ORIGINAL ARTICLE

Supplementation of Korean Red Ginseng improves behavior deviations in animal models of autism

Edson Luck T. Gonzales^1,2,3#, Jong-Hwa Jang^4#, Darine Froy N. Mabunga^1,2,3, Ji-Woon Kim^1,2,3, Mee Jung Ko^1,2,3, Kyu Suk Cho^1,2,3, Geon Ho Bahn⁵, Minha Hong⁶, Jong Hoon Ryu⁷, Hee Jin Kim⁸, Jae Hoon Cheong⁸ and Chan Young Shin^1,2,3*

¹Department of Neuroscience, School of Medicine, Konkuk University, Seoul, Korea; ²Neuroscience Research Center, IABS, Konkuk University, Seoul, Korea; ³KU Open Innovation Center, Konkuk University, Seoul, Korea; ⁴Department of Dental Hygiene, Hanseo University, Seosan, Korea; ⁵Department of Neuropsychiatry, School of Medicine, Kyung Hee University, Seoul, Korea; ⁶Department of Psychiatry, School of Medicine, Dankook University Hospital, Cheonan, Korea; ⁷Department of Oriental Medicine, Kyung Hee University, Seoul, Korea; ⁸Department of Pharmacy, Sahmyook University, Seoul, Korea

Abstract

Background: Autism spectrum disorder (ASD) is heterogeneous neurodevelopmental disorders that primarily display social and communication impairments and restricted/repetitive behaviors. ASD prevalence has increased in recent years, yet very limited therapeutic targets and treatments are available to counteract the incapacitating disorder. Korean Red Ginseng (KRG) is a popular herbal plant in South Korea known for its wide range of therapeutic effects and nutritional benefits and has recently been gaining great scientific attention, particularly for its positive effects in the central nervous system.

Objectives: Thus, in this study, we investigated the therapeutic potential of KRG in alleviating the neurobehavioral deficits found in the valproic acid (VPA)-exposed mice models of ASD.

Design: Starting at 21 days old (P21), VPA-exposed mice were given daily oral administrations of KRG solution (100 or 200 mg/kg) until the termination of all experiments. From P28, mice behaviors were assessed in terms of social interaction capacity (P28–29), locomotor activity (P30), repetitive behaviors (P32), short-term spatial working memory (P34), motor coordination (P36), and seizure susceptibility (P38).

Results: VPA-exposed mice showed sociability and social novelty preference deficits, hyperactivity, increased repetitive behavior, impaired spatial working memory, slightly affected motor coordination, and high seizure susceptibility. Remarkably, long-term KRG treatment in both dosages normalized all the ASD-related behaviors in VPA-exposed mice, except motor coordination ability.

Conclusion: As a food and herbal supplement with various known benefits, KRG demonstrated its therapeutic potential in rescuing abnormal behaviors related to autism caused by prenatal environmental exposure to VPA.

Keywords: Panax ginseng; nutraceutical; autistic behaviors; Korean Red Ginseng; prenatal VPA exposure

Autism spectrum disorders (ASD) is a range of neurodevelopmental disorders that generally characterize social communication and social interaction difficulties accompanied by restrictive and repetitive behaviors (1). ASD continues to gain much attention in the society because of its increasing diagnoses and heterogeneity but still has unexplained proposed etiologies and limited therapeutic entities (2). Thus, many researchers, including us, have given much focus on how to uncover this formidable condition by finding the possible etiologies, tracing the pathologic mechanisms and discovering therapeutic treatments of ASD in many animal models (3–13). Most importantly, the current medication used for the treatment of ASD is only symptomatic and limited to the alleviation of repetitive behaviors, as well as other comorbid symptoms such as depression, seizure, aggression, and sleep or gastrointestinal disturbances. While a tough race continues for the development of small molecular therapeutic entities against ASD, one possible approach is to investigate the beneficial effects of functional food items as well as herbal medicines.

Ginseng, belonging to the genus Panax, is a popular herbal plant widely used for its variety of effects in both traditional and research-based medicines. For many years, ginseng gained a lot of attention everywhere for its adaptogenic, aphrodisiac (14) [including treatment of erectile dysfunction (15)], anticarcinogenic (16–18), antioxidant (17, 19, 20), and antiobesity (21) properties, among others, in its several ginsenoside compounds that can be found extensively in Korean Red Ginseng (KRG). Red ginseng is a sun-dried and steamed form of the harvested root of the plant. Choi published an ample review about the characteristics and pharmacological and medicinal components of ginseng (22).

In addition to its known benefits, studies of KRG related to the central nervous system (CNS) have been gaining knowledge as well [see for review the works of Kim, HJ (23)]. In both human and animal studies, KRG, in different preparations and extractions, showed considerable benefits in Alzheimer’s disease (24), cerebral blood flow, inhibition of superoxide production (25), ischemic injury (26, 27) and learning and memory (28, 29), among others. Thus, it must be interesting to investigate the effect of KRG on neurodevelopmental disorders such as ASD and others.

Valproic acid (VPA) and other valproate products are known antiepileptic drugs (AED) (30) and are also used for maintenance treatment of manic type bipolar disorder (31) and migraine headache (32). Over the years, VPA used during pregnancy was found to cause varying degrees of cognitive deficits (33), birth defects (34, 35), and increased risk of autism in human offspring (36). Thus, VPA-exposed animal models have been widely used by many researchers including us to possibly explain the underlying pathologic pathways of ASD and apply existing and novel therapeutic entities for ASD based on the impaired pathway [see for review (37)].

VPA-exposed models of ASD show neural tube defects (observed as crooked tails), neurochemical alterations, and behavioral deficits such as social impairments (37). Previously, we also showed the preventive effect of chronic KRG treatment given at embryonic days 10–15 (E10–E15) from the deleterious effect of prenatal VPA exposure at E12 and rescued the neural tube defects, social impairments, and increased seizure susceptibility in offspring rats (13). These results paved a way to further study the therapeutic potentials of postnatal KRG treatment in VPA-exposed ASD animal models which could have a stronger clinical importance in a real-world situation. In this study, we asked whether long-term KRG treatment to offspring mice could alleviate the social and other behavioral impairments after in utero VPA exposure. The impact of the result of this study can lead to more promising therapeutic opportunities of KRG for the rising diagnosis but limited treatment of ASD clinically.

Materials and methods

Animals

Animal treatment and maintenance were carried out in agreement with the Animal Care and Use Guidelines of Sahmyook University and Konkuk University, Korea, and in accordance with the 14th article of the Korean Animal Protection Law as well as the Principle of Laboratory Animal Care (NIH publication No. 85–23, revised 1985) (38). All efforts were made to minimize the number of animals as well as their suffering. Ten female ICR mice at gestational day 5 were purchased from OrientBio (Gyeonggi-do, Korea). They were maintained on a 12:12-h circadian cycle with lights on at 07:00 and off at 19:00, at a constant temperature (22±2°C) and humidity (55±5%). Treatments were administered according to the schedule (Fig. 1), and behavior tests were carried out in designated rooms. Subjects were given ample time to rest in between experiments.

Fig. 1.   The experiment scheme shows the main step periods from in utero VPA exposure to long-term postnatal KRG treatment along with the behavior tests employed.

Subcutaneous injection of VPA to pregnant mice

Sodium VPA (Sigma, St. Louis, MO) was dissolved in 0.9% saline at a concentration of 50 mg/mL, pH 7.3. Pregnant mice were subcutaneously injected with either 300 mg/kg of VPA or saline solution in the loose skin area of the neck on gestational day 10 (E10). VPA-exposed male offspring were randomly assigned in groups to distribute each litter equally to different treatment conditions.

KRG preparation and treatment of male offspring mice

The KRG extract used in this study was manufactured from the roots of a 6-year-old fresh ginseng, Panax ginseng Meyer, harvested in the Republic of Korea by Korea Ginseng Corporation, Seoul. Red ginseng was made and extracted as described previously (13) in a standardized method from previous studies (39–41). From the VPA- and saline-treated mothers, male offspring were randomly selected from each litter to be assigned in a designated group. The groups include the control, VPA, VPA+KRG in 100 mg/kg, and VPA+KRG in 200 mg/kg. Each group was given daily oral administrations of either distilled water or KRG solution from P21 and continued until the termination of all experiments. KRG extracts were freshly prepared by dissolving in distilled water before administration. All in all, a total of 10 mice in each group (Control, VPA, VPA+KRG100, VPA+KRG200) were utilized for behavior testing. Behavior assessment of each group started at P28 until P40 with the following tests described below and as summarized in Fig. 1.

Three-chamber sociability and social novelty preference tests

The modified sociability and social novelty preference tests as described previously in our lab (12) was originally established by Moy et al. (42). The test was performed in a three-chamber rectangular cage (23×40×22 cm in each compartment). Two 10 cm² openings provided access to the three chambers. The central compartment was the starting area of each trial and both the side compartments contain a round wire cage of 10 cm diameter wherein conspecific stranger mice were put (during social novelty preference test) and one left empty during sociability test. Stranger mice were of the same age and gender and had never encountered the subject mice previously. Stranger mice were habituated to the wire cage twice for 2 days before the actual test. Testing period takes approximately 30 min including 5-min habituation, 10-min sociability test, 10-min social novelty preference test, and few minutes for cleaning, consecutively.

Sociability tests determine the social interaction of subject mice to the stranger mouse in the wire cage (SM) versus the empty cage (EC). The duration in each compartment was tracked by EthoVision software (EthoVision 3.1, Noldus Information Technology, the Netherlands), and sniffing duration to each wire cage was measured by ‘blind observers’ 2 m away from the box. Sociability index (SI; SM compartment duration/EC compartment duration) was further calculated. Different mice groups were tested in every consecutive trial to avoid time difference bias.

Immediately after a sociability test, social novelty preference test began to assess the preference subject mouse to explore a novel mouse/object over an already familiar one. The previous SM had become the familiar mouse (FM), while a novel stranger mouse (NM) was introduced in the previously empty wire cage. The subject mouse was put back in the central compartment to begin the test. The duration in each compartment, social preference index (NM compartment duration/FM compartment duration), and sniffing duration in each cage were measured in the same way as the sociability test. At the end of each complete trial, animals were returned to their respective cages and the box surface was wiped and cleaned with 70% ethanol solution. Tests were performed between 9:00 and 17:00 hours. EthoVision software connected through an overhead camera tracked the movement of each subject in every compartment.

Open-field test

An open-field test was performed to assess the spontaneous locomotor activity of mice. Mice were exposed for the first time in an open box (42×42×42 cm). Five boxes are available to test five animals simultaneously for video recording and behavior tracking using EthoVision software. The animals were allowed to habituate for 5 min after their introduction to the central area, and locomotor behavior was measured for 20 min. The distance moved, and movement duration was calculated. The surfaces of each box were cleaned and wiped with 70% ethanol at the end of each trial.

Marble-burying test

This test was performed to assess the stereotyped and repetitive behaviors of VPA-exposed mice and was described previously, which we slightly modified in our study (43). Twenty marbles (15 mm diameter) were evenly arranged (5×4) on the bedding surface after 10-min habituation to the plastic home cages (12×26×12 cm) filled with 5-cm deep and lightly pressed corncob bedding. Each mouse was put back in the marble-filled cage and left for 20 min. Afterward, mice were carefully and swiftly removed in each cage, and the number of marbles covered with bedding up to more than 50% its surface was counted by a ‘blind observer’.

Y-maze test

Spatial working memory through spontaneous alternations can be assessed in a Y-maze test (44). This procedure is based on the knowledge that a normal animal (the subject) has an innate inclination to visit an arm, which was not previously explored (45). The maze was made of black polyvinyl chloride with three arms (5 cm×35 cm×10 cm) forming a Y shape in equal angles. Each mouse was introduced in one of the arms facing the central area. Total entries and spontaneous alternations (total alternations/[total arm entries – 2]×100) of arm entries were recorded for 8 min (46). The animals were removed, and the surfaces were cleaned with 70% ethanol at the end of each trial.

Rotarod test

The rotarod apparatus (Ugo Basile Srl, Comerio, Italy) is made of a corrugated drum (3 cm diameter) divided into five compartments by round plastic walls. In this study, we employed the fixed speed rotarod as previously described (47), which we slightly modified. The rotation speed was set at 36 rpm constantly, and animals were trained in this situation for 5 min a day prior to the actual test. The latency to fall and falling frequency were recorded during the 20-min trial duration in each mouse.

Measurement of electroshock seizure threshold

As previously described (12, 13, 48), electroshock seizure threshold was measured in 5-week old mice with slight modifications. The seizure was induced by a current stimulator (ECT unit, Ugo Basile, Italy) attached as ear clips or auricular electrodes, and the full-scale seizure was observed by full hind limb extension. Convulsive current 50 (CC₅₀), as defined by convulsion in 50% of animals, was determined through ‘staircase’ procedure and calculated by the Litchfield–Wilcoxon II method (49). The current settings were at 100 Hz frequency, 0.5 ms pulse width, 2 sec shock duration with a primary current set at 20 mA. One seizure induction was given to each mouse and if it showed full hind limb extension, the next mouse was induced with a current 2 mA lower than the previous setting. Otherwise, the current was increased by 2 mA if no full seizure response was observed.

Statistical analysis

All data were presented as the means±standard error of mean (SEM), and statistics were analyzed using a non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test for all pairwise comparisons. Unpaired Student’s t-test was also used to compare the difference of responses in behaviors between the control and the VPA-exposed mice. Statistical significance was set to a p value of less than 5% (p<0.05). All analyses were calculated using GraphPad Prism version 5 software.

Results

Long-term KRG treatment improved the VPA-induced impairments in sociability of mice offspring

VPA injection at E10 in pregnant ICR mice has presented a socially impaired phenotype in the male offspring. Figure 2a revealed that the VPA-exposed mice had lower stay duration in the SM compartment compared to control (p<0.01). Interestingly, KRG treatments normalized the VPA-induced impairment to control levels (p>0.05) and were significantly different to VPA-only group (100 mg/kg, p<0.01; 200 mg/kg, p<0.001). Conversely, duration in the empty wire cage compartment was higher among the VPA-exposed mice as compared to the control (p<0.01) and the KRG-treated VPA-exposed mice groups (100 mg/kg, p<0.05; 200 mg/kg, p<0.001). Meanwhile, time spent in the central area did not differ among groups.

Fig. 2.   Effects of long-term KRG treatment on impaired sociability of VPA-exposed mice in the three-chamber apparatus by measuring the duration in each compartment (a), sniffing duration in the wire cages with or without stranger mouse (b), and the social preference index by comparing the duration between the stranger and empty compartments (c). Bars indicate the mean±SEM. n=10 mice per group. *p<0.05, **p<0.01, and ***p<0.001.

In Fig. 2b, we further compared the sniffing behavior (quantified by time) of each group to the SM or the empty wire cage (50). Results showed a lower sniffing duration of the VPA-exposed mice than that of the SM as compared to the control (p<0.05), while KRG treatment rescued this impairment (100 mg/kg, p<0.05; 200 mg/kg, p<0.001) to control level (p>0.05). Sniffing duration of the VPA-exposed mice in the empty wire cage showed a trend that was slightly higher than the control and the KRG treated groups (p>0.05). The 200 mg/kg dose of KRG also had a significantly higher sniffing duration in the SM than 100 mg/kg dose (p<0.05) but not against the control group (p>0.05). SI further confirmed that the VPA-exposed mice have a significantly lower index than the control (p<0.01) and the KRG-treated groups (p<0.05) (Fig. 2c). Taken together, the results suggest that prenatal VPA exposure can cause a significant decrease in sociability behavior of mice offspring and long-term treatment of KRG using 100 mg/kg and 200 mg/kg dosages, which could rescue this impairment to control levels.

Long-term KRG treatment improved the VPA-induced impairments in social novelty preference in mice offspring

Social novelty preference test immediately followed the sociability test for each mouse. As shown in Fig. 3a, duration in the FM (previously SM) compartment was higher for the VPA-exposed mice as compared to the control (p<0.001) and the KRG-treated VPA-exposed mice (100 mg/kg, p<0.01; 200 mg/kg, p<0.001). In addition, duration in the novel mouse compartment (previously EC) was significantly lower for the VPA-exposed mice than the control (p<0.001) and the KRG-treated VPA-exposed mice (100 mg/kg, p<0.01; 200 mg/kg, p<0.001). Time spent in the central compartment did not differ among all groups. The result also showed that VPA-induced social preference impairment was normalized to control levels by long-term KRG treatment. Interestingly, there was a significant difference between the KRG-treated VPA-exposed mice groups, with the 200 mg/kg dose group having a higher duration in novel mouse compartment and lower duration in FM compartment than the 100 mg/kg dose group (p<0.05).

Fig. 3.   Effects of long-term KRG treatment on impaired social preference of VPA-exposed mice in the three-chamber apparatus by measuring the duration in each compartment (a), sniffing duration in the wire cages with novel or familiar mouse (b), and the sociability index by comparing the duration between the novel and familiar compartments (c). Bars indicate the mean±SEM. n=10 mice per group. *p<0.05, **p<0.01, and ***p<0.001.

In accordance with the duration in the compartments, sniffing duration may further show a more sensitive and ethological measure of social preference in mice (50) (Fig. 3b). The VPA-exposed mice showed a higher sniffing duration to the FM than the control (p<0.001) and the KRG-treated VPA-exposed mice groups (100 mg/kg p<0.01; 200 mg/kg, p<0.05). On the other hand, sniffing duration of the VPA-exposed mice to the novel mouse significantly differed only against the 200 mg/kg KRG-treated VPA-exposed mice group (p<0.01) but not against the control (p>0.05) or the 100 mg/kg KRG-treated VPA-exposed mice group (p>0.05). Social novelty preference index (SPI) was evidently lower in the VPA-exposed mice than the control group (p<0.05) and the 200 mg/kg KRG-treated VPA-exposed mice group (p<0.01) but not in the 100 mg/kg KRG-treated VPA-exposed mice group (p>0.05) (Fig. 3c), which was encouraging. Overall, we have quantified that the VPA-exposed mice showed an impaired social novelty preference and long-term KRG treatment can further rescue this impairment.

Long-term KRG treatment alleviated the hyperactivity phenotype in the VPA-exposed mice offspring

The VPA-exposed mice exhibit a hyperactive phenotype when compared to the control group as measured by the distance moved (Fig. 4a) in the open field (p<0.001). However, there was no significant difference between the VPA-exposed mice and the control mice in the movement duration parameter (Fig. 4b) (p>0.05). Interestingly, KRG treatment normalized the VPA-induced hyperactivity phenotype to control levels (100 mg/kg, p<0.01; 200 mg/kg, p<0.01) in the distance moved parameter (Fig. 4a). Furthermore, only the 200 mg/kg VPA dosage showed a significant decrease in the increased movement duration (Fig. 4b) of the VPA-exposed mice (p<0.01).

Fig. 4.   Effects of long-term KRG treatment on hyperactivity phenotype of VPA-exposed mice in the open-field apparatus by measuring the distance moved (a) and movement duration (b). Bars indicate the mean±SEM. n=10 mice per group. ***p<0.001 vs. control group and ^##p<0.01 vs. VPA only group.

Long-term KRG treatment alleviated the increased marble burying and normalized the decreased spontaneous alternation behavior in the VPA-exposed mice offspring

In Fig. 5a, the VPA-exposed mice showed increased marble-burying behavior as compared to the control group (p<0.01). Interestingly, long-term KRG treatment decreased the burying behavior of the VPA-exposed mice (100 mg/kg, p<0.01; 200 mg/kg, p<0.05) to control levels. In addition, spontaneous alternation of the VPA-exposed mice was significantly lower than the control group (p<0.05) in the Y-maze test (Fig. 5b). Remarkably, KRG treatment of the VPA-exposed mice normalized the decreased spontaneous alternations (100 mg/kg, p<0.05; 200 mg/kg, p<0.01) to control levels.

Fig. 5.   Effects of long-term KRG treatment on increased repetitive behavior of VPA-exposed mice in the marble-burying test (a) and impaired spontaneous alternations in the Y-maze test (b). Bars indicate the mean±SEM. n=10 mice per group. *p<0.05, ***p<0.01 vs. control group and ^#p<0.05, ^##p<0.01 vs. VPA only group.

Tendency of motor balance and coordination impairment in the VPA-exposed offspring

As shown in Fig. 6, there was a tendency but no significant difference between the VPA-exposed mice and the control group in the latency to fall and falling frequency performance in the rotarod test (p>0.05). Furthermore, there was no effect found in the KRG treatment in the overall rotarod performance.

Fig. 6.   Effects of long-term KRG treatment on impaired motor coordination and balance of VPA-exposed mice in the fixed speed rotarod apparatus by measuring the latency to fall (a) and falling frequency (b) from the rotarod. No significance was observed. Bars indicate the mean±SEM. n=10 mice per group.

Long-term KRG treatment rescued the decreased seizure threshold in the VPA-exposed mice offspring

VPA-exposed mice showed a significantly lower threshold against electrically induced seizure as compared to control group (Fig. 7). Interestingly, however, KRG treatment dose dependently normalized this impairment to control levels with 200 mg/kg dosage being highly effective. Each point in Fig. 7a shows the percentage of rats that exhibited full hind limb extension response to the electric currents employed.

Fig. 7.   Effects of long-term KRG treatment on decreased electroshock seizure threshold of VPA-exposed mice by determining the convulsive current (CC₅₀) using the ‘staircase’ method and calculating the electroconvulsive rate in percentage (a) and mean EC50 current threshold (b). Data are expressed in the non-linear fit graph (a) and a bar graph that indicates the mean current of seizure response of each group (b). n=10 mice per group. ***p<0.001 vs. control group and ^###p<0.001 vs. VPA-only group.

Discussion

The present study reveals the potential therapeutic effects of KRG when treated postnatally in VPA-exposed mice offspring models of ASD. Here, we showed that long-term KRG treatment in VPA-exposed mice rescued the various behavioral impairments related to ASD including sociability and social preference, locomotor activity, marble burying, spontaneous alternation, and electroshock seizure threshold. The order of experiments was planned considering the validation of ASD symptoms and the degree of stress they can induce in animals. We first conducted the social test, as this is crucial to determine whether the VPA-induced mice show a validated core symptom of autism. We followed it up with open-field test for the measurement of locomotor activity and marble-burying test for repetitive behaviors based on their relevance to ASD and the complexity of the experimental methods. Y-maze and rotarod tests determine the variable symptoms accompanying the ASD in terms of spatial working memory and motor coordination, respectively. The experiments ended with the electroshock seizure threshold test, which was obviously most stressful to the animals.

KRG treatment was initiated on 3-week-old VPA-exposed mice and was given a duration of 7 days for treatment prior to the start of behavioral assessments. Furthermore, KRG treatment continued until all behavior tests were done. We considered this as long-term treatment of KRG, which showed therapeutic effects in previous studies with different pathologic conditions in mice (51, 52) and humans (53, 54). The KRG dosages that we employed in this study were based on their efficacy in alleviating the impairments found in our previous study (13). Indeed, these dosages are well within a range to show a therapeutic effect in the behavior of VPA-treated animals because other dosages, such as 50 mg/kg, did not show a robust efficacy in our preliminary study (data not shown).

To roughly depict the clinical scenario of the treatment regimen of KRG in autistic patients, we used juvenile mice to represent school-age and puberty stages in humans. Previous research studied the ages of mice in comparison to human life stages showing that a 1-month-old mouse is equivalent to a 12.5-year-old child (55). Thus, the age of mice when KRG treatment started (P21) was approximately equivalent to the beginning of the school-age stage in humans. Furthermore, the experimental period between P28 and P38 could be highly similar to the puberty stage of human development in which the treatment regimen was still ongoing. Thus, the treatment period in this study represents a clinical set-up where long-term treatment of KRG started at an age when a child is usually diagnosed with ASD.

The sociability and social novelty preference deficits that we found in this study are in agreement with previous results using rats (10, 12, 13) and mice models (56–59). One of the primary diagnostic criteria for ASD is social impairment (1), and we have presented the validity of using the VPA-exposed animal models of ASD in this study. Thus, we investigated whether KRG could rescue the ASD-related impairments brought about by prenatal VPA exposure. Indeed, the long-term administration of KRG revealed its promising therapeutic properties in ameliorating the social deficit found in VPA-exposed mice. This enlightens our previous question on whether KRG effect, on the prevention of the neural tube defect when given during pregnancy, is also directly related to the behaviors in animal offspring (13). Our present results remarkably showed that the effect of KRG in reversing the social impairments goes beyond structural protection as it rescued the socially impaired phenotype in the developmentally affected VPA-exposed mice. Ultimately, studying the brain of VPA-exposed mice after KRG treatments and behavioral improvements would be an important step in elucidating the effects of KRG in the brain structural and neurochemical levels.

Hyperactivity phenotype in VPA-exposed animal models is well known in studies using rats (6, 13). However, a previous study using mice did not find differences in locomotor activity between control and VPA-exposed groups (58), but it should be noted that the length of the trial was only 5 min in that study. In another study, no significant difference in locomotor activity for 30-min testing period was also observed when they tested the locomotor behavior of VPA-exposed and control animals from ages P56 to P64, while VPA exposure was given during E13 to pregnant mothers (60). In previous studies as well as our present study, we performed an open-field test for 20 min in juvenile mice where we found hyperactivity in the VPA-exposed mice group (59). ASD and its comorbid attention deficit hyperactivity disorder (ADHD)-like behavior, especially the hyperactivity and impulsivity phenotypes, are more noticeable in younger children. Thus, assessing the locomotor behavior of animal models at an earlier age would be appropriate. We suggest and validate that the VPA-exposed mice models of ASD also show hyperactivity compared with the rat models in previous studies (6, 13). Remarkably, long-term KRG treatment normalized the hyperactivity of the VPA-exposed mice to control levels, which are reminiscent of our previous reports demonstrating the anti-hyperactivity effects of KRG in an animal model of ADHD (61).

Other relevant symptoms observed in ASD are restrictive, repetitive behaviors. The VPA-exposed mice models were validated previously to show increased repetitive behavior as measured by self-grooming and marble-burying assays (60). On a positive note, we found an increased marble-burying behavior of the VPA-exposed mice in the present study. Of interest, long-term KRG treatment alleviated the increased marble-burying behaviors in the VPA-exposed mice. A role of mGluR antagonism on the alleviation of repetitive behaviors in the VPA-exposed mice models was previously demonstrated (60). In addition, we observed a dysregulation of mGluR pathways in the VPA-exposed rat models of ASD (62). It is therefore of great interest to investigate the effect of KRG related to glutamatergic functions in the brain, such as mGluR5 antagonism in the future investigation.

Working memory is known to be declined in children and adolescents with idiopathic autism and 22q11.2 hemizygosity as found in previous studies (63–65). Indeed, a genetic model of autism, known as the Tbx1 heterozygous (HT) mice in which the mutated Tbx1 gene is part of the chromosome region 22q11.2 in humans, has shown a decreased T-maze spontaneous alternation along with ASD-related social interaction and ultrasonic vocalization impairments (66). Interestingly, our VPA mice model of ASD also showed a decreased spontaneous alternation in the Y-maze paradigm. Thus, our VPA mice model agrees with the previous genetic model of ASD having impairments in working memory and social interaction. Furthermore, while the VPA-exposed mice show impaired Y-maze spontaneous alternations, KRG treatment reversed the condition to control levels. It must be interesting and revealing to study the working memory-related hippocampal region of the VPA-exposed mice and the effect of KRG in the pathologic process involved in ASD.

Previously, we have consistently shown the decreased electroshock seizure threshold phenotype in the VPA-exposed rat models of ASD (10, 12, 13). This condition was suggested to be related to the excitatory/inhibitory imbalance in the brain (10, 67) through upregulated excitatory processes but reduced GABAergic receptor system (10, 68, 69). The result of the present study, therefore, confirms the previous results and extends the knowledge that increased seizure susceptibility is also true for VPA-exposed mice. Certainly, studying the brains of VPA-exposed mice will be needed to confirm and compare the molecular results found in rats and postmortem human brains. Here, we further showed the potential therapeutic effects of KRG in increasing the seizure threshold in the VPA-exposed mice. With regards to the well-studied GABAergic system reduction in ASD patients and rat models, studying the GABAergic system in the VPA-exposed mice will also be needed for confirmation and elucidation of how KRG achieved its therapeutic mechanism.

The mechanism of actions underlying the therapeutic effect of long-term KRG treatment in the postnatal conditions is highly essential; these steps should be investigated in the near future. In addition, further studies are needed to isolate and identify the specific ginsenosides of KRG that are responsible for the alleviation of various behaviors in VPA-exposed models. In any case, we believe that the postnatal therapeutic effects of KRG provide additional lines of potential options, on top of the growing list of candidate molecules and targets that could treat the core symptoms of ASD even after brain development, which could be more clinically relevant. Whether the therapeutic effects of KRG are mediated by neurochemical or microstructural changes in the brain would be an additional important question to be answered, which could provide hints on the pathophysiological mechanisms of ASD. Finally, this study importantly showed the wide array of benefits in alleviating behavior impairments in animal models of ASD through supplementation of an herbal or nutraceutical product found in KRG.

Acknowledgements

This work was supported by a grant from The Korean Society of Ginseng funded by Korea Ginseng Corporation (CY Shin).

References

1. American Psychiatric Association (2013). The diagnostic and statistical manual of mental disorders: DSM 5. Arlington, VA: American Psychiatric Association.
2. Lord C, Bishop SL. Autism spectrum disorders. Soc Policy Rep 2010; 24: 3–16.
3. Markram K, Rinaldi T, La Mendola D, Sandi C, Markram H. Abnormal fear conditioning and amygdala processing in an animal model of autism. Neuropsychopharmacology 2008; 33: 901–12. PubMed Abstract | Publisher Full Text
4. Chomiak T, Hu B. Alterations of neocortical development and maturation in autism: insight from valproic acid exposure and animal models of autism. Neurotoxicol Teratol 2013; 36: 57–66. PubMed Abstract | Publisher Full Text
5. Bristot Silvestrin R, Bambini-Junior V, Galland F, Daniele Bobermim L, Quincozes-Santos A, Torres Abib R, et al. Animal model of autism induced by prenatal exposure to valproate: altered glutamate metabolism in the hippocampus. Brain Res 2013; 1495: 52–60. PubMed Abstract | Publisher Full Text
6. Schneider T, Przewlocki R. Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism. Neuropsychopharmacology 2005; 30: 80–9. PubMed Abstract | Publisher Full Text
7. Silverman JL, Yang M, Lord C, Crawley JN. Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci 2010; 11: 490–502. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
8. Schneider T, Turczak J, Przewłocki R. Environme ntal enrichment reverses behavioral alterations in rats prenatally exposed to valproic acid: issues for a therapeutic approach in autism. Neuropsychopharmacology 2005; 31: 36–46.
9. Schneider T, Roman A, Basta-Kaim A, Kubera M, Budziszewska B, Schneider K, et al. Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid. Psychoneuroendocrinology 2008; 33: 728–40. PubMed Abstract | Publisher Full Text
10. Kim KC, Kim P, Go HS, Choi CS, Park JH, Kim HJ, et al. Male-specific alteration in excitatory post-synaptic development and social interaction in pre-natal valproic acid exposure model of autism spectrum disorder. J Neurochem 2013; 124: 832–43. PubMed Abstract | Publisher Full Text
11. Ingram JL, Peckham SM, Tisdale B, Rodier PM. Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism. Neurotoxicol Teratol 2000; 22: 319–24. PubMed Abstract | Publisher Full Text
12. Kim KC, Kim P, Go HS, Choi CS, Yang S-I, Cheong JH, et al. The critical period of valproate exposure to induce autistic symptoms in Sprague–Dawley rats. Toxicol Lett 2011; 201: 137–42. PubMed Abstract | Publisher Full Text
13. Kim P, Park JH, Kwon KJ, Kim KC, Kim HJ, Lee JM, et al. Effects of Korean red ginseng extracts on neural tube defects and impairment of social interaction induced by prenatal exposure to valproic acid. Food Chem Toxicol 2013; 51: 288–96. PubMed Abstract | Publisher Full Text
14. Nocerino E, Amato M, Izzo AA. The aphrodisiac and adaptogenic properties of ginseng. Fitoterapia 2000; 71: S1–5. PubMed Abstract | Publisher Full Text
15. Jang D-J, Lee MS, Shin B-C, Lee Y-C, Ernst E. Red ginseng for treating erectile dysfunction: a systematic review. Br J Clin Pharmacol 2008; 66: 444–50. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
16. Yun TK, Lee YS, Lee YH, Kim SI, Yun HY. Anticarcinogenic effect of Panax ginseng CA Meyer and identification of active compounds. J Korean Med Sci 2001; 16(Suppl): S6–18. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
17. Keum Y-S, Park K-K, Lee J-M, Chun K-S, Park JH, Lee SK, et al. Antioxidant and anti-tumor promoting activities of the methanol extract of heat-processed ginseng. Cancer Lett 2000; 150: 41–8. PubMed Abstract | Publisher Full Text
18. Xiaoguang C, Hongyan L, Xiaohong L, Zhaodi F, Yan L, Lihua T, et al. Cancer chemopreventive and therapeutic activities of red ginseng. J Ethnopharmacol 1998; 60: 71–8. PubMed Abstract | Publisher Full Text
19. Lee BM, Lee SK, Kim HS. Inhibition of oxidative DNA damage, 8-OHdG, and carbonyl contents in smokers treated with antioxidants (vitamin E, vitamin C,β-carotene and red ginseng). Cancer Lett 1998; 132: 219–27. PubMed Abstract | Publisher Full Text
20. Zhang D, Yasuda T, Yu Y, Zheng P, Kawabata T, Ma Y, et al. Ginseng extract scavenges hydroxyl radical and protects unsaturated fatty acids from decomposition caused by iron-mediated lipid peroxidation. Free Radic Biol Med 1996; 20: 145–50. PubMed Abstract | Publisher Full Text
21. Kim JH, Hahm DH, Yang DC, Kim JH, Lee HJ, Shim I. Effect of crude saponin of Korean Red Ginseng on high fat diet-induced obesity in the rat. J Pharmacol Sci 2005; 97: 124–31. PubMed Abstract | Publisher Full Text
22. Choi K-T. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng CA Meyer. Acta Pharmacol Sin 2008; 29: 1109. PubMed Abstract | Publisher Full Text
23. Kim HJ, Kim P, Shin CY. A comprehensive review of the therapeutic and pharmacological effects of ginseng and ginsenosides in central nervous system. J Ginseng Res 2013; 37: 8. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
24. Heo JH, Lee ST, Chu K, Oh M, Park HJ, Shim JY, et al. An open-label trial of Korean red ginseng as an adjuvant treatment for cognitive impairment in patients with Alzheimer’s disease. Eur J Neurol 2008; 15: 865–8. PubMed Abstract | Publisher Full Text
25. Kim CS, Park JB, Kim K-J, Chang SJ, Ryoo S-W, Jeon BH. Effect of Korea red ginseng on cerebral blood flow and superoxide production. Acta Pharmacol Sin 2002; 23: 1152–6. PubMed Abstract
26. Bae E-A, Hyun Y-J, Choo M-K, Oh JK, Ryu JH, Kim D-H. Protective effect of fermented red ginseng on a transient focal ischemic rats. Arch Pharm Res 2004; 27: 1136–40. PubMed Abstract | Publisher Full Text
27. Lee JS, Choi HS, Kang SW, Chung J-H, Park HK, Ban JY, et al. Therapeutic effect of Korean red ginseng on inflammatory cytokines in rats with focal cerebral ischemia/reperfusion injury. Am J Chin Med 2011; 39: 83–94. PubMed Abstract | Publisher Full Text
28. Jin S-H, Park J-K, Nam K-Y, Park S-N, Jung N-P. Korean red ginseng saponins with low ratios of protopanaxadiol and protopanaxatriol saponin improve scopolamine-induced learning disability and spatial working memory in mice. J Ethnopharmacol 1999; 66: 123–9. PubMed Abstract | Publisher Full Text
29. Petkov V, Mosharrof A. Effects of Standarized Ginseng extract on learning, memory and physical capabilites. Am J Chin Med 1987; 15: 19–29. PubMed Abstract | Publisher Full Text
30. Davis R, Peters DH, McTavish D. Valproic acid. A reappraisal of its pharmacological properties and clinical efficacy in epilepsy. Drugs 1994; 47: 332–72. PubMed Abstract | Publisher Full Text
31. Macritchie K, Geddes J, Scott J, Haslam D, Goodwin G. Valproic acid, valproate and divalproex in the maintenance treatment of bipolar disorder. Cochrane Database Syst Rev 2001; 3: CD003196. PubMed Abstract
32. Freitag F. Divalproex in the treatment of migraine. Psychopharmacol Bull 2002; 37: 98–115.
33. Meador KJ, Baker GA, Browning N, Cohen MJ, Bromley RL, Clayton-Smith J, et al. Fetal antiepileptic drug exposure and cognitive outcomes at age 6 years (NEAD study): a prospective observational study. Lancet Neurol 2013; 12: 244–52. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
34. Jentink J, Loane MA, Dolk H, Barisic I, Garne E, Morris JK, et al. Valproic acid monotherapy in pregnancy and major congenital malformations. N Engl J Med 2010; 362: 2185–93. PubMed Abstract | Publisher Full Text
35. Robert E, Guibaud P. Maternal valproic acid and congenital neural tube defects. Lancet 1982; 320: 937. Publisher Full Text
36. Christensen J, Grønborg TK, Sørensen MJ, Schendel D, Parner ET, Pedersen LH, et al. Prenatal valproate exposure and risk of autism spectrum disorders and childhood autism. JAMA 2013; 309: 1696–703. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
37. Roullet FI, Lai JK, Foster JA. In utero exposure to valproic acid and autism – a current review of clinical and animal studies. Neurotoxicol Teratol 2013; 36: 47–56. PubMed Abstract | Publisher Full Text
38. National Institutes of Health (1985). Guide for the care and use of laboratory animals. USA: National Academies.
39. Kim J, Park J, Kang H, Kim O, Lee J. Beneficial effects of Korean red ginseng on lymphocyte DNA damage, antioxidant enzyme activity, and LDL oxidation in healthy participants: a randomized, double-blind, placebo-controlled trial. Nutr J 2012; 11: 47. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
40. Lee JH, Cho SH. Korean red ginseng extract ameliorates skin lesions in NC/Nga mice: an atopic dermatitis model. J Ethnopharmacol 2011; 133: 810–17. PubMed Abstract | Publisher Full Text
41. Park H-M, Kim S-J, Kim J-S, Kang H-S. Reactive oxygen species mediated ginsenoside Rg3-and Rh2-induced apoptosis in hepatoma cells through mitochondrial signaling pathways. Food ChemToxicol 2012; 50: 2736–41. Publisher Full Text
42. Moy SS, Nadler JJ, Perez A, Barbaro RP, Johns JM, Magnuson TR, et al. Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice. Genes Brain Behav 2004; 3: 287–302. PubMed Abstract | Publisher Full Text
43. Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology 2009; 204: 361–73. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
44. Maurice T, Phan VL, Noda Y, Yamada K, Privat A, Nabeshima T. The attenuation of learning impairments induced after exposure to CO or trimethyltin in mice by sigma (σ) receptor ligands involves both σ1 and σ2 sites. Brit J Pharmacol 1999; 127: 335–42. Publisher Full Text
45. Wey MC, Fernandez E, Martinez PA, Sullivan P, Goldstein DS, Strong R. Neurodegeneration and motor dysfunction in mice lacking cytosolic and mitochondrial aldehyde dehydrogenases: implications for Parkinson’s disease. PLoS One 2012; 7: e31522. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
46. Sarter M, Bodewitz G, Stephens DN. Attenuation of scopolamine-induced impairment of spontaneous alternation behaviour by antagonist but not inverse agonist and agonist β-carbolines. Psychopharmacology 1988; 94: 491–5. PubMed Abstract | Publisher Full Text
47. Monville C, Torres EM, Dunnett SB. Comparison of incremental and accelerating protocols of the rotarod test for the assessment of motor deficits in the 6-OHDA model. J Neurosci Methods 2006; 158: 219–23. PubMed Abstract | Publisher Full Text
48. Park HG, Yoon SY, Choi JY, Lee GS, Choi JH, Shin CY, et al. Anticonvulsant effect of wogonin isolated from Scutellaria baicalensis. Eur J Pharmacol 2007; 574: 112–19. PubMed Abstract | Publisher Full Text
49. Litchfield JT Jr, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J Pharmacol Exp Ther 1949; 96: 99–113. PubMed Abstract
50. Crawley JN. Mouse behavioral assays relevant to the symptoms of autism. Brain Pathol 2007; 17: 448–59. PubMed Abstract | Publisher Full Text
51. Yun T, Yun Y, Han I. Anticarcinogenic effect of long-term oral administration of red ginseng on newborn mice exposed to various chemical carcinogens. Cancer Detect Prev 1982; 6: 515–25.
52. Attele AS, Zhou YP, Xie JT, Wu JA, Zhang L, Dey L, et al. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component. Diabetes 2002; 51: 1851–8. PubMed Abstract | Publisher Full Text
53. Cho YK, Sung H, Lee HJ, Joo CH, Cho GJ. Long-term intake of Korean red ginseng in HIV-1-infected patients: development of resistance mutation to zidovudine is delayed. Int Immunopharmacol 2001; 1: 1295–305. PubMed Abstract | Publisher Full Text
54. Flurkey K, Currer J, Harrison D. Mouse models in aging research. In: Fox JG, Barthold S, Davisson M, Newcomer C, Quimby F, Smith A, eds. The mouse in biomedical research. 2nd ed. Burlington, MA: American College Laboratory Animal Medicine (Elsevier); 2007, p. 637–72.
55. Hong B, Ji YH, Hong JH, Nam KY, Ahn TY. A double-blind crossover study evaluating the efficacy of Korean red ginseng in patients with erectile dysfunction: a preliminary report. J Urol 2002; 168: 2070–3. PubMed Abstract | Publisher Full Text
56. Gandal MJ, Edgar JC, Ehrlichman RS, Mehta M, Roberts TP, Siegel SJ. Validating gamma oscillations and delayed auditory responses as translational biomarkers of autism. Biol Psychiatry 2010; 68: 1100–6. PubMed Abstract | Publisher Full Text
57. Kataoka S, Takuma K, Hara Y, Maeda Y, Ago Y, Matsuda T. Autism-like behaviours with transient histone hyperacetylation in mice treated prenatally with valproic acid. Int J Neuropsychopharmacol 2013; 16: 91–103. PubMed Abstract | Publisher Full Text
58. Roullet FI, Wollaston L, Decatanzaro D, Foster JA. Behavioral and molecular changes in the mouse in response to prenatal exposure to the anti-epileptic drug valproic acid. Neuroscience 2010; 170: 514–22. PubMed Abstract | Publisher Full Text
59. Kim J-W, Seung H, Kwon KJ, Ko MJ, Lee EJ, Oh HA, et al. Subchronic treatment of donepezil rescues impaired social, hyperactive, and stereotypic behavior in valproic acid-induced animal model of autism. PLoS One 2014; 9: e104927. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
60. Mehta MV, Gandal MJ, Siegel SJ. mGluR5-antagonist mediated reversal of elevated stereotyped, repetitive behaviors in the VPA model of autism. PLoS One 2011; 6: e26077. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
61. Kim HJ, Joo SH, Choi I, Kim P, Kim MK, Park SH, et al. Effects of Red Ginseng on neonatal hypoxia-induced hyperacitivity phenotype in rats. J Ginseng Res 2010; 34: 8–16. Publisher Full Text
62. Kim KC, Choi CS, Kim J-W, Han S-H, Cheong JH, Ryu JH, et al. MeCP2 modulates sex differences in the postsynaptic development of the valproate animal model of autism. Mol Neurobiol 2014: 1–17.
63. Campbell LE, Azuma R, Ambery F, Stevens A, Smith A, Morris RG, et al. Executive functions and memory abilities in children with 22q11.2 deletion syndrome. Aust N Z J Psychiatry 2010; 44: 364–71. PubMed Abstract | Publisher Full Text
64. Sobin C, Kiley-Brabeck K, Daniels S, Khuri J, Taylor L, Blundell M, et al. Neuropsychological characteristics of children with the 22q11 deletion syndrome: a descriptive analysis. Child Neuropsychol 2005; 11: 39–53. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
65. Lewandowski KE, Shashi V, Berry PM, Kwapil TR. Schizophrenic-like neurocognitive deficits in children and adolescents with 22q11 deletion syndrome. Am J Med Genet B Neuropsychiatr Genet 2007; 144B: 27–36. PubMed Abstract | Publisher Full Text
66. Hiramoto T, Kang G, Suzuki G, Satoh Y, Kucherlapati R, Watanabe Y, et al. Tbx1: identification of a 22q11.2 gene as a risk factor for autism spectrum disorder in a mouse model. Hum Mol Genet 2011; 20: 4775–85. PubMed Abstract | PubMed Central Full Text | Publisher Full Text
67. Rubenstein JL, Merzenich MM. Model of autism: increased ratio of excitation/inhibition in key neural systems. Genes Brain Behav 2003; 2: 255–67. PubMed Abstract | Publisher Full Text
68. Blatt GJ, Fitzgerald CM, Guptill JT, Booker AB, Kemper TL, Bauman ML. Density and distribution of hippocampal neurotransmitter receptors in autism: an autoradiographic study. J Autism Dev Disord 2001; 31: 537–43. PubMed Abstract | Publisher Full Text
69. Fatemi SH, Halt AR, Stary JM, Kanodia R, Schulz SC, Realmuto GR. Glutamic acid decarboxylase 65 and 67 kDa proteins are reduced in autistic parietal and cerebellar cortices. Biol Psychiatry 2002; 52: 805–10. PubMed Abstract | Publisher Full Text

Footnote

^#These authors contributed equally to this work.