Pitna Kim

The critical period of valproate exposure to induce autistic symptoms in Sprague–Dawley rats

Prenatal exposure to valproic acid (VPA) induces neural tube defects and impairment in social behaviors related to autistic spectrum disorder in newborns, which make it a useful animal model of autism. In this study, we compared the effects of different time window of prenatal valproic acid exposure for inducing the altered social behaviors relevant to autism from embryonic day 7 to embryonic day 15 in Sprague-Dawley rats to determine the critical periods for the impairment. Compared to E7, E9.5 and E15 exposure, VPA exposure at E12 showed most significant changes in behaviors over control animals with reduced sociability and social preference. E9.5 exposure to valproic acid showed strong reproductive toxicity including decrease in the number of live birth. In general, exposure at E15 showed only marginal effects on reproduction and social behaviors. Finally, VPA-exposed rats at E12 were more sensitive to electric shock than VPA-exposed rats at any other periods. These results suggested that E12 is the critical period in rats when valproate exposure has prominent effects for inducing the altered social behavior similar to human autistic behavior.

A comprehensive review of the therapeutic and pharmacological effects of ginseng and ginsenosides in central nervous system

Ginseng is one of the most widely used herbal medicines in human. Central nervous system (CNS) diseases are most widely investigated diseases among all others in respect to the ginseng’s therapeutic effects. These include Alzheimer’s disease, Parkinson’s disease, cerebral ischemia, depression, and many other neurological disorders including neurodevelopmental disorders. Not only the various types of diseases but also the diverse array of target pathways or molecules ginseng exerts its effect on. These range, for example, from neuroprotection to the regulation of synaptic plasticity and from regulation of neuroinflammatory processes to the regulation of neurotransmitter release, too many to mention. In general, ginseng and even a single compound of ginsenoside produce its effects on multiple sites of action, which make it an ideal candidate to develop multi-target drugs. This is most important in CNS diseases where multiple of etiological and pathological targets working together to regulate the final pathophysiology of diseases. In this review, we tried to provide comprehensive information on the pharmacological and therapeutic effects of ginseng and ginsenosides on neurodegenerative and other neurological diseases. Side by side comparison of the therapeutic effects in various neurological disorders may widen our understanding of the therapeutic potential of ginseng in CNS diseases and the possibility to develop not only symptomatic drugs but also disease modifying reagents based on ginseng.

Male‐specific alteration in excitatory post‐synaptic development and social interaction in pre‐natal valproic acid exposure model of autism spectrum disorder

Autism spectrum disorder (ASD) is a pervasive developmental disorder characterized by three main behavioral symptoms including social deficits, impaired communication, and stereotyped and repetitive behaviors. ASD prevalence shows gender bias to male. Prenatal exposure to valproic acid (VPA), a drug used in epilepsy and bipolar disorder, induces autistic symptoms in both human and rodents. As we reported previously, prenatally VPA‐exposed animals at E12 showed impairment in social behavior without any overt reproductive toxicity. Social interactions were not significantly different between male and female rats in control condition. However, VPA‐exposed male offspring showed significantly impaired social interaction while female offspring showed only marginal deficits in social interaction. Similar male inclination was observed in hyperactivity behavior induced by VPA. In addition to the ASD‐like behavioral phenotype, prenatally VPA‐exposed rat offspring shows crooked tail phenotype, which was not different between male and female groups. Both male and female rat showed reduced GABAergic neuronal marker GAD and increased glutamatergic neuronal marker vGluT1 expression. Interestingly, despite of the similar increased expression of vGluT1, post‐synaptic marker proteins such as PSD‐95 and α‐CAMKII expression was significantly elevated only in male offspring. Electron microscopy showed increased number of post‐synapse in male but not in female at 4 weeks of age. These results might suggest that the altered glutamatergic neuronal differentiation leads to deranged post‐synaptic maturation only in male offspring prenatally exposed to VPA. Consistent with the increased post‐synaptic compartment, VPA‐exposed male rats showed higher sensitivity to electric shock than VPA‐exposed female rats. These results suggest that prenatally VPA‐exposed rats show the male preponderance of ASD‐like behaviors including defective social interaction similar to human autistic patients, which might be caused by ectopic increase in glutamatergic synapses in male rats.

Pax6-Dependent Cortical Glutamatergic Neuronal Differentiation Regulates Autism-Like Behavior in Prenatally Valproic Acid-Exposed Rat Offspring

Imbalance in excitatory/inhibitory signal in the brain has been proposed as one of the main pathological features in autism spectrum disorders, although the underlying cellular and molecular mechanism is unclear yet. Because excitatory/inhibitory imbalance can be induced by aberration in glutamatergic/GABAergic neuronal differentiation, we investigated the mechanism of dysregulated neuronal differentiation between excitatory and inhibitory neurons in the embryonic and postnatal brain of prenatally valproic acid-exposed rat offspring, which is often used as an animal model of autism spectrum disorders. Transcription factor Pax6, implicated in glutamatergic neuronal differentiation, was transiently increased in embryonic cortex by valproate exposure, which resulted in the increased expression of glutamatergic proteins in postnatal brain of offspring. Chromatin immunoprecipitation showed increased acetylated histone binding on Pax6 promoter region, which may underlie the transcriptional up-regulation of Pax6. Other histone deacetylase (HDAC) inhibitors including TSA and SB but not valpromide, which is devoid of HDAC inhibitor activity, induced Pax6 up-regulation. Silencing Pax6 expression in cultured rat primary neural progenitor cells demonstrated that up-regulation of Pax6 plays an essential role in valproate-induced glutamatergic differentiation. Blocking glutamatergic transmission with MK-801 or memantine treatment, and to a lesser extent with MPEP treatment, reversed the impaired social behaviors and seizure susceptibility of prenatally valproate-exposed offspring. Together, environmental factors may contribute to the imbalance in excitatory/inhibitory neuronal activity in autistic brain by altering expression of transcription factors governing glutamatergic/GABAergic differentiation during fetal neural development, in conjunction with the genetic preload.

Chronic exposure to ethanol of male mice before mating produces attention deficit hyperactivity disorder‐like phenotype along with epigenetic dysregulation of dopamine transporter expression in mouse offspring

Preconception exposure to EtOH through the paternal route may affect neurobehavioral and developmental features of offspring. This study investigates the effects of paternal exposure to EtOH before conception on the hyperactivity, inattention, and impulsivity behavior of male offspring in mice. Sire mice were treated with EtOH in a concentration range approximating human binge drinking (0–4 g/kg/day EtOH) for 7 weeks and mated with untreated females mice to produce offspring. EtOH exposure to sire mice induced attention deficit hyperactivity disorder (ADHD)‐like hyperactive, inattentive, and impulsive behaviors in offspring. As a mechanistic link, both protein and mRNA expression of dopamine transporter (DAT), a key determinant of ADHD‐like phenotypes in experimental animals and humans, were significantly decreased by paternal EtOH exposure in cerebral cortex and striatum of offspring mice along with increased methylation of a CpG region of the DAT gene promoter. The increase in methylation of DAT gene promoter was also observed in the sperm of sire mice, suggesting germline changes in the epigenetic methylation signature of DAT gene by EtOH exposure. In addition, the expression of two key regulators of methylation‐dependent epigenetic regulation of functional gene expression, namely, MeCP2 and DNMT1, was markedly decreased in offspring cortex and striatum sired by EtOH‐exposed mice. These results suggest that preconceptional exposure to EtOH through the paternal route induces behavioral changes in offspring, possibly via epigenetic changes in gene expression, which is essential for the regulation of ADHD‐like behaviors.

Effects of ethanol exposure during early pregnancy in hyperactive, inattentive and impulsive behaviors and MeCP2 expression in rodent offspring.

Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system.

Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL.

BackgroundAt the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis.Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs.MethodsNeural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C.) diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay.ResultsIn this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL.ConclusionTo the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally critical periods through the degradation of IκBα and the activation of NF-κB signaling. The reduced NPCs death might underlie the neurodevelopmental defects collectively called fetal valproate syndrome, which shows symptoms such as mental retardation and autism-like behavior.

Effects of Korean red ginseng extracts on neural tube defects and impairment of social interaction induced by prenatal exposure to valproic acid

Ginseng is one of the most widely used medicinal plants, which belongs to the genus Panax. Compared to uncured white ginseng, red ginseng has been generally regarded to produce superior pharmacological effects with lesser side/adverse effects, which made it popular in a variety of formulation from tea to oriental medicine. Using the prenatal valproic acid (VPA)-injection model of autism spectrum disorder (ASD) in rats, which produces social impairrment and altered seizure susceptibility as in human ASD patients as well as mild neural tube defects like crooked tail phenotype, we examined whether chronic administration of red ginseng extract may rescue the social impairment and crooked tail phenotype in prenatally VPA-exposed rat offspring. VPA-induced impairment in social interactions tested using sociability and social preference paradigms as well as crooked tail phenotypes were significantly improved by administration of Korean red ginseng (KRG) in a dose dependent manner. Rat offspring prenatally exposed to VPA showed higher sensitivity to electric shock seizure and increased locomotor activity in open-field test. KRG treatment reversed abnormal locomotor activity and sensitivity to electric shock to control level. These results suggest that KRG may modulate neurobehavioral and structural organization of nervous system adversely affected by prenatal exposure to VPA.

Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

BackgroundPrenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models.MethodsNeural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis.ResultsReduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67.ConclusionsThese results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.

A simple behavioral paradigm to measure impulsive behavior in an animal model of attention deficit hyperactivity disorder (ADHD) of the spontaneously hypertensive rats.

Impulsiveness is an important component of many psychiatric disorders including Attention-deficit/hyperactivity disorder (ADHD). Although the neurobiological basis of ADHD is unresolved, behavioral tests in animal models have become indispensable tools for improving our understanding of this disorder. In the punishment/extinction paradigm, impulsivity is shown by subjects that persevere with responding despite punishment or unrewarded responses. Exploiting this principle, we developed a new behavioral test that would evaluate impulsivity in the most validated animal model of ADHD of the Spontaneously Hypertensive rat (SHR) as compared with the normotensive “control” strain, the Wistar Kyoto rat (WKY). In this paradigm we call the Electro-Foot Shock aversive water Drinking test (EFSDT), water-deprived rats should pass over an electrified quadrant of the EFSDT apparatus to drink water. We reasoned that impulsive animals show increased frequency to drink water even with the presentation of an aversive consequence (electro-shock). Through this assay, we showed that the SHR was more impulsive than the WKY as it demonstrated more “drinking attempts” and drinking frequency. Methylphenidate, the most widely used ADHD medication, significantly reduced drinking frequency of both SHR and WKY in the EFSDT. Thus, the present assay may be considered as another behavioral tool to measure impulsivity in animal disease models, especially in the context of ADHD.