Mechanistic study of the anti-excitatory amino acid toxicity of Bushen Zhichan decoction for Parkinson's disease based on the transcriptional regulation of EAAT1 by YY1.

ETHNOPHARMACOLOGICAL RELEVANCE: Bushen Zhichan decoction (BSZCF) is derived from Liuwei Dihuang Pill, a famous Chinese herbal formula recorded in the book Key to Therapeutics of Children's Diseases. It has been widely used as a basic prescription for nourishing and tonifying the liver and kidneys to treat Parkinson's disease (PD), but its mechanism remains to be explored. AIM OF THE STUDY: BSZCF, a Chinese herbal formula comprising five herbs: Rehmannia glutinosa (Gaertn.) DC., Dioscorea oppositifolia L., Cornus officinalis Siebold & Zucc., Fallopia multiflora (Thunb.) Haraldson and Cistanche tubulosa (Schenk) Wight, is used clinically to treat PD. In vivo and in vitro experiments were designed to elucidate the mechanism of BSZCF in the protection of dopamine (DA) neurons and the treatment of PD. The toxicity of excitatory amino acids (EAA) may be attenuated by inhibiting the transcription factor Yin Yang 1 (YY1) and up-regulating the expression of excitatory amino acid transporter 1 (EAAT1). MATERIALS AND METHODS: IN VIVO: After 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into specific pathogen free (SPF) C57BL/6J mice, model mice were intragastrically given adamantane hydrochloride tablets (AHT) or different doses of BSZCF for 14 days. Both open field and pole-climbing tests were conducted to assess behavioral changes. In vitro: 1-Methyl-4-phe-nylpyridiniumiodide (MPP+)-injured human neuroblastoma cells (SH-SY5Y) were utilized to construct PD cell models. Primary astrocytes were transfected with EAAT1 and YY1 lentiviruses for EAAT1 gene knockout and YY1 gene knockout astrocytes, respectively. The high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of BSZCF was performed to control the quality of blood drugs. The optimal concentration and time of PD cell models treated by BSZCF were determined by the use of Cell Counting Kit-8 (CCK8). Enzyme-linked immunosorbent assay (ELISA) was used for measuring glutamate (Glu) in the peripheral blood and cells of each group. Western blotting (WB) and real-time quantitative polymerase chain reaction (qPCR) were used to detect tyrosine hydroxylase (TH), dopamine transporters (DAT), EAAT1 and YY1 protein and mRNA. After the blockade of EAAT1, immunofluorescence (IF) assay was used to detect the TH protein in each group. RESULTS: In vivo research showed that BSZCF improved the behavioral symptoms of PD mice, and reduced the death of DA neurons and the level of Glu. The mechanism may be related to the decrease of YY1 expression and the increase of EAAT1 levels. In vitro experiments showed that the anti-excitatory amino acid toxicity of BSZCF was achieved by inhibiting YY1 expression and regulating EAAT1. CONCLUSIONS: By inhibiting YY1 to increase the expression of EAAT1 and attenuating the toxicity of Glu, BSZCF exerts the effect of protecting DA neurons and treating PD-like symptoms in mice.

Astrocyte-specific deletion of the transcription factor Yin Yang 1 in murine substantia nigra mitigates manganese-induced dopaminergic neurotoxicity.

Manganese (Mn)-induced neurotoxicity resembles Parkinson's disease (PD), but the mechanisms underpinning its effects remain unknown. Mn dysregulates astrocytic glutamate transporters, GLT-1 and GLAST, and dopaminergic function, including tyrosine hydroxylase (TH). Our previous in vitro studies have shown that Mn repressed GLAST and GLT-1 via activation of transcription factor Yin Yang 1 (YY1). Here, we investigated if in vivo astrocytic YY1 deletion mitigates Mn-induced dopaminergic neurotoxicity, attenuating Mn-induced reduction in GLAST/GLT-1 expression in murine substantia nigra (SN). AAV5-GFAP-Cre-GFP particles were infused into the SN of 8-week-old YY1 flox/flox mice to generate a region-specific astrocytic YY1 conditional knockout (cKO) mouse model. 3 weeks after adeno-associated viral (AAV) infusion, mice were exposed to 330 µg of Mn (MnCl2 30 mg/kg, intranasal instillation, daily) for 3 weeks. After Mn exposure, motor functions were determined in open-field and rotarod tests, followed by Western blotting, quantitative PCR, and immunohistochemistry to assess YY1, TH, GLAST, and GLT-1 levels. Infusion of AAV5-GFAP-Cre-GFP vectors into the SN resulted in region-specific astrocytic YY1 deletion and attenuation of Mn-induced impairment of motor functions, reduction of TH-expressing cells in SN, and TH mRNA/protein levels in midbrain/striatum. Astrocytic YY1 deletion also attenuated the Mn-induced decrease in GLAST/GLT-1 mRNA/protein levels in midbrain. Moreover, YY1 deletion abrogated its interaction with histone deacetylases in astrocytes. These results indicate that astrocytic YY1 plays a critical role in Mn-induced neurotoxicity in vivo, at least in part, by reducing astrocytic GLAST/GLT-1. Thus, YY1 might be a potential target for treatment of Mn toxicity and other neurological disorders associated with dysregulation of GLAST/GLT-1.

Effects of Combined Bushen Zhichan Recipe and Levodopa in a Rodent Model of Parkinson Disease: Potential Mechanisms.

BACKGROUND Parkinson disease is characterized by the loss of neurons in the substantia nigra, and under pathological conditions, glutamate can produce excitotoxic effects on nerve cells. The astrocytic excitatory amino acid transporter (EAAT) 1 can be functionally upregulated and targeted to functional compartments, resulting in reduced excitotoxicity. levodopa is the gold standard for the treatment of Parkinson disease, but prolonged levodopa treatment often leads to the development of abnormal involuntary movements. Numerous studies suggest the potential beneficial effects of traditional Chinese medicine on Parkinson disease. MATERIAL AND METHODS We validated the efficacy of a Bushen Zhichan recipe combined with levodopa in a rodent Parkinson disease model and explored its possible mechanisms. RESULTS Rats in the combined levodopa and Bushen Zhichan recipe group performed significantly better than the control group in the open field and forelimb function experiments. The number of midbrain dopaminergic neurons in rats in the levodopa and Bushen Zhichan recipe group was greater compared to controls. The levodopa and Bushen Zhichan recipe group exhibited decreased glutamate receptors and increased γ-aminobutyric acid receptors in the striatum. At the same time, EAAT1 was increased and EAAT2 was synchronized with the number of glutamate receptors. CONCLUSIONS Our results indicate that levodopa combined with Bushen Zhichan recipe significantly improves behavior and protects dopaminergic neurons in a rodent Parkinson disease model, and suggest that the mechanism involves the decrease of excitatory amino acid toxicity and the increase in the expression of EAAT1.

Bisphenol A exposure disrupts aspartate transport in HepG2 cells.

The liver is the organ responsible for bisphenol A (BPA) metabolism, an environmental chemical agent. Exposure to this toxin is associated with liver abnormalities and dysfunction. An important role played by excitatory amino acid transporters (EAATs) of the slc1 gene family has been reported in liver injuries. To gain insight into a plausible effect of BPA exposure in the liver glutamate/aspartate transport, using the human hepatoblastoma cell line HepG2, we report a BPA-dependent dynamic regulation of SLC1A3 and SLC1A2. Through the use of radioactive [3 H]- d-aspartate uptake experiments and immunochemical approaches, we characterized time and dose-dependent regulation of the protein levels and function of these transporters after acute exposure to BPA. An increase in nuclear Yin Yang 1 was found. These results suggest an important involvement of the EAATs in liver physiology and its disruption after acute BPA exposure.

Xiaoyaosan exerts antidepressant-like effects by regulating the functions of astrocytes and EAATs in the prefrontal cortex of mice.

BACKGROUND: Mounting evidence indicates that the cerebral cortex is an important physiological system of emotional activity, and its dysfunction may be the main cause of stress. Glutamate is the primary excitatory neurotransmitter in the central nervous system (CNS), which initiates rapid signal transmission in the synapse before its reuptake into the surrounding glia, specifically astrocytes (ASTs). The astrocytic excitatory amino acid transporters 1 (EAAT1) and 2 (EAAT2) are the major transporters that take up synaptic glutamate to maintain optimal extracellular glutamic levels, thus preventing accumulation in the synaptic cleft and ensuing excitotoxicity. Growing evidence has shown that excitotoxicity is associated with depression. Therefore, we hypothesized that the underlying antidepressant-like mechanism of Xiaoyaosan (XYS), a Chinese herbal formula, may be related to the regulation of astrocytic EAATs. Therefore, we studied the antidepressant mechanism of XYS on the basis of EAAT dysfunction in ASTs. METHODS: Eighty adult C57BL/6 J mice were randomly divided into 4 groups: a control group, a chronic unpredictable mild stress (CUMS) group, a Xiaoyaosan (XYS) treatment group and a fluoxetine hydrochloride (Flu) treatment group. Except for the control group, mice in the other groups all received chronic unpredictable mild stress for 21 days. Mice in the control and CUMS groups received gavage administration with 0.5 mL of normal saline (NS) for 21 days, and mice in the XYS and Flu treatment groups were administered dosages of 0.25 g/kg/d and 2.6 mg/kg/d by gavage. The effects of XYS on the depressive-like behavioral tests, including the open field test (OFT), forced swimming test (FST) and sucrose preference test (SPT), were examined. The glutamate (Glu) concentrations of the prefrontal cortex (PFC) were detected with colorimetry. The morphology of neurons in the PFC was observed by Nissl staining. The expression of glial fibrillary acidic protein (GFAP), NeuN, EAAT1 and EAAT2 proteins in the PFC of mice was detected by using Western blotting and immunohistochemistry. Quantitative real-time PCR (qPCR) was used to detect the expression of the GFAP, NeuN, EAAT1 and EAAT2 genes in the PFC of mice. RESULTS: The results of behavioral tests showed that CUMS-induced mice exhibited depressive-like behavior, which could be improved in some tests with XYS and Flu treatment. Immunohistochemistry and Western blot analysis showed that the protein levels of GFAP, NeuN, EAAT1 and EAAT2 in the PFC of CUMS mice were significantly lower than those in the control group, and these changes could be reversed by XYS and Flu. The results of qPCR analysis showed that the expression of GFAP, NeuN, EAAT1 and EAAT2 mRNAs in the PFC of CUMS mice was not significantly changed, with the exception of EAAT2, compared with that of the control group, while the expression of the above mRNAs was significantly higher in the XYS and Flu groups than that in the CUMS group. CONCLUSION: XYS may exert antidepressant-like effects by improving the functions of AST and EAATs and attenuating glutamate-induced neuronal damage in the frontal cortex.

Involvement of the glutamate/glutamine cycle and glutamate transporter GLT-1 in antidepressant-like effects of Xiao Yao san on chronically stressed mice.

BACKGROUND: Xiao Yao San (XYS) is an herbal prescription which is used in the treatment of depression for thousands of years from Song dynasty in China (960-1127 A.D.), and is the bestselling and most popular herb formula for treating major depression. This study aimed to assess the chronic antidepressant effects of XYS and fluoxetine in depressed mice induced by chronic unpredictable mild stress (CUMS) and its association with  alterations in glutamate/glutamine cycle and glutamate transporters. METHODS: Mice in the control and model group were given 0.5 ml physiological saline by intragastric administration. Mice in two treatment groups were given XYS (0.25 g/kg/d) and fluoxetine (2.6 mg/kg/d), respectively. The depressive-like behaviors such as forced swim test (FST), sucrose preference test (SPT) and novelty-suppressed feeding (NSF) test were measured after mice exposed to CUMS for 21 days. Body weight, contents of glutamate and glutamine, glutamine/glutamate ratio that is usually thought to reflect glutamate/glutamine cycle, and the protein and mRNA expressions of glutamate transporters (excitatory amino acid transporter 1-2,GLAST/EAAT1 and GLT-1/EAAT2) were measured. The immunoreactivities of GLAST and GLT-1 in the hippocampus were also investigated. RESULTS: After CUMS exposure, mice exhibited depressive-like behaviors, body weight loss, increased glutamate level, decreased glutamine level, elevated glutamine/glutamate ratio, decreased GLT-1 protein expression and mRNA level, and decreased average optical density (AOD) of GLT-1 in the CA1, CA3 and DG in the hippocampus. These abnormalities could be effectively reversed by XYS or fluoxetine treatment. In addition, the study also found that GLAST expression in the hippocampus could not be altered by 21-d CUMS. CONCLUSION: The studies indicated that XYS may have therapeutic actions on depression -like behavior s induced by CUMS in mice possibly mediated by modulation of glutamate/glutamine cycle and glutamate transporter GLT-1 in the hippocampus.

Flavonoid constituents of Dobera glabra leaves: amelioration impact against CCl4-induced changes in the genetic materials in male rats.

CONTEXT: Dobera glabra (Forssk.) Poir (Salvadoraceae) is a highly valued tree with diverse importance as special mineral sourced feed and a folkloric tool for forecasting droughts. However, there are no reports on its phytochemical and biological investigations. OBJECTIVE: Phytochemical investigation of D. glabra leaves and its protective potential against CCl4 inducing changes in the genetic materials. MATERIALS AND METHODS: D. glabra extract, DGE (70% MeOH/H2O), was applied to polyamide column chromatography, eluting with MeOH/H2O of decreasing polarities, followed by preparative chromatographic tools, yielded seven compounds. Three DGE doses (50, 100 and 200 mg/kg bw/d) were administrated for 8 weeks intragastrically to male albino rats prior treated with CCl4 (0.5 mL/kg/bw). The reactive oxygen species (ROS) levels, expression changes of glutamate transporters (GLAST, GLT-1 and SNAT3) mRNA, DNA fragmentation and glutathione peroxidase (GPx) activity were investigated in the liver tissues of these rats. RESULTS: Isorhamnetin-3-O-ß-glucopyranoside-7-O-α-rhamnopyranoside, isorhamnetin-3-O-α-rhamnopyranoside-7-O-ß-glucopyranoside, kaempferol-3,7-di-O-α-rhamnopyranoside, isorhamnetin-3-O-ß-glucopyranoside, kaempferol-3-O-ß-glucopyranoside, isorhamnetin and kaempferol were identified. DGE (200 mg/kg bw) + CCl4 exhibited the most significant reduction in ROS levels and DNA fragmentation with 251.3% and141% compared to 523.1% and 273.2% for CCl4, respectively. Additionally, it increased significantly the mRNA expression of GLAST, GLT-1 and SNAT3 to 2.16-, 1.72- and 2.09-fold, respectively. Also, GPx activity was increased to 4.8 U/mg protein/min compared to CCl4 (1.8 U/mg protein/min). DISCUSSION AND CONCLUSION: Flavonoid constituents, antioxidant effect and genotoxic protection activity of D. glabra were first reported. DGE may be valuable in the treatment and hindrance of hepatic oxidative stress and genotoxicity.

Tanycytes and a differential fatty acid metabolism in the hypothalamus.

Although the brain controls all main metabolic pathways in the whole organism, its lipid metabolism is partially separated from the rest of the body. Circulating lipids and other metabolites are taken up into brain areas like the hypothalamus and are locally metabolized and sensed involving several hypothalamic cell types. In this study we show that saturated and unsaturated fatty acids are differentially processed in the murine hypothalamus. The observed differences involve both lipid distribution and metabolism. Key findings were: (i) hypothalamic astrocytes are targeted by unsaturated, but not saturated lipids in lean mice; (ii) in obese mice labeling of these astrocytes by unsaturated oleic acid cannot be detected unless ß-oxidation or ketogenesis is inhibited; (iii) the hypothalamus of obese animals increases ketone body and neutral lipid synthesis while tanycytes, hypothalamic cells facing the ventricle, increase their lipid droplet content; and (iv) tanycytes show different labeling for saturated or unsaturated lipids. Our data support a metabolic connection between tanycytes and astrocytes likely to impact hypothalamic lipid sensing. GLIA 2017;65:231-249.

Electroacupuncture relieves neuropathic pain via upregulation of glutamate transporters in the spinal cord of rats.

Glutamate transports (GTs), the only vehicle for removal of glutamate from the extracellular fluid, is reported to be related to chronic pain. To investigate whether the glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) participate in electroacupuncture (EA) analgesia, the EA effect was observed with paw withdraw threshold in a rat model of spared nerve injury. The expression levels of GLAST and GLT-1 were determined with Western Blot and RT-PCR. The results showed significantly upregulated GLAST and GLT-1, along with the relieved pain behaviors after EA treatment. In addition, intrathecal injection of GTs inhibitor, l-trans-pyrrolidine-2-4-dicarboxylate, attenuated the EA-induced analgesic effect. The experiment demonstrates that EA can increase the GTs of neuropathic pain rats, which might be one of the mechanisms underlying its effectiveness in the neuropathic pain.

1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells.

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.

Tea Polyphenols Protect Against Methylmercury-Induced Cell Injury in Rat Primary Cultured Astrocytes, Involvement of Oxidative Stress and Glutamate Uptake/Metabolism Disorders.

Methylmercury (MeHg) is an extremely dangerous environmental contaminant, accumulating preferentially in CNS and causing a series of cytotoxic effects. However, the precise mechanisms are still incompletely understood. The current study explored the mechanisms that contribute to MeHg-induced cell injury focusing on the oxidative stress and Glu uptake/metabolism disorders in rat primary cultured astrocytes. Moreover, the neuroprotective effects of tea polyphenols (TP), a natural antioxidant, against MeHg cytotoxicity were also investigated. Astrocytes were exposed to 0, 2.5, 5, 10, and 20 µM MeHgCl for 6-30 h, or pretreated with 50, 100, 200, and 400 µM TP for 1-12 h; cell viability and LDH release were then determined. For further experiments, 50, 100, and 200 µM of TP pretreatment for 6 h followed by 10 µM MeHgCl for 24 h were performed for the examination of the responses of astrocytes, specifically addressing NPSH levels, ROS generation, ATPase activity, the expressions of Nrf2 pathway as well as Glu metabolism enzyme GS and Glu transporters (GLAST and GLT-1). Exposure of MeHg resulted in damages of astrocytes, which were shown by a loss of cell viability, and supported by high levels of LDH release, morphological changes, apoptosis rates, and NPSH depletion. In addition, astrocytes were sensitive to MeHg-mediated oxidative stress, a finding that is consistent with ROS overproduction; Nrf2 as well as its downstream genes HO-1 and γ-GCSh were markedly upregulated. Moreover, MeHg significantly inhibited GS activity, as well as expressions of GS, GLAST, and GLT-1. On the contrary, pretreatment with TP presented a concentration-dependent prevention against MeHg-mediated cytotoxic effects of astrocytes. In conclusion, the findings clearly indicated that MeHg aggravated oxidative stress and Glu uptake/metabolism dysfunction in astrocytes. TP possesses some abilities to prevent MeHg cytotoxicity through its antioxidative properties.

Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses.

Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw)) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.

Transcriptional Regulation of the Astrocytic Excitatory Amino Acid Transporter 1 (EAAT1) via NF-κB and Yin Yang 1 (YY1).

Astrocytic glutamate transporter excitatory amino acid transporter (EAAT) 1, also known as glutamate aspartate transporter (GLAST) in rodents, is one of two glial glutamate transporters that are responsible for removing excess glutamate from synaptic clefts to prevent excitotoxic neuronal death. Despite its important role in neurophysiological functions, the molecular mechanisms of EAAT1 regulation at the transcriptional level remain to be established. Here, we report that NF-κB is a main positive transcription factor for EAAT1, supported by the following: 1) EAAT1 contains two consensus sites for NF-κB, 2) mutation of NF-κB binding sites decreased EAAT1 promoter activity, and 3) activation of NF-κB increased, whereas inhibition of NF-κB decreased EAAT1 promoter activity and mRNA/protein levels. EGF increased EAAT1 mRNA/protein levels and glutamate uptake via NF-κB. The transcription factor yin yang 1 (YY1) plays a role as a critical negative regulator of EAAT1, supported by the following: 1) the EAAT1 promoter contains multiple consensus sites for YY1, 2) overexpression of YY1 decreased EAAT1 promoter activity and mRNA/protein levels, and 3) knockdown of YY1 increased EAAT1 promoter activity and mRNA/protein levels. Manganese decreased EAAT1 expression via YY1. Epigenetic modifiers histone deacetylases (HDACs) served as co-repressors of YY1 to further decrease EAAT1 promoter activity, whereas inhibition of HDACs reversed manganese-induced decrease of EAAT1 expression. Taken together, our findings suggest that NF-κB is a critical positive regulator of EAAT1, mediating the stimulatory effects of EGF, whereas YY1 is a negative regulator of EAAT1 with HDACs as co-repressors, mediating the inhibitory effects of manganese on EAAT1 regulation.

Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

Postnatal stress alters stress responses for life, with serious consequences on the central nervous system (CNS), involving glutamatergic neurotransmission and development of voluntary alcohol intake. Several drugs of abuse, including alcohol and cocaine, alter glutamate transport (GluT). Here, we evaluated effects of chronic postnatal stress (CPS) on alcohol intake and brain glutamate uptake and transporters in male adolescent Wistar rats. For CPS from postnatal day (PD) 7, pups were separated from their mothers and exposed to cold stress (4 °C) for 1 h daily for 20 days; controls remained with their mothers. Then they were exposed to either voluntary ethanol (6%) or dextrose (1%) intake for 7 days (5-7 rats per group), then killed. CPS: (1) increased voluntary ethanol intake, (2) did not affect body weight gain or produce signs of toxicity with alcohol exposure, (3) increased glutamate uptake by hippocampal synaptosomes in vitro and (4) reduced protein levels (Western measurements) in hippocampus and frontal cortex of glial glutamate transporter-1 (GLT-1) and excitatory amino-acid transporter-3 (EAAT-3) but increased glutamate aspartate transporter (GLAST) levels. We propose that CPS-induced decrements in GLT-1 and EAAT-3 expression levels are opposed by activation of a compensatory mechanism to prevent excitotoxicity. A greater role for GLAST in total glutamate uptake to prevent enlarged extracellular glutamate levels is inferred. Although CPS strongly increased intake of ethanol, this had little impact on effects of CPS on brain glutamate uptake or transporters. However, the impact of early life adverse events on glutamatergic neurotransmission may underlie increased alcohol consumption in adulthood.

Chemically functionalized single-walled carbon nanotubes enhance the glutamate uptake characteristics of mouse cortical astrocytes.

Using a radioactive glutamate uptake assay and immunolabeling, we report that single-walled carbon nanotubes, chemically functionalized with polyethylene glycol (SWCNT-PEG), delivered as a colloidal solute, cause an increase in the uptake of extracellular glutamate by astrocytes and an increase in the immunoreactivity of the glutamate transporter GLAST on their cell surface, which is likely a consequence of an increase in the immunoreactivity of glial fibrillary acidic protein. Additional corollary is that astrocytes exposed to SWCNT-PEG became larger and stellate, morphological characteristics of maturation and heightened activity of these glial cells. These results imply that SWCNT-PEG could potentially be used as a viable candidate for neural prosthesis applications, perhaps to alleviate the death toll of neurons due to glutamate excitotoxicity, a pathological process observed in brain and spinal cord injuries.

Adaptive changes of extracellular amino acid concentrations in mouse dorsal striatum by 4-AP-induced cortical seizures.

The striatum is a major target of cerebral cortical output. The cortico-striatal projection has been well described, however, the neurochemical changes that occur in the striatum after prolonged cortical hyperactivation remain to be investigated. In this study, extracellular levels of glutamate, GABA, and alanine levels were measured in the dorsal striatum using microdialysis in anesthetized mice at resting condition and during 4-aminopyridine (4-AP)-induced cortical seizures. After topical application of 4-AP on the primary motor cortex that induced cortical seizures, the extracellular level of striatal GABA increased by 40% in 60 min. By contrast, the extracellular level of striatal glutamate decreased by 20%. Moreover, the surface amounts of striatal glutamate/aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1), the major astrocytic high-affinity glutamate transporters, tended to increase by cortical seizures in 60 min, suggesting a recruitment of the glutamate transporters from internal stores. 4-AP also resulted in a steady increase of alanine levels which are thought to reflect glutamate and pyruvate metabolism in neurons and astrocytes. These observations possibly delineate adaptive changes of striatal metabolism by severe cortical seizures.

Alteration in 5-HT2C, NMDA receptor and IP3 in cerebral cortex of epileptic rats: restorative role of Bacopa monnieri.

Bacopa monnieri is effective in stress management, brain function and a balanced mood. 5-HT2C receptors have been implicated in stress whereas NMDA receptors and mGlu5 play crucial role in memory and cognition. In the present study, we investigated the role of B. monnieri extract in ameliorating pilocarpine induced temporal lobe epilepsy through regulation of 5-HT2C and NMDA receptors in cerebral cortex. Our studies confirmed an increased 5-HT2C receptor function during epilepsy thereby facilitating IP3 release. We also observed an decreased NMDA receptor function with an elevated mGlu5 and GLAST gene expression in epileptic condition indicating the possibility for glutamate mediated excitotoxicity. These alterations lead to impaired behavioural functions as indicated by the Elevated Plus maze test. Carbamazepine and B. monnieri treatments to epileptic rats reversed the alterations in 5-HT2C, NMDA receptor functions and IP3 content thereby effectively managing the neurotransmitter balance in the cerebral cortex.

Yokukansan, a traditional Japanese medicine, adjusts glutamate signaling in cultured keratinocytes.

Glutamate plays an important role in skin barrier signaling. In our previous study, Yokukansan (YKS) affected glutamate receptors in NC/Nga mice and was ameliorated in atopic dermatitis lesions. The aim of this study was to assess the effect of YKS on skin and cultured human keratinocytes. Glutamate concentrations in skin of YKS-treated and nontreated NC/Nga mice were measured. Then, glutamate release from cultured keratinocytes was measured, and extracellular glutamate concentrations in YKS-stimulated cultured human keratinocytes were determined. The mRNA expression levels of NMDA receptor 2D (NMDAR2D) and glutamate aspartate transporter (GLAST) were also determined in YKS-stimulated cultured keratinocytes. The glutamate concentrations and dermatitis scores increased in conventional mice, whereas they decreased in YKS-treated mice. Glutamate concentrations in cell supernatants of cultured keratinocytes increased proportionally to the cell density. However, they decreased dose-dependently with YKS. YKS stimulation increased NMDAR2D in a concentration-dependent manner. Conversely, GLAST decreased in response to YKS. Our findings indicate that YKS affects peripheral glutamate signaling in keratinocytes. Glutamine is essential as a transmitter, and dermatitis lesions might produce and release excess glutamate. This study suggests that, in keratinocytes, YKS controls extracellular glutamate concentrations, suppresses N-methyl-D-aspartate (NMDA) receptors, and activates glutamate transport.

[Effect of electroacupuncture intervention on behavioral changes and hippocampal excitatory amino acid transporter mRNA expression in depression rats].

OBJECTIVE: To observe the influence of music-electroacupuncture (EA) and pulse EA on locomotor activity and hippocampal neuron numbers and excitatory amino acid transporters (EAATs) mRNA expression in depression rats, so as to explore their mechanisms underlying improvement of depression in acupuncture clinic. METHOD: Sixty SD rats were randomly divided into blank control, depression model, medication, pulse EA and music-EA groups, with 12 rats in each group. Depression model was established by using chronic unpredictable mild stress protocols combined with solitary feeding. Rats of the medication group were treated by oral administration of fluoxetine (a standard antidepressant, 2 mg/kg). EA was applied to "Baihui" (GV 20) and "Yintang" (EX-HN 3) for 20 min, once a day for 21 days. The animals' ethological changes were detected by using field tests for recording the number of crossing squares and the number of rearing. The number of hippocampal neurons were calculated under microscope after Nissl's staining of the hippocampal tissue section. Hippocampal EAAT 1 and EAAT 2 mRNA expression levels were assayed by fluorescent quantitative real-time POR. RESULTS: In comparison with the blank control group, the numbers of crossing and rearing movements, as well as the expression levels of hippocampal EAAT 1 and EAAT 2 mRNA were significantly lower in the model group (P < 0.01), while compared with the model group, the numbers of both crossing and rearing movements of the medication, pulse EA and music-EA groups were obviously increased (P < 0.01). Correspondingly, the expression levels of hippocampal EAAT 1 and EAAT 2 mRNA were considerably upregulated in the pulse EA and music-EA groups in comparison with the model group (P < 0.01). No significant differences were found among the medication, pulse EA and music-EA groups (P > 0.05). Results of Nissl's staining displayed that in the model group, the hippocampal neurons were loose in arrangement and some of them were absent, but in the medication, pulse EA and music-EA groups, the neurons were clear in shape and lined in order. CONCLUSION: Both pulse EA and music-EA can effectively improve the locomotor activity and hippocampal neuronal structure in depression rats which may be closely associated with their effects in upregulating hippocampal EAAT 1 and EAAT 2 mRNA expression.

α-Tanycytes of the adult hypothalamic third ventricle include distinct populations of FGF-responsive neural progenitors.

Emerging evidence suggests that new cells, including neurons, can be generated within the adult hypothalamus, suggesting the existence of a local neural stem/progenitor cell niche. Here, we identify α-tanycytes as key components of a hypothalamic niche in the adult mouse. Long-term lineage tracing in vivo using a GLAST::CreER(T2) conditional driver indicates that α-tanycytes are self-renewing cells that constitutively give rise to new tanycytes, astrocytes and sparse numbers of neurons. In vitro studies demonstrate that α-tanycytes, but not ß-tanycytes or parenchymal cells, are neurospherogenic. Distinct subpopulations of α-tanycytes exist, amongst which only GFAP-positive dorsal α2-tanycytes possess stem-like neurospherogenic activity. Fgf-10 and Fgf-18 are expressed specifically within ventral tanycyte subpopulations; α-tanycytes require fibroblast growth factor signalling to maintain their proliferation ex vivo and elevated fibroblast growth factor levels lead to enhanced proliferation of α-tanycytes in vivo. Our results suggest that α-tanycytes form the critical component of a hypothalamic stem cell niche, and that local fibroblast growth factor signalling governs their proliferation.