these underlying pathophysiological problems through the use of HBOT will lead to improvements in FXS symptoms.
Fig. 8. (a) The number of approaches. (b) Contact time (sec). Stranger 1 is the familiar mouse in session II. Bars indicate means standard error of the mean for each parameter. HBOT group, n = 11; control group, n = 14; *p < 0.05, **p < 0.01. The results suggested that HBOT group mice showed increased levels of social interaction and made more active social behaviors than the control group to partner mice in this test.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments
Anxiety is a dominating behavioral characteristic of FXS. Particularly, many FXS patients exhibit shyness or so-called ‘‘social anxiety” and are easily upset by novel environment or people [8,23]. They appear to have a particular kind of anxiety involving social and novel situations or environments [8–9]. The threechambered social approach test has a potential social component, though it is also an exploration-based test. KO mice display abnormal social responses that may be considered consistent with ‘‘social anxiety” [19,26]. In the three-chambered social approach test, HBOT group mice exhibited a significantly higher number of approaches to the wire cup containing an acquaintance mouse and the wire cup containing the object than control group mice. These mice demonstrated increased social and non-social behaviors than the control group to partner mice in the test. These results seem to be similar to those obtained by El-Baz F et al, who reported an increase in social behaviors in autistic children after hyperbaric oxygen therapy [16].The presented results suggest that HBOT-treated animals display decreased ‘‘social anxiety” compared with control mice. In this study, the duration of time spent engaging in social behavior differed to a lesser extent. We agree with the view of Spencer et al. [23] that the most consistent and robust difference is the number of active approaches rather than the time spent in interactions initiated by the subject mouse. Their hypothesis is that once the approach has occurred, the duration of interaction depends on the cooperation of both the mouse that initiated the interaction and the recipient mouse. Thus, regardless of the interest of the initiating mouse, if the partner is not cooperative, the duration of the interaction will be shorter[23].Therefore, the recipient mouse in our study, also an Fmr1 KO mouse, untreated with hyperbaric oxygen therapy, maybe displayed lower levels of interest with the recipient interaction mouse. Although the social responses of Fmr1 KO mice are complex and influenced by experience with partners and test environment, the present findings demonstrate that HBOT-treated animals display decreased ‘‘social anxiety” compared with control mice.
We thank Professor B. A. Oostra (Cellular Biology and Genetics Research Center, Erasmus University, Rotterdam, Netherlands) very much for providing the FVB Fmr1 knockout mice. This work was supported by the National Natural Science Foundation of China, China (Grant No. 31771327), the Science and Technology Plan Project of Guangdong Province, China (Grant No. 2015A0030302090), the innovative Academic team project of Guangzhou Bureau of Education, China (Grant No. 1201610032), the Science and Technology project of Guangzhou Municipal Health Bureau, China (Grant No. 20151A010129 and 20151A011080), the China College Students’ Innovation and Entrepreneurship Training program, China (Grant No. 201810570017), the Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation, China (Grant No. pdjhb0420) and by the Guangzhou Medical University Students’’ Scientific and Technological Innovation Project, China (Grant No. 2017A059). We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jocn.2020.02.004. References [1] Phillips JP, Wilson GA. Fragile X syndrome. Indian J Pediatrics 1998;65:181–91. https://doi.org/10.1007/BF02752293. [2] Bassell GJ, Warren ST. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron 2008;60:201–14. https://doi.org/ 10.1016/j.neuron.2008.10.004. [3] O’Donnell WT, Warren ST. A decade of molecular studies of fragile X syndrome. Annu Rev Neurosci 2002;25:315–38. https://doi.org/10.1146/annurev. neuro.25.112701.142909.
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Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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Experimental study
Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy Chao-Wen Luo a,1, Xiao-Yan Deng a,1, Jin-Ling Cheng a, Dan-Xian Xiao a, Chu-Yu Zhang a, Jia-Xin Feng a, Sheng-Qiang Chen b,⇑, Nan Hu b a b
KingMed school of Laboratory Medicine, Guangzhou Medical University, Guangzhou 510182 China The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260 China
a r t i c l e
i n f o
Article history: Received 15 July 2019 Accepted 8 February 2020 Available online xxxx Keywords: Hyperbaric oxygen therapy Fragile X Syndrome Fmr1 Anxiety Social behaviors
a b s t r a c t Fragile X syndrome (FXS) is a common mental retardation syndrome. Anxiety and abnormal social behaviors are prominent features of FXS in humans. To better understand the effects of hyperbaric oxygen therapy (HBOT) on these behaviors, we analyzed anxiety-related and social behaviors in Fmr1 knockout mice treated by HBOT. In the open field test, HBOT group mice preferred the periphery to central areas and tended to run or walk along the wall. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group. In the elevated plus maze test, the percentage of distance traveled was significantly increased in the open arm and significantly decreased in the closed arm for HBOT group mice compared with control group mice. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm and exhibited fewer anxiety-related behaviors. In the three-chambered social approach test, the HBOT group mice made more approaches to the wire cup containing an acquaintance mouse than control group mice in the sociability test and made more approaches to the wire cup containing a stranger mouse than control group mice in the social novelty preference test. The results suggested that HBOT group mice showed increased levels of social interaction and decreased ‘‘social anxiety” than the control group to partner mice in this test. Our findings indicated that HBOT resulted in altered anxiety and social behavior in Fmr1 knockout mice and could possibly be used as a treatment for FXS. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability, caused by the loss of fragile X mental retardation (Fmr1) gene function [1,2], primarily due to abnormal methylation of expanded CGG trinucleotide repeats in the 50 untranslated region of the gene [3,4].The protein product of the Fmr1 gene (FMRP), highly expressed in the brain, is an RNAbinding protein related to the intracellular transport and translation of mRNAs, many of which are significant for synaptic function [3,5]. Individuals with FXS present with specific physical and behavioral phenotypes. Physically, male FXS patients have mild facial anomalies including a long narrow face, protruding ears and prominent jaw, hyper-extensible joints, flat feet and, in postpubescent males, macro-orchidism. Behaviorally, male FXS ⇑ Corresponding author at: Institute of Neuroscience, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, China. E-mail address: [email protected] (S.-Q. Chen). 1 Equal contributors.
patients have features including cognitive impairment, attention deficits, hyperactivity, sensory hypersensitivity and hyperarousal, anxiety and social isolation and autistic-like behaviors, including gaze avoidance, and perseverative language and hand stereotypies [1,6–7]. Additionally, individuals with FXS are also commonly diagnosed with autism, and Fragile X syndrome is the most commonly known cause of autism [8–10]. The mechanism by which this occurs is unclear. Although clearly two different syndromes, there are also many commonalities between the two disorders [9]. The most common autistic features in Fragile X boys are repetitive motor behaviors, sensitivity to sensory stimuli of all modalities, and social anxiety. Abnormal social behaviors, especially shyness and social withdrawal, can be a prominent feature of FXS [8–10]. Several authors have speculated on a possible link between Fmr1 and autism. Hyperbaric oxygen therapy (HBOT) is the use of greater than atmospheric pressure oxygen as a pharmacological treatment for basic disease processes and their associated diseases [11,12]. HBOT has a variety of physiological effects depending on the chronicity of
https://doi.org/10.1016/j.jocn.2020.02.004 0967-5868/Ó 2020 Elsevier Ltd. All rights reserved.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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the underlying pathology [13]. In previous reports, HBOT has been found to potentially improve abnormal social behaviors in autistic individuals [14–16], but no one has yet investigated these processes in fragile X syndrome. Fmr1 knockout mice, which have undetectable levels of Fmr1 mRNA and FMRP, exhibit several of the behavioral and physical characteristics of the human syndrome [17,18]. In addition, it’s important to point out that the anxiety responses observed in FXS patients are mainly observed in social settings. Especially shyness and social withdrawal, abnormal social behaviors can be a prominent feature of FXS. Social anxiety is frequently observed in high-functioning males with solely mild or moderate deficits in FMRP and is often the presenting problem in females with FXS [8,19]. The present study was attempted to develop an animal model of aspects of the human case series to experimentally examine the efficacy of HBOT in a mouse model of FXS, and to provide information about anxiety-related and social behaviors improvement leading by HBOT in human patients. Therefore, we designed a series of experiments to further characterize anxiety-related responses and social interactions in Fmr1 KO mice treated with hyperbaric oxygen therapy. Our findings indicated that HBOT group mice displayed (a) hyperactivity with thigmotaxis in the open field test (b) a decrease in anxiety-related response in the elevated plus maze test and (c) more active social behaviors than the control group to partner mice in the three-chambered social test. 2. Materials and methods 2.1. Subjects Animal behavioral testing was performed on a colony of Fmr1 KO mice bred at Guangzhou Medical University from stock provided by S. Q. Chen, which originated from 129/OLA ES cells from B. Oostra. The genetic background and breeding strategy used follow the guidelines detailed in Crusio WE, Goldowitz D, Holmes A, Wolfer D (2009) Standards for the publication of mouse mutant studies. Mice (two to five per cage) were housed in a room with a 12-hours light/dark cycle ——lights on at 6:00 a.m., off at 18:00p.m.—— with access to food and water ad libitum. In general, behavioral testing was performed between 9:00 a.m. and15:00p. m.. All experiments were carried out in accordance with the guidelines laid down by the National Institutes of Health in the USA regarding the care and use of animals for experimental procedures and with the European Communities Council Directive of 24 November 1986 (86/609/EEC). Correct targeting and integration were confirmed by genotyping using polymerase chain reaction. There were 11 male mice in HBOT group and 14 male mice in control group. The HBOT group was treated with hyperbaric oxygenation for a total of 21 days over two courses while the altitude control group did receive any treatment. After a total of 52 day, 25 mice in two groups were tested with a series of behavioral tests. The control group was tested at the same ages as the HBOT treated group. 2.2. Hyperbaric oxygen therapy HBOT was conducted as previously described [14,16] and similar to treatments used clinically in human patients in The Second Affiliated Hospital of Guangzhou Medical University. HBOT was delivered in a NG90IIIB mono-place chamber with 100% oxygen commencing 30 days after born. All mice receiving hyperbaric oxygen treatment were placed in the home cage of the animals. The cages were slid into the hyperbaric chamber on a gurney. During treatment sessions, the pressure in the chamber was slowly increased to 0.20 MPa(2.0 ATA) over the course of 20 min, followed
by a period of 40 min at the target pressure, and finally by a period of 15 min in which the pressure was gradually decreased back to ambient pressure. Oxygen flow rate was 5 L/min. Total treatment time was 80 min. Mice were carefully observed throughout the dives for behavioral changes or signs of distress. The group received a total of two HBOT courses (one session/day, 10 days a course, 1 day in between, then the second course). 2.3. Visual placing responses Poor visual acuity may influence the results of most behavioral tests. Before the detailed behavioral assays were conducted, as a result, visual acuity was evaluated using visual placing responses [20]. When suspended by the tail and lowered toward the table, all the Fmr1 KO mice raised their heads and reached out their forelimbs for the surface of the table, indicating no difference in visual acuity between the groups. Therefore, we used the Fmr1 KO mice for the following behavioral assays in consecutive order. 2.4. Open field test The open field test was conducted as described previously [17,20]. Each test mouse was placed in the center of an open field arena (70 70 cm each) illuminated at 600 lx and allowed to explore for 5 min. Each parameter within 5 min was evaluated with a computerized video-tracking system (Smart v 2.5.21; Panlab, Spain). 2.5. Elevated plus maze test The elevated plus maze test was conducted as previously described [17,20,21]. The elevated plus maze was elevated to a height of 50 cm above the floor and consisted of two enclosed arms surrounded by 15 cm high walls and two open arms. These arms (30 5 cm each) extended from a central area (5 5 cm). Each mouse was placed in the central area facing one of the open arms and allowed to explore for 5 min. Each parameter was analyzed using a computerized video-tracking system (Smart v 2.5.21). 2.6. Three-chambered social approach test To investigate sociability and social novelty preference, the three-chambered social approach test was conducted as described previously [18,22]. Sociability was defined by how the test mice made social contact with a familiar mouse versus an inanimate object. Social novelty preference was defined as how the test mice engaged in social contact with a novel stranger versus a familiar mouse. The three-chamber apparatus was a transparent plastic box with two transparent acrylic partitions with a rectangular opening (6 6 cm). The acrylic partitions divided the box into three chambers (left, center, and right; 20 40 23 cm each), which were illuminated at 120 lx. Each side chamber contained a cuboid wire cage (7.2 7.7 17 cm each) in the corner to hold a stimulus mouse. The wire cage consisted of wire bars, allowing minimal contact between the mice and to prevent fighting. A white bottle was placed on the top of the wire cage to prevent the test mouse from climbing to the top of the cage. All stimulus mice were of the same background, age, sex, and weight, and were acclimated to the wire cages for 10 min before beginning the test. The test consisted of three phases: acclimation, sociability, and social novelty preference. During the acclimation phase, a test mouse was placed in the center chamber and allowed to freely investigate and habituate to all three chambers and wire cages for 10 min. A familiar littermate was then placed in one of the wire cages. A wire cage containing a small ball was placed in the other side of threechamber as a non-social and inanimate object. During the second
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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phase, the test mouse was placed in the center chamber and allowed to freely investigate all three chambers for 10 min. The second phase was the sociability test. Subsequently, a novel unfamiliar mouse introduced as a novel stranger mouse was replaced for the small ball within another wire cage in the other side of the three-chamber apparatus. During the third phase, the test mouse was placed in the center chamber and allowed to freely investigate all three chambers for 10 min. The third phase was the social novelty preference test. Movement of the test mouse was recorded with a video camera and movement tracks were analyzed using Smart v 2.5.21. Parameters, including the number of events, duration, and mean time per event per test, were measured. 2.7. Statistics Statistical analyses were performed using the Statistical Package for Social Sciences version 13.0 (SPSS Inc., Chicago, IL USA). Unless noted otherwise, two-tailed P values were determined. Data are presented as mean ± SEM. One-way ANOVA was used to assess for differences between the HBOT and the control group. A probability value of p < 0.05 was considered statistically significant.
Fig. 1. The open field test.
3. Results 3.1. Open field test To evaluate spontaneous motor activity, exploratory behaviors, and emotional responses in a novel environment, we performed the open field test (Fig. 1). During the 5 min test session, both in the central (Fig. 3b, F1, 23 = 9.49, p < 0.01) and boundary areas (Fig. 3b, F1, 23 = 21.83, p < 0.01), the velocity traveled was increased in HBOT group mice compared with the control group mice. Distance traveled in the boundary (Fig. 3a, F1,23 = 21.53, p < 0.01) was also significantly increased in HBOT group mice. In addition, although time mice spent in areas did not significantly differ between groups, there were trends towards increased time spent in the boundary area (Fig. 3c, F1,23 = 0.98, p ＞ 0.05) and reduced time in the central area (Fig. 3c, F1,23 = 1.51, p ＞ 0.05) in HBOT compared with control group mice. Distance traveled in the center (Fig. 3a, F1,23 = 1.17, p ＞ 0.05) was increased in HBOT group mice, but did not significantly differ between groups. In a novel, brightly lit arena, mice preferred the periphery to the central areas and tend to run or walk along the wall, a behavior called thigmotaxis. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group mice Fig. 2. 3.2. The elevated plus maze test To investigate anxiety-related behaviors, we performed the elevated plus maze test (Fig. 4). We observed that the number of entrances was significantly increased in the open arm (Fig. 6a, F1,21 = 15.93, p < 0.01) in HBOT compared with control group mice. In addition, the distance traveled was significantly increased in the open arm (Fig. 6b, F1,21 = 22.91, p < 0.01) and significantly decreased in the closed arm (Fig. 6b, F1,21 = 4.58, p < 0.05) in HBOT group mice compared with control group mice. We also found that HBOT group mice spent significantly more time in the open arm than control group mice (Fig. 6c, F1,21 = 6.70, p < 0.05). However, the number of entrances in the closed arm and the time mice spent in the closed arm did not differ significantly between groups, but there were trend towards reduced the number of entrances in the closed arm (Fig. 6a, F1,21 = 2.78, p＞0.05) and reduced time in the closed arm (Fig. 6c, F1,21 = 3.87, p＞0.05) in the HBOT group
Fig. 2. The track of the HBOT group (left) and control group mice (right) in the open field test.
mice compared with control group mice. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm with fewer anxiety-related behaviors Fig. 5. 3.3. Social interaction test To examine social behaviors, including sociability and social novelty preference, we first performed the three-chambered social approach test in the Fmr1 knockout mice treated by hyperbaric oxygen therapy (Fig. 7). HBOT group mice exhibited a significantly higher number of approaches to the wire cup containing an acquaintance mouse (Fig. 8a, F1,23 = 9.89, p < 0.01) and the wire cup containing the object (Fig. 8a, F1,23 = 6.76, p < 0.05) than control group mice. For the duration of social interaction initiated by the subjects, HBOT group mice tended to spend more time around the wire cup containing an acquaintance mouse than control group mice (Fig. 8b, F1,23 = 3.95, p ＞ 0.05), although this result was not statistically significant. In addition, HBOT group mice spent significantly more time around the wire cup containing the object (Fig. 8b, F1,23 = 4.62, p < 0.05) than the control group mice. In the social novelty preference test, HBOT group mice also exhibited significantly more approaches to the wire cup containing a stranger mouse (Fig. 8a, F1,23 = 16.73, p < 0.01) and the wire cup containing an acquaintance mouse (Fig. 8a, F1,23 = 13.76, p < 0.01) than control group mice, but there were no significant differences between two groups for the duration of social interaction initiated by the subjects.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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Fig. 3. (a) Distance traveled in areas (cm). (b) Velocity traveled in areas (cm/s). (c) Time in areas (sec). Bars indicate means standard error of the mean for each parameter. HBOT group, n = 11; control group, n = 14; *p < 0.05,**p < 0.01. The results suggested that thigmotaxis was significantly increased in the HBOT group compared with the control group.
Fig. 5. The track of HBOT group (left) and control group mice (right) in the elevated plus maze test.
Fig. 4. The elevated plus maze test.
4. Discussion FXS is the most common inherited cause of intellectual disability that affects all major ethnic groups and races. The actual worldwide prevalence is estimated to range between 1:5000–7000 men and 1:4000–6000 women [1,7]. Thus, it is crucial to find the safe and effective treatment for FXS due to the rapidly growing patient population and the consequent huge burden on affected individuals, their families and care givers, and society as a whole. Fmr1
knockout mice, which have undetectable levels of Fmr1 mRNA and FMRP, exhibit several of the physical and behavioral characteristics of the human syndrome [18,23]. These mice are very useful in studies to elucidate mechanisms by which loss of FMR1 gene function alters behaviors and to test potential therapies to ameliorate these responses [23]. In additions, the X-linked nature of the disease results in considerable variation in the severity of symptoms when comparing males and females in human patients [19,24]. The symptoms in male patients tend to be more severe than in female patients with fragile X. 10% of males diagnosed with FXS are able to live independently compared with 44% of females [19,24]. As a result, the present study was attempted to develop an animal model of aspects of the human case series to experimen-
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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Fig. 6. (a) Number of entrances into arm. (b) Distance traveled in arm (cm). (c) Time in arm (sec). Bars indicate means standard error of the mean for each parameter. HBOT group, n = 9; control group, n = 14; *p < 0.05, **p < 0.01. These results suggested that HBOT group mice displayed enhanced motor activity in the open arm and exhibited fewer anxiety-related behaviors.
Fig. 7. The three-chambered social approach test.
tally examine the efficacy of HBOT in the Fmr1 KO male mouse model of FXS, and to provide information about anxiety-related and social behaviors improvement leading by HBOT in human patients. To evaluate exploratory behaviors, spontaneous motor activity, and emotional responses in a novel environment, we performed the open field test. In the open field test, the Fmr1 knockout mice treated with hyperbaric oxygen therapy showed, contrary to
expectations, hyperactivity with thigmotaxis, suggesting a potential anxiogenic state[17,2315–16]. However, we find that distance traveled in the center with fewer anxiety-related behaviors was increased in HBOT group mice, although did not significantly differ between groups. The contradictory of the results suggests the need to conduct additional tests of anxiety to assess anxiety-like behavior in the mice. The Fmr1 KO mice are hyperactive, so increased locomotor activity could confound assessment of anxiety in these tests [25]. To further assess anxiety-related responses in Fmr1 KO mice treated with hyperbaric oxygen therapy, we performed the elevated plus maze test. In the present study, we find it interesting that the Fmr1 knockout mice treated with hyperbaric oxygen therapy enhanced motor activity in the open arm, suggesting a potential decrease in anxiety-like behavior. The anxiety-related behaviors of Fmr1 KO mice are complicated and may even appear to be contradictory. In some previous studies, Fmr1 KO mice showed decreased anxiety-like behavior both in the open-field and in the light/dark tests but increased anxiety-related responses in the mirrored chamber [17,23,25]. Therefore, it is likely to be an important factor that type of test chosen to evaluate anxietyrelated responses in FXS mouse models [23,26]. Given that both the open-field and the elevated plus maze test are tests for exploratory based anxiety. Currently, the responses of Fmr1 KO mice in non-exploratory tests of anxiety, such as light enhanced startle or stress induced hyperthermia, are being studied in our laboratory, which will be meaningful to further understand the relationship between anxiety responses and exploration in these mice.
Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004
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From these data, we can draw the following conclusions. The present results suggest that HBOT changed anxiety and social behaviors in the Fmr1 knockout mouse and could possibly be used as a treatment for FXS. There are several possible explanations for this pattern of results. HBOT has been used in several cerebral hypoperfusion conditions and can compensate for decreased blood flow by increasing the oxygen content of plasma and body tissues [12,13]. There is evidence that HBOT can reduce oxidative stress through the upregulation of antioxidant enzymes [12,13,27]. HBOT has been reported to possess strong anti-inflammatory properties and has been shown to improve immune function [12,13]. HBOT can also increase the function and production of mitochondria and improve neurotransmitter abnormalities [13,27]. In addition, enzymes can be upregulated with HBOT to help with detoxification problems [13]. Further, dysbiosis, commonly found in autistic children, can be improved by HBOT [13]. Finally, HBOT has been shown to mobilize stem cells from bone marrow to systemic circulation. Previous research showed that stem cells can enter the brain and form new neurons, astrocytes, and microglia in humans [13,28]. It is expected that amelioration of these underlying pathophysiological problems through the use of HBOT will lead to improvements in FXS symptoms.
Fig. 8. (a) The number of approaches. (b) Contact time (sec). Stranger 1 is the familiar mouse in session II. Bars indicate means standard error of the mean for each parameter. HBOT group, n = 11; control group, n = 14; *p < 0.05, **p < 0.01. The results suggested that HBOT group mice showed increased levels of social interaction and made more active social behaviors than the control group to partner mice in this test.
Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments
Anxiety is a dominating behavioral characteristic of FXS. Particularly, many FXS patients exhibit shyness or so-called ‘‘social anxiety” and are easily upset by novel environment or people [8,23]. They appear to have a particular kind of anxiety involving social and novel situations or environments [8–9]. The threechambered social approach test has a potential social component, though it is also an exploration-based test. KO mice display abnormal social responses that may be considered consistent with ‘‘social anxiety” [19,26]. In the three-chambered social approach test, HBOT group mice exhibited a significantly higher number of approaches to the wire cup containing an acquaintance mouse and the wire cup containing the object than control group mice. These mice demonstrated increased social and non-social behaviors than the control group to partner mice in the test. These results seem to be similar to those obtained by El-Baz F et al, who reported an increase in social behaviors in autistic children after hyperbaric oxygen therapy [16].The presented results suggest that HBOT-treated animals display decreased ‘‘social anxiety” compared with control mice. In this study, the duration of time spent engaging in social behavior differed to a lesser extent. We agree with the view of Spencer et al. [23] that the most consistent and robust difference is the number of active approaches rather than the time spent in interactions initiated by the subject mouse. Their hypothesis is that once the approach has occurred, the duration of interaction depends on the cooperation of both the mouse that initiated the interaction and the recipient mouse. Thus, regardless of the interest of the initiating mouse, if the partner is not cooperative, the duration of the interaction will be shorter[23].Therefore, the recipient mouse in our study, also an Fmr1 KO mouse, untreated with hyperbaric oxygen therapy, maybe displayed lower levels of interest with the recipient interaction mouse. Although the social responses of Fmr1 KO mice are complex and influenced by experience with partners and test environment, the present findings demonstrate that HBOT-treated animals display decreased ‘‘social anxiety” compared with control mice.
We thank Professor B. A. Oostra (Cellular Biology and Genetics Research Center, Erasmus University, Rotterdam, Netherlands) very much for providing the FVB Fmr1 knockout mice. This work was supported by the National Natural Science Foundation of China, China (Grant No. 31771327), the Science and Technology Plan Project of Guangdong Province, China (Grant No. 2015A0030302090), the innovative Academic team project of Guangzhou Bureau of Education, China (Grant No. 1201610032), the Science and Technology project of Guangzhou Municipal Health Bureau, China (Grant No. 20151A010129 and 20151A011080), the China College Students’ Innovation and Entrepreneurship Training program, China (Grant No. 201810570017), the Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation, China (Grant No. pdjhb0420) and by the Guangzhou Medical University Students’’ Scientific and Technological Innovation Project, China (Grant No. 2017A059). We thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jocn.2020.02.004. References [1] Phillips JP, Wilson GA. Fragile X syndrome. Indian J Pediatrics 1998;65:181–91. https://doi.org/10.1007/BF02752293. [2] Bassell GJ, Warren ST. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron 2008;60:201–14. https://doi.org/ 10.1016/j.neuron.2008.10.004. [3] O’Donnell WT, Warren ST. A decade of molecular studies of fragile X syndrome. Annu Rev Neurosci 2002;25:315–38. https://doi.org/10.1146/annurev. neuro.25.112701.142909.
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Please cite this article as: C.-W. Luo, X. Y. Deng, J. L. Cheng et al., Altered anxiety and social behaviors in a mouse model of Fragile X syndrome treated with hyperbaric oxygen therapy, Journal of Clinical Neuroscience, https://doi.org/10.1016/j.jocn.2020.02.004