Background
Attention deficit hyperactivity disorder (ADHD) is a common childhood and adolescent neurodevelopmental disorder characterized by core symptoms such as hyperactivity, inattention, and impulsivity. Moreover, ADHD have a high incidence rate of psychiatric comorbidity disorder, which is accompanied by other developmental and behavioral symptoms including sociability and leaning deficit [
1]. The symptoms of ADHD usually decline with growth; however, it is a serious problem that about two-thirds of patients are not completely cured even in adulthood [
2]. It has been suggested that dysfunctions of dopaminergic systems in the brain contribute to the pathogenetic basis of ADHD, because the amount of dopamine is reduced in the striatum and prefrontal cortex of ADHD patients [
3], and methylphenidate (MPH), a dopamine transporter inhibitor, reverses the down-regulation of dopamine in these areas and improves abnormal behavior in ADHD patients [
4]. Moreover, serotonergic and cholinergic dysfunctions also contribute to the development of ADHD [
5,
6]. There are genetic and environmental factors for the onset of ADHD; the former includes various genetic variations such as in the dopamine transporter [
7‐
9] and receptors [
10,
11], serotonin 5-HT
2A receptor [
12], and N-methyl-D-aspartate glutamate receptor subunit [
13]. Moreover, environmental factors appear to alter the epigenetic regulation of the disorder [
14].
We have previously demonstrated that post-weaning social isolation (ISO) rearing of rodents causes behavioral abnormalities similar to those of ADHD patients such as attention deficit-like behaviors, increased aggressive responses, hyperactivity and fear memory deficits [
15‐
18] and that ISO-induced attention deficit-like behaviors were reversed by MPH [
18]. Moreover, our neurochemical studies have suggested that ISO-induced fear memory deficits are in part mediated by a decrease in the expression level of phosphorylated forms of neuroplasticity-related signaling molecules, such as CaMKII and CREB, since the administration of tacrine, an acetylcholinesterase inhibitor, reverses the down-regulated expression of these phosphorylated forms in the hippocampus by mechanisms relevant to improvement of fear memory deficits in ISO mice [
19,
20]. Moreover, we found that ISO mice also showed impairment of sociability [
20]. On the other hand, clinical evidence indicates that deficits in sociability are one of the core symptoms of autism spectrum disorder (ASD) and that a considerable number of patients with ASD exhibit comorbid symptoms of ADHD [
21,
22]. Taken together, our previous findings suggest that ISO mice provide a putative animal model of developmental disorders including the comorbidity of ADHD and ASD [
23,
24] to explore drugs with therapeutic and/or preventive potentials for the disorders.
Traditional herbal medicines such as Kampo and Chinese medicines have been used to treat the symptoms of many diseases. Moreover, clinical studies in patients with psychological disorders have shown that some traditional herbal medicines can ameliorate the positive and negative syndrome scale of schizophrenia, and the symptoms of inattention, hyperactivity, and impulsivity in children and adolescents [
25,
26]. These studies suggest that novel agents effective for treating neuropsychological disorders may be found in traditional herbal remedies. Recently, Iwasaki et al. have reported that yokukansan (YKS), a traditional herbal medicine that has long been used for patients with neurosis, insomnia, and irritability in children, can improve the behavioral and psychological symptoms of dementia patients such as hallucinations, agitation, and aggression in patients with Alzheimer’s disease and other forms of senile dementia [
27,
28]. Moreover, it has been reported that YKS also improves not only memory disturbances but also abnormal social interaction, such as increased aggressive behavior and decreased social behavior, in amyloid precursor protein transgenic mice [
29], suggesting that YKS can exert a beneficial effect on symptoms of several neuropsychological disorders. These clinical and experimental findings prompted us to investigate whether YKS has therapeutic and preventive/delaying potentials for neurodevelopmental disorders. In the present study, we employed ISO mice as a putative animal model of developmental disorders with comorbid features of ADHD and ASD and elucidated the pharmacological effects of YKS on ISO-induced behavioral abnormalities and neurochemical alterations in the brain. We also used keishito (KST) as a reference Kampo prescription because the clinical use of KST is different from that of YKS. Indeed, KST is prescribed for the initial symptoms of a cold with a headache and fever in children. In this study, we compared the effects of YKS with those of KST to elucidate whether these prescriptions for children exert different pharmacological effects in ISO mice.
Discussion
Our previous studies have demonstrated that social isolation rearing of mice from early weaning induces behavioral abnormalities such as hyperactivity, reduced sociability, spatial attention deficit, and impaired fear memory performance. Considering the face and predictive validities of ISO mice, we have proposed that ISO from early weaning offers an animal model of comorbid developmental disorder with ADHD and ASD symptoms, and that the ISO model is useful for screening drugs for the treatment of such disorders [
18,
20]. In this study, we investigated the therapeutic and preventive/delaying potentials of YKS and KST against ISO-induced behavioral and pharmacological abnormalities in order to elucidate the possible efficacy of Kampo medicines for the treatment of neurodevelopmental disorders. Our findings suggested that YKS and KST may be useful for the prevention or delaying of onset in developmental disorders.
In the experiments we first conducted to elucidate the therapeutic potentials of YKS and KST for treating ISO-induced behavioral abnormalities, the administration of YKS significantly improved impairments of attention-like behavior in the water finding test and context-dependent fear memory performance in the fear conditioning test, but not sociability in the 3-chamber test, whereas KST had no significant effects on these behavioral indices. This finding suggests that YKS can exert a therapeutic action against some symptoms related to attention deficit and memory impairment in the ISO model. In Kampo medicine, YKS is prescribed particularly to target neuropsychiatric symptoms in children, while KST is used to improve the initial symptoms of children with a headache and fever. Therefore, the differences in such utilization of YKS and KST are likely relevant to the differences in the therapeutic activity observed between YKS and KST. In previous studies, we demonstrated that acute administration of MPH ameliorated ISO-induced spatial attention deficits in the water finding test in part via central cholinergic systems, and that dysfunction of central cholinergic systems was involved in deficits of contextual and conditional fear memory caused by ISO from early weaning [
19,
20]. Moreover, several lines of experimental evidence indicate that YKS is able to improve behaviors related to spatial cognitive dysfunction in animal models of dementia via central cholinergic systems [
30]. Taken together, the present findings suggest that the ameliorative effects of YKS on spatial cognition-related behaviors in the water-finding and fear conditioning tests may be due to the distinctive pharmacological profiles of this Kampo medicine, which in part involves central cholinergic mechanisms.
It is of interest that in the experiments where the preventive/delaying potentials of YKS and KST were examined by administration in week 0 of the ISO period, both YKS and KST improved ISO-induced deficits in sociability- and attention-like behaviors as well as impairment of context-dependent fear memory. These findings indicate that both YKS and KST can exert preventive/delaying actions against behavioral abnormalities induced by ISO. We previously reported that MPH ameliorated sociability deficits caused by ISO, and that the effect of MPH was diminished by pre-treatment with SCH23390, a dopamine D
1 receptor antagonist, indicating that sociability deficits in ISO mice are at least in part attributable to alterations of dopaminergic function in the brain [
20]. However, in the present study, the preventive/delaying effect of YKS or KST on sociability deficits in ISO mice was unaltered by SCH23390, suggesting that, unlike MPH, a mechanism(s) independent of dopaminergic systems may be involved in the preventive/delaying effects of YKS and KST.
The present study also demonstrated that YKS and KST exhibited preventive/delaying effects on ISO-induced deficits of attention-like behavior in a manner reversed by scopolamine. This finding indicates the involvement of central cholinergic systems in the effects of these Kampo formulae, and supports at least the aforementioned therapeutic effect of YKS on ISO-induced deficits in attention-like behavior. This is supported by previous findings reported by Ouchi et al. [
18]. They found that MPH amelioration of attention-like behavior in ISO animals could be reversed by scopolamine, suggesting the involvement of cholinergic dysfunction in impaired attention-like behavior. Several mechanisms may account for the preventive/delaying effects of YKS and KST. First, it is likely that the administration of YKS and KST during the ISO period facilitates the function of central cholinergic systems and thereby ameliorates attention-like behavior deficits in ISO animals. This possibility seems plausible for YKS rather than KST since, as discussed above, KST failed to exert therapeutic actions against ISO-induced behavioral abnormalities including impaired attention-like behavior. Second, both YKS and KST may be able to protect central cholinergic systems from dysfunction, which is induced from the early stages of ISO [
18]. This mechanism is very likely to be implicated in the effect of KST, since KST ameliorated ISO-induced behavioral deficits when the administration was started from week 0, but not from week 2 after starting ISO. Moreover, chemical constituents of YKS and KST have been reported to have neuroprotective effects [
33‐
36], suggesting that these compounds protect central cholinergic systems from ISO-induced dysfunction. This hypothesis is currently under investigation in our laboratory.
Importantly, the present neurochemical studies conducted according to the experimental schedule in Fig.
1b clearly revealed that daily administration of YKS and KST during the entire duration of ISO reversed the down-regulated expression of phosphorylated forms of CaMKII and CREB, neuroplasticity-related signaling proteins, in the hippocampus. We analyzed these proteins in the hippocampus because of a couple of reasons. First, synaptic plasticity-related signaling in the hippocampus is an important molecular biological basis of learning and memory including conditional fear memories [
19,
37,
38]. Secondly, our previous study demonstrated that ISO of mice for 2 weeks after a weaning period impaired the consolidation process of contextual and auditory fear memories via causing dysfunction of hippocampal synaptic plasticity-related neuro-signaling, and that tacrine, an acetylcholinesterase inhibitor, ameliorated ISO-induced deficits in fear memories and hippocampal neuro-signaling function [
19,
20]. Our previous findings have suggested that ISO-induced fear memory deficits and dysfunction of hippocampal synaptic plasticity-related neuro-signaling are in part due to impairment of central cholinergic systems, and that amelioration or prevention of cholinergic deficit contributes to the improvement of these impairments induced by ISO [
20]. Considering the present data that daily administration of YKS and KST during the entire duration of the ISO period ameliorated not only contextual fear memory deficits, but also impairment of hippocampal neuro-signaling in ISO mice as well as tacrine, the present findings raise the possibility that YKS and KST may be able to prevent cholinergic systems from ISO-induced brain dysfunction via normalizing neuroplasticity-related hippocampal neuro-signaling, and thereby improve fear memory deficits. This idea is supported by previous reports that YKS ameliorated olfactory bulbectomy-induced deficits in spatial memory deficit, and that the effects were at least partly mediated by muscarinic receptor stimulation and the normalization of central cholinergic systems [
30,
39].
One of the most interesting findings in this study is that not only YKS but also KST exerted significant preventive/delaying effect on ISO-induced behavioral abnormalities and hippocampal neuro-signaling deficits, because the clinical applications of KST significantly differ from those of YKS. The mechanism(s) and chemical constituent(s) involved in the actions of KST are still unclear. However, considering the fact that when administration of KST was conducted for almost a same period by the therapeutic procedure, KST did not affect ISO-induced behavioral abnormalities, it is very likely that KST may be able to block processes including epigenetic alteration, which is triggered early after starting ISO. Recent findings reported by Araki et al. have demonstrated that ISO induces epigenetic changes in promoter coding regions of the
GABA
B1a
receptor [
40] and
srd5a1 [
41], a gene coding for the biosynthetic enzyme neurosteroid allopregnanolone, which is down-regulated by ISO [
16,
42]. Moreover, our preliminary study indicated that inhibition of allopregnanolone biosynthesis in the brain caused impairment of sociability behavior in mice in a manner reversible by systemic administration of allopregnanolone (unpublished data). Therefore, ISO mice may show behavior abnormalities through the epigenetic regulation, and it is possible that ISO-induced epigenetic alterations of the aforementioned genes and others are prevented by the administration of KST from an early stage of ISO. Further studies are needed to examine this possibility involved in the preventive/delaying effect of KST.
Acknowledgments
This study was in part supported by a Grant-in-Aid for Challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to KM (#15 K15269), a 2014 Grant-in-Aid from Kobayashi International Scholarship Foundation (KM), and a Grant-in-Aid for the 2014 and 2015 Cooperative Research Project I from the Institute of Natural Medicine, University of Toyama (TY and KM).