Synergistic efficacy and diminished adverse effect profile of composite treatment of several ADHD medications.

Although attention-deficit/hyperactivity disorder (ADHD) is widely studied, problems regarding the adverse effect risks and non-responder problems still need to be addressed. Combination pharmacotherapy using standard dose regimens of existing medication is currently being practiced mainly to augment the therapeutic efficacy of each drug. The idea of combining different pharmacotherapies with different molecular targets to alleviate the symptoms of ADHD and its comorbidities requires scientific evidence, necessitating the investigation of their therapeutic efficacy and the mechanisms underlying the professed synergistic effects. Here, we injected male ICR mice with MK-801 to induce ADHD behavioral condition. We then modeled a "combined drug" using sub-optimal doses of methylphenidate, atomoxetine, and fluoxetine and investigated the combined treatment effects in MK-801-treated mice. No sub-optimal dose monotherapy alleviated ADHD behavioral condition in MK-801-treated mice. However, treatment with the combined drug attenuated the impaired behavior of MK-801-treated animals. Growth impediment, sleep disturbances, or risk of substance abuse were not observed in mice treated subchronically with the combined drugs. Finally, we observed that the combined ADHD drug rescued alterations in p-AKT and p-ERK1/2 levels in the prefrontal cortex and hippocampus, respectively, of MK-801-treated mice. Our results provide experimental evidence of a possible new pharmacotherapy option in ameliorating the ADHD behavioral condition without the expected adverse effects. The detailed mechanism of action underlying the synergistic therapeutic efficacy and reduced adverse reaction by combinatorial drug treatment should be investigated further in future studies.

Green tea extract containing enhanced levels of epimerized catechins attenuates scopolamine-induced memory impairment in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea has been used as a traditional medicine to control brain function and digestion. Recent works suggest that drinking green tea could prevent cognitive function impairment. During tea manufacturing processes, such as brewing and sterilization, green tea catechins are epimerized. However, the effects of heat-epimerized catechins on cognitive function are still unknown. To take this advantage, we developed a new green tea extract, high temperature processed-green tea extract (HTP-GTE), which has a similar catechin composition to green tea beverages. AIM OF THE STUDY: This study aimed to investigate the effect of HTP-GTE on scopolamine-induced cognitive dysfunction and neuronal differentiation, and to elucidate its underlying mechanisms of action. MATERIALS AND METHODS: The neuronal differentiation promoting effects of HTP-GTE in SH-SY5Y cells was assessed by evaluating neurite length and the expression level of synaptophysin. The DNA methylation status at the synaptophysin promoter was determined in differentiated SH-SY5Y cells and in the hippocampi of mice. HTP-GTE was administered for 10 days at doses of 30, 100 and 300 mg/kg (p.o.) to mice, and its effects on cognitive functions were measured by Y-maze and passive avoidance tests under scopolamine-induced cholinergic blockade state. RESULTS: HTP-GTE induced neuronal differentiation and neurite outgrowth via the upregulation of synaptophysin gene expression. These beneficial effects of HTP-GTE resulted from reducing DNA methylation levels at the synaptophysin promoter via the suppression of DNMT1 activity. The administration of HTP-GTE ameliorated cognitive impairments in a scopolamine-treated mouse model. CONCLUSIONS: These results suggest that HTP-GTE could alleviate cognitive impairment by regulating synaptophysin expression and DNA methylation levels. Taken together, HTP-GTE would be a promising treatment for the cognitive impairment observed in dysfunction of the cholinergic neurotransmitter system.

Subchronic treatment of donepezil rescues impaired social, hyperactive, and stereotypic behavior in valproic acid-induced animal model of autism.

Autism spectrum disorder (ASD) is a group of pervasive developmental disorders with core symptoms such as sociability deficit, language impairment, and repetitive/restricted behaviors. Although worldwide prevalence of ASD has been increased continuously, therapeutic agents to ameliorate the core symptoms especially social deficits, are very limited. In this study, we investigated therapeutic potential of donepezil for ASD using valproic acid-induced autistic animal model (VPA animal model). We found that prenatal exposure of valproic acid (VPA) induced dysregulation of cholinergic neuronal development, most notably the up-regulation of acetylcholinesterase (AChE) in the prefrontal cortex of affected rat and mouse offspring. Similarly, differentiating cortical neural progenitor cell in culture treated with VPA showed increased expression of AChE in vitro. Chromatin precipitation experiments revealed that acetylation of histone H3 bound to AChE promoter region was increased by VPA. In addition, other histone deacetyalse inhibitors (HDACIs) such as trichostatin A and sodium butyrate also increased the expression of AChE in differentiating neural progenitor cells suggesting the essential role of HDACIs in the regulation of AChE expression. For behavioral analysis, we injected PBS or donepezil (0.3 mg/kg) intraperitoneally to control and VPA mice once daily from postnatal day 14 all throughout the experiment. Subchronic treatment of donepezil improved sociability and prevented repetitive behavior and hyperactivity of VPA-treated mice offspring. Taken together, these results provide evidence that dysregulation of ACh system represented by the up-regulation of AChE may serve as an effective pharmacological therapeutic target against autistic behaviors in VPA animal model of ASD, which should be subjected for further investigation to verify the clinical relevance.

Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3ß/ß-catenin pathway.

Autism is a spectrum of neurodevelopmental disorders characterized by social isolation and lack of interaction. Anatomically, autism patients often show macrocephaly and high neuronal density. To investigate the mechanism underlying the higher neuronal populations seen in ASD, we subcutaneously injected VPA (400 mg/kg) into pregnant Sprague-Dawley rats on E12, an animal model often used in ASD study. Alternatively, cultured rat neural progenitor cells were treated with VPA. Until E18, VPA induced NPC proliferation and delayed neurogenesis in fetal brain, but the subsequent differentiation of NPCs to neurons increased brain neuronal density afterward. Similar findings were observed with NPCs treated with VPA in vitro. At a molecular level, VPA enhanced Wnt1 expression and activated the GSK-3ß/ß-catenin pathway. Furthermore, inhibition of this pathway attenuated the effects of VPA. The findings of this study suggest that an altered developmental process underlies the macrocephaly and abnormal brain structure observed in the autistic brain.

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.

ATP induced microglial cell migration through non-transcriptional activation of matrix metalloproteinase-9.

In response to brain insults, microglia, the resident inflammatory cells in CNS, migrate into injured sites to initiate inflammatory responses in brain. ATP, released from apoptotic or necrotic cells induce chemoattractive responses but the mechanism is not clear yet. In this study, we investigated whether ATP modulates microglial migration by regulating the activity of matrix metalloproteinases (MMPs). ATP induced rapid microglial migration and increased the activity of MMP-9 in the culture supernatants (secreted compartments) in a concentration-dependent manner. The increased activity of secreted MMP-9 is due to the increased protein secretion, but not by the increased MMP-9 mRNA and protein expression. Inhibition of MMP-9 activity by treatment with specific inhibitors including GM6001 and SB-3CT prevented ATP-induced microglial migration. ATP-induced microglial migration was also inhibited by P2Y receptor antagonists including clopidogrel as well as PI3K inhibitor such as wortmanin. Taken together, ATP non-transcriptionally increased MMP-9 activity by activation of P2Y and PI3K. The results from the present investigation may provide further insights into the regulation of the activity of MMP-9 during microglial migration, which may play essential role in the regulation of inflammatory responses in pathological situations such as neurodegenerative disorders.