Summary of medical doctoral thesis: Research on establishing an autism model using sodium valproate and ameliorative effects of enriched environment on behaviors in white mice

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This research with two aims: Establishing an animal model of autism using Swiss white mice by prenatal exposure to sodium valproate. Evaluating the effects of enriched environment on behaviors in modelled white mice

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Nội dung Text: Summary of medical doctoral thesis: Research on establishing an autism model using sodium valproate and ameliorative effects of enriched environment on behaviors in white mice

MINISTRY OF MINISTRY OF DEFENCE EDUCATION AND TRAINING VIETNAM MILITARY MEDICAL UNIVERSITY DAO THU HONG RESEARCH ON ESTABLISHING AN AUTISM MODEL USING SODIUM VALPROATE AND AMELIORATIVE EFFECTS OF ENRICHED ENVIRONMENT ON BEHAVIORS IN WHITE MICE Speciality: Physiology Code: 62720107 SUMMARY OF MEDICAL DOCTORAL THESIS HANOI - 2018
This research was carried out in Vietnam Military Medical University Supervisors: 1. Tran Hai Anh, M.D., Ph.D., Assoc.Prof. 2. Can Van Mao, M.D., Ph.D., Assoc.Prof. Reviewer 1: Reviewer 2: Reviewer 3: This thesis will be defended in doctoral examination council of Vietnam Military Medical University at….on the date of …………….. This thesis is available at: 1. National Library 2. Library of Vietnam Military Medical University 3. ………………………………………………….
1 INTRODUCTION 1. Imperativeness Autism is a neurodevelopmental disorder whose diagnosis is based on three behavioral criteria: unusual reciprocal social interactions, deficits in communication, and stereotyped repetitive behaviors with restricted interests. The etiology and pathophysiology of autism are unclear. Nowadays, autism is known as a modern-day phenomenon with the prevalence increase all over the world. The studies of CDC shown that the prevalence of autism was 0.5 per 1000 children in 1985, increased 12 per 1000 children in 2012. Prenatal exposure to valproate (VPA) leads abnormal neurodevelopment and increased risk of autism spectrum disorders. In animal model, prenatal VPA-exposure also lead abnormal behaviors and pathology similar to that observed in autism individuals. For that reason, valproate is used to induce animal model of autism in order to investigate the mechanism of biological changes and to evaluate the effect of interventions for autism. In Vietnam, there are several researches about some risk factors, some clinical features in autistic patients. However, there is no research about animal model of autism and pathophysiological mechanism for autism. So, we conducted this research with two aims: 1. Establishing an animal model of autism using Swiss white mice by prenatal exposure to sodium valproate. 2. Evaluating the effects of enriched environment on behaviors in modelled white mice. 2. Scientific significance The thesis has provided data about behavioral alterations of Swiss mice prenatally exposed to sodium valproate (VPA) with dose 300-
2 500mg/kg i.p., creating animal model of autism in Swiss mice by using sodium valproate i.p. at dose 500mg/kg. Providing data about the ameliorating of enriched environment on behaviors in VPA- exposed mice. 3. Practical significance Using that VPA-exposed mice to determine neurobiological alterations related to the pathogenesis of autism and assess the effects of drugs or treatments on autism. 4. Structure of the thesis The thesis consists 124 pages. 2 pages Introduction; Chapter 1 (Documentary Overview) 32 pages; Chapter 2 (Subject and methods) 14 pages; Chapter 3 (Results) 41 pages; Chapter 4 (Discussion) 33 pages; 1 page Conclusions and 1 page Recommendations. The thesis has 22 tables (2 appendix tables), 35 figuress (5 appendix figures) and 147 references (7 Vietnamese and 140 English references). Chapter 1. DOCUMENTARY OVERVIEW 1.1. Terminology and brief history of autism Autism was first mentioned from the 1900s, derived from the Greek "Autos" - "self", described patients with isolated, social withdrawal. Leo Kanner (1943) used the term "autism" to describe a group of children with abnormal development such as social interaction deficits, impairment in language development, strange and repetitive behaviors, early onset before 3 years of age. Hans Asperger (1944) described a milder form of autism called Asperger's syndrome. In 1960 – 1970s, several studies suggested that autism was caused by abnormal brain structures, or by biochemical and metabolic changes. In 1999, autism was classified as pervasive developmental disorder. Accordingly, autism is a pervasive
3 developmental disorder, affecting many aspects of the development but the most impact on communication skills and social relations. 1.2. Diagnostic criteria for autism Currently, there are two autism diagnostic criteria systems: "Diagnostic and Statistical manual of Mental disorders" (DSM) of The American Psychiatric Association and "International Classification of Diseases" (ICD) of The World Health Organization. In which the DSM criteria are applied fairly common. 1.3. Pathophysiology of autism The mechanisms that lead to autism are poorly understood, however scientists focus around the disruption of normal cerebral development and its subsequent implications on the functional brain unit. Others hypotheses are discussed the mechanisms of autism related to a role of environmental factors in association with genetic factors and epigenetics is a candidate biological mechanism for gene- environment interactions in autism. 1.4. Methods to model autistic features in animal Animal models of autism is based on the mimics the causal factors in human disease. The same causal factors, obtain from animal manifestations and pathological behaviors similar to autism describe in human. The animal model of autism is divided to three main groups: - Brain lesion models of autism: models obtain when damaging brain areas such as the cerebellum, amygdala, medial prefrontal cortex. - Environmental factors models of autism: exposure during pregnancy or early postnatal period with anticonvulsants and inflammatory factors. - Genetic models of autism: mutagenic models of genes involved in autism, genetic model diseases are associated with autism.
4 1.5. Test for behavioral evaluation in autism modelled animals Behavioral assays for animal model of autism are a battery of tests based on cored symptoms and associated symptoms of autism. The cored symptoms include abnormal reciprocal social interactions, impaired communication, and repetitive behaviors, restricted interests. Associated symptoms include seizures, anxiety, mental retardation, hyper-reactivity and hypo-reactivity to sensory stimuli, sleep disruption and gastrointestinal distress… 1.5.1. Tests for social interaction To measure social interaction, we use three-chambered test or maternal behaviors test. 1.5.2. Tests for communication Responses to social, non-social odors, emitted ultrasonic vocalizations in different contexts are means of assessing communication in animals. 1.5.3. Tests for repetitive behaviors To count the number of repetitive behaviors such as rotating, digging, grooming or use T maze test, Y maze test. 1.5.4. Tests for associated symptoms To measure seizures, sleep disorder using EEG recording. Anxiety-related behaviors can be evaluated by elevated plus-maze test or light–dark exploration. Assess learning and memory by using Morris water maze test, object recognition and operant discrimination tasks. Assess hypersensitivity to sensory stimuli by acoustic startle. Assess motor clumsiness by using rotarod test. 1.6. Treatments for autism Autism is a developmental disorder include abnormal behaviors, impairment in communication and social interaction. To intervene
5 effectively, we should have a specialist to assess impairments and capacity of children with autism. The intervention areas include psychology, language, education, medical and therapeutic activities...which focuses on the skills deficit compare with the same age children. 1.7. Effects of enriched environment on behaviors in animals modelled for autism Enriched environment (EE) is a laboratory condition in which the environment is "riched" more than the standard conditions in the laboratory. The enriched environments was first described by Donald Hebb (1947) who noted qualitative differences in behavior between the rats he brought home for his children to play with, and the rats who were cage-kept in the laboratory. Until the 1960s, biochemical and structural changes were found in the brains of rats exposed to enriched environments. Autism is as a neurodevelopmental disorder, using enriched environment having proven to improve the defects caused by neurodevelopmental disorder. 1.8. Research about autism in Vietnam Recent years in Vietnam, children with autism were examined and treated increases quickly. Some study have focus on epidemiology, clinical features, no study in animal model of autism… The diagnosis and treatment for children with autism are difficult because they have not been properly invested. So, many children with autism have not diagnosed and treated, leading children with autism difficult to integrate society.
6 Chapter 2. SUBJECTS AND METHODS 2.1. Subjects Swiss white offsprings of parent mice offer by National Institute of Hygiene and Epidemiology. * Research aim 1: Establishing an animal model of autism in Swiss white mice. Mice were divided into control group (Control) n=35, prenatal VPA exposure groups at doses of 300, 400 and 500 mg/kg body weight (VPA300) n=31, (VPA400) n=32 and (VPA500) n=35. * Research aim 2: Evaluation effects of enriched environment on behaviors in modelled white mice. Mice were divided into: control group raised up in standard environment (Control-SE) n=69, control group raised up in enriched environment (Control-EE) n=72, VPA group raised up in standard environment (VPA-SE) n=66 and VPA group raised up in enriched environment (VPA-EE) n=65. The care and use of laboratory animals were in accordance with the guidelines established by Laboratory Animal Production Center in Institute of Hygiene and Epidemiology and Laboratory Animal Board in Vietnam Military Medical University. 2.2. Methods 2.2.1. Research design A prospective with intervention study. 2.2.2. Materials The chemicals used in the study included sodium valproate (Sigma Aldrich, Germany) and 0.9% sodium chloride solution (B.BRAUN, Vietnam). Mice were kept stable at 25 ± 1°C, humidity 60-70% and 12/12 hour dark light cycle. Control-SE and VPA-SE mice were housed in a standardized environment (SE), while the Control-EE and VPA-EE mice were housed in enriched environment (EE). SE was a cage with size 30x20x15 cm (L x W x H), freely accessed to pelleted food,
7 water, and nesting material. EE was a large cage with size 50x30x30 cm (L x W x H), divided into two layers, including components: running wheels, ladder, vines, turtles, sleepers, tunnel, rolling ball, freely accessed to pelleted food, water and nesting material. A quiet laboratory room at a temperature of 25 ± 1°C had an experimental chamber for conducting behavioral assessments. The round cylindrical experimental chamber (2-m in diameter, 2-m in hight) was enclosed around the ceiling and made of thick black fabric and used 25 watt incandescent light bulbs. Equipments for behavior analysis included: incline surface, ultrasonic sound recording and analysis system, open field, rotarod, elevated plus maze, three-chamber apparatus, Morris water maze, and Any-maze behavioral recording and analysis system. 2.2.3. Research procedure * Research aim 1: Establishing an animal model of autism in Swiss white mice. Female adult Swiss mice with controlled fertility cycle were mated overnight and the next morning when spermatozoa were found was designated as the first day of gestation. At day 12.5 of pregnancy, dams were randomly divided into groups. Control dams received a single intraperitoneal (i.p.) injection of physiological saline at dose 0.01 ml/g b.w., VPA dams received a single intraperitoneal injection of sodium valproate at dose 300 mg/kg, 400 mg/kg, 500 mg/kg and 600 mg/kg b.w. With these VPA doses, we conducted a dose/ toxicity test. Because the VPA 600 mg/kg dose was lethal/ toxic to the exposed pups during the first week postpartum, the exposed pups at VPA 600 mg/kg dose were evaluated behaviors limited. Therefore, the present study used doses of 300-500 mg/kg and experimental results corresponding to these doses.
8 The offsprings were divided into control group or VPA300, VPA400, and VPA500 groups according to the dose exposure in utero. Behavioral examinations of mice were scheduled as follows: ultrasonic vocalization recording at 3 to 10 days of age; negative geotaxis test at 6 to 8 days of age; open field test, three-chambered test, elevated pluss maze test, rotarod test, Morris water maze test at 49 to 61 days of age. Results were compared amon groups of mice, then the most effective dose of VPA was chosen for modelling autism in mice. * Research aim 2: Evaluation effects of enriched environment on behaviors in modelled white mice. Modelling autism in Swiss mice by injecting the dose of VPA 500 mg/kg b.w. The offspring were weaned on PND 21 and housed separately in a standard environmental cage (SE) or an enriched environmental cage (EE) for four weeks, with 3 to 6 animals per cage by gender. Assess offspring’s behaviors before housing in the SE or EE: included ultrasonic vocalization recording at 3 to 10 days of age, and negative geotaxis test at 6 to 8 days of age. Enriched environment housing procedure: EE cages were classified into samples labeled with ordinal numbers, each sample included five objects and changed every three days including a new replacement litter material, washed and dried the objects, a new replacement object and changed positions of two objects. Assess offspring’s behaviors after housing in the SE or EE: using open field test, three-chambered test, elevated plus maze test, rotarod test, Morris water maze test at 49 to 61 days of age. Behavioral data were analyzed and compared among the groups to evaluate effects of enriched environment on behaviors in VPA-exposed mice.
9 2.2.4. Data analysis The results were represented by the mean, standard deviation, and percentage. The difference between percentages of groups were compared using Chi-square test, the difference between means of groups were analyzed by T-test/ANOVA (normal distribution) or Mann-Whitney test/ Kruskal-Walis test (non-standard distribution). The difference among means at different times were analyzed by Mix ANOVA. The data of research was processed by IBM SPSS software version 20.0. 2.2.5. Research location The research was conducted at Laboratory of Physiology in Vienam Military Medical University. Chapter 3. RESULTS 3.1. Establishing an animal model of autism in Swiss white mice by prenatal exposure to sodium valproate 3.1.1. Effects of prenatal exposure to sodium valproate on motor coordination development in negative geotaxis test Figure 3.1. 1800 rotating time in control and VPA groups at PND 6 to 8. ** p
10 3.1.2. Effects of prenatal exposure to sodium valproate on social communication by emitting ultrasonic vocalization (USV) Figure 3.7. Number of calls in control and VPA groups at PND 3 to 10 in below 35 kHz bandwidth. The number of calls in VPA groups were lower than that in the control group, which was clearly shown in the VPA500 group (p < 0.001). 3.1.3. Effects of prenatal exposure to sodium valproate on locomotor and exploratory activity in open field test There were no significant difference between group (p > 0.05) for distance and speed travel. 3.1.4. Effects of prenatal exposure to sodium valproate on social interaction in three-chambered test Table 3.4. Number of entries and duration in chambers in session 1 in control and VPA groups. Parameters Group n ± SD p a.Control 35 14.51 ± 5.89 Number of pa,b > 0.05 b.VPA300 31 13.26 ± 7.06 entries in pa,c < 0.01 c.VPA400 32 9.45 ± 4.65 chamber 1 pa,d < 0.001 d.VPA500 30 9.10 ± 6.69 a.Control 35 295.61 ± 67.91 Time in b.VPA300 31 270.02 ± 55.48 chamber 1 > 0.05 c.VPA400 32 291.33 ± 102.41 (s) d.VPA500 30 262.10 ± 122.78 a.Control 35 91.61 ± 42.93 Time in pa,b > 0.05 b.VPA300 31 89.78 ± 30.05 central pa,c > 0.05 c.VPA400 32 118.71 ± 116.55 chamber (s) pa,d < 0.05 d.VPA500 30 133.22 ± 99.27
11 Number of entries in strange mouse chamber in control group was higher than that in the VPA400 and VPA500 groups (p < 0.01 to p < 0.001). Duration in central chamber in control group was lower than that in the VPA500 group (p < 0.05). 3.1.5. Effects of prenatal exposure to sodium valproate on anxiety- related behaviors in elevated-plus maze test The results in Table 3.8 showed that number of entries in opened arms in control group was higher than that in the VPA300 and VPA400 groups (p < 0.05 and p < 0.01, respectively). Duration in opened arms in control group was higher than that in the VPA300, VPA400 and VPA500 groups (p < 0.05 and p
12 A B Figure 3.13. Distance (A) and duration (B) to find the hidden platform in control and VPA groups over six days of training. Distance to find the hidden platform in control group was shorter than that in the VPA300 and VPA500 groups (p < 0.05 to p < 0.01). Time to find the hidden platform in control group was shorter than that in the VPA500 group (p < 0.01). 3.2.2. Effects of enriched environment on behaviors in mice prenatally exposure to sodium valproate at dose of 500 mg/kg b.w. 3.2.2.1. Effects of enriched environment on locomotor and exploratory activity of autism-modelled mice in the open field Table 3.11. Activities in open field in control and VPA500 groups housed in the standard and enriched environment. Parameters Group n ± SD p a.Control-SE 62 20.82 ± 11.72 Number of 18.72 ± 10.84 pa,b > 0.05 b.Control-EE 72 entries in pa,c < 0.05 central zone c.VPA-SE 66 16.22 ± 9.22 pc,d < 0.001 d.VPA-EE 65 22.82 ± 10.27 a.Control-SE 62 25.65 ± 22.18 Time in b.Control-EE 72 30.08 ± 28.38 pa,b > 0.05 central zone pa,c > 0.05 (s) c.VPA-SE 66 20.70 ± 13.20 pc,d < 0.01 d.VPA-EE 65 29.79 ± 19.01 Number of entries and duration in central zone in VPA-SE group were higher than those in the VPA-EE group (p < 0.01 to p < 0.001, respectively).
13 3.2.2.2. Effects of enriched environment on social interaction behaviours in autism-modelled mice Table 3.12. Number of entries and duration in chambers in session 1 in control and VPA500 groups housed in the standard and enriched environment. Parameters Group N ± SD p a.Control 69 14.75 ± 9.98 Number of pa,b < 0.001 b.Control-EE 72 8.76 ± 4.07 entries in pa,c < 0.01 c.VPA 66 10.86 ± 8.66 chamber 1 pc,d > 0.05 d.VPA-EE 64 9.34 ± 4.53 a.Control 69 314.33 ± 95.94 Time in pa,b < 0.05 b.Control-EE 72 360.03 ± 145.93 chamber 1 pa,c > 0.05 c.VPA 66 307.22 ± 113.21 (s) pc,d > 0.05 d.VPA-EE 64 342.63 ± 138.99 a.Control 69 85.71 ± 51.68 Time in pa,b < 0.01 b.Control-EE 72 80.95 ± 93.41 central pa,c < 0.05 c.VPA 66 116.92 ± 91.61 chamber (s) pc,d < 0.01 d.VPA-EE 64 83.48 ± 76.40 Number of entries in chamber 1 in control-SE group was higher than that of the control-EE group (p < 0.001) and VPA-SE group (p < 0.01). Duration in chamber 1 in control-SE group was lower than that of the control-EE group (p < 0.05). Duration in central chamber of control-SE group was lower than that of the VPA-SE group (p < 0,05), in EE groups were lower than that of the SE groups (p < 0.01). 3.2.2.3. Effects of enriched environment on anxiety-related behaviours in autism-modelled mice The results in Table 3.18 showed that number of entries in opened arms in control-SE group was higher than that of the control-EE group (p < 0.05) and VPA-SE groups (p < 0.01); in VPA-SE group was lower than that of the VPA-EE group (p < 0.05). Duration in opened arms in control-SE group was higher than that of the control- EE group (p < 0.01) and VPA-SE group (p < 0.01).
14 3.2.2.4. Effects of enriched environment on motor coordination, balance in rotarod test in autism-modelled mice Figure 3.27. Latency to fall off the rotating rod in control and VPA500 groups house in SE and EE. ***: p < 0.001, compared with that of Control-SE; +++: p < 0.001, compared with that of VPA-SE. The latency to fall off the rotating rod in control-SE group was shorter than that in the control-EE group; in VPA-SE group was shorter than that in the VPA-EE group (p < 0.001). 3.2.2.5. Effects of enriched environment on spatial learning and memory in autism-modelled mice B Figure 3.32. Distance (A) and duration to find the hidden platform (B) rod in control and VPA500 groups house in standard environment and enriched environment over six days of training.
15 Distance to find the hidden platform in VPA-SE group was longer than that of the VPA-EE group (p < 0.001). Duration to find the hidden platform of control-SE group was longer than that of the Control-EE group (p < 0.001), in VPA-SE group was longer than that of the VPA-EE group (p < 0.01). Chapter 4. DISCUSSION 4.1. Making animal model of autism in Swiss white mice by prenatal injection of sodium valproate There are three approaches to model autism in animal, as genetic models, environmental factors models, and brain lesion models. Each approach has its own advantages and disadvantages [9],[67],[68]. Researchers chose the method for modeling autism based on research purposes and research facilities. In Viet Nam, there are currently no technique to develop genetic models and/or brain lesion models for autism. The present study chose modeling autism in mice by exposure to sodium valproate (VPA) on day 12.5 of gestation based on previous studies showed that VPA was an environmental factor that altered neurodevelopment especially around the neural tube closure and the completion of neural structures. Many studies used the VPA dose of 100-800 mg/kg b.w, oral, subcutaneous, intraperitoneal injection during day 9 to 15 of gestation. The determination of day 12.5 was based on monitoring estrous cycle and mating overnight at proestrus and estrus stage. The next morning, determining the presence of sperm in the female mice vagina was the first day of gestation (day 0.5 from the night before to the next morning). Female mice after mating were separately care, monitored the sign of pregnancy. In the morning of day 13 (day 12.5), determined the pregnant mice for injection. The time for injection
16 VPA was similar to that in the previous studies, which was successful to create animal model of autism in mice. The choice for a battery of tests to evaluate behaviors in mice based on behaviors in autistic mice corresponding to autistic traits in human was suggested by Crawley, Wöhr, etc. 4.1.1. Effects of prenatal exposure to sodium valproate on motor coordination development in negative geotaxis test Negative geotaxis test evaluates function of the vestibular and motor coordinative development. Cheaha et al. studied on autistic Swiss mice by using subcutaneous VPA dose of 600mg/kg on day 13 of pregnancy found that the 1800 rotating time in negative geotaxis test at PND3-10 in VPA group was longer than that in the control group. Wöhr et al. studied on Shank1 gene mutant mice (autism- related genes), coding for shelving postsynaptic protein, also pointed out in the period 2-12 days old, Shank1-/- mice was prolonger the 1800 rotating time in the negative geotaxis test than Shank1+/- mice and control mice (Shank1+/+). The results in the preset study showed that the 1800 rotating time in VPA group was longer than that in control group, which was clearly in VPA500 group, suggesting the development of motor function of VPA500 group was slower than of the control group. Our study is similar to previous studies in rats and mice on motor function. 4.1.2. Effects of prenatal exposure to sodium valproate on communication by emitted ultrasonic vocalization (USV) Mice emit USV in different contexts during development and maturation. Research on USV is a tool for behavioural phenotyping of mouse models of neurodevelopmental disorders, including autism.
17 Cheaha et al. studied on autistic Swiss mice by using VPA found that number of calls per minute of VPA group was lower than control group in the period 3-10 days old. Wöhr et al. studied on Shank1 gene mutant mice showed that at PND8, in Shank1-/- mice number of calls was lower, total call time was shorter, peak frequency was higher, and frequency modulation was lower than those of the Shank1+/- and Shank1+/+ groups, but no difference in characteristics of calls by gender. In our study, the VPA mice (particularly VPA500) reduced emitting USV ratio of above 35 kHz bandwidth, reducing number of calls, duration of call, and frequency of call below 35 kHz bandwidth. So, prenatal exposure to VPA leads to reduce social communication on 3-10 days old mice. 4.1.3. Effects of prenatal exposure to sodium valproate on locomotor and exploratory activity in open field Open field test use to evaluate locomotor activity and exploratory behavior. In this study, there were no difference in distance and speed travelled in open field test among groups, indicating that VPA had no effect on locomotor activity. Number of entries and time in central zone in the VPA500 group tended lower than those in the control group showed a partly impairment exploration and restrict interest in the VPA500 group. Roullet et al. studied on mice exposed to VPA dose of 800 mg/kg b.w orally on day 11 of pregnancy found that no difference in locomotor activity in open field between VPA and control group. Mehta et al. studied on mice exposed to VPA dosed 600 mg/kg b.w subcutaneously on day 13 of pregnancy also found that VPA had no effect on spontaneous motor activity, there were no difference in distance and speed in open field between VPA and control group, but number of entries and time in central zone in
18 VPA500 groups was lower than those of the control group. Thus, VPA group increased anxiety-related behaviours and reduced exploratory. These changes may relate to a decrease in number of Purkinje cells in cerebellum or abnormal in prefrontal cortex and amygdala. The results of activity in open field test in our study were similar to these hypotheses. 4.1.4. Effects of prenatal exposure to sodium valproate on social interaction behaviors in the three-chambered tests In session 1, VPA500 group decreased number of entries to novelty mouse chamber, decreased number of contact with novelty mouse, increased time in the central chamber; VPA400 group decreased the number of entries to novelty mouse chamber; VPA300 group decreased number and time contact with novelty mouse. In session 2, VPA500 group decreased number of entries to novelty mouse chamber and number of contact with novelty mouse, increased time in the central chamber; VPA300 group decreased number of contact with novelty mouse. These results expressed impairment in social interaction and social recognition in mice prenatally exposed to VPA. Cheaha et al. studied on Swiss mice model autism by using VPA also found that time contact with strange mouse in VPA group was lower than control group in session 1 of three-chambered test. Markram et al. studied on rats model autism by using VPA dose 500 mg/kg on day 12.5 of pregnancy found that VPA group exhibited less play behavior, explored each other less, as indicated by sniffing and touching each other and avoided interaction by hiding more than control group. The results of in vitro slice electrophysiology showed that the basolateral amygdala network of VPA group was hyperreactive to stimulation and