Glutamine Supplementation Ameliorates Chronic Stress-induced Reductions in Glutamate and Glutamine Transporters in the Mouse Prefrontal Cortex

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.

Pharmacological Dissection of Intrinsic Optical Signal Reveals a Functional Coupling between Synaptic Activity and Astrocytic Volume Transient

The neuronal activity-dependent change in the manner in which light is absorbed or scattered in brain tissue is called the intrinsic optical signal (IOS), and provides label-free, minimally invasive, and high spatial (~100 µm) resolution imaging for visualizing neuronal activity patterns. IOS imaging in isolated brain slices measured at an infrared wavelength (>700 nm) has recently been attributed to the changes in light scattering and transmittance due to aquaporin-4 (AQP4)-dependent astrocytic swelling. The complexity of functional interactions between neurons and astrocytes, however, has prevented the elucidation of the series of molecular mechanisms leading to the generation of IOS. Here, we pharmacologically dissected the IOS in the acutely prepared brain slices of the stratum radiatum of the hippocampus, induced by 1 s/20 Hz electrical stimulation of Schaffer-collateral pathway with simultaneous measurement of the activity of the neuronal population by field potential recordings. We found that 55% of IOSs peak upon stimulation and originate from postsynaptic AMPA and NMDA receptors. The remaining originated from presynaptic action potentials and vesicle fusion. Mechanistically, the elevated extracellular glutamate and K⁺ during synaptic transmission were taken up by astrocytes via a glutamate transporter and quinine-sensitive K2P channel, followed by an influx of water via AQP-4. We also found that the decay of IOS is mediated by the DCPIB- and NPPB-sensitive anion channels in astrocytes. Altogether, our results demonstrate that the functional coupling between synaptic activity and astrocytic transient volume change during excitatory synaptic transmission is the major source of IOS.

A Patient Diagnosed with Spinocerebellar Ataxia Type 5 associated with SPTBN2: Case Report / 대한소아신경학회지

Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative disorders which disrupt the afferent and efferent pathways of the cerebellum that cause cerebellar ataxia. Spectrin beta non-erythrocytic 2 (SPTBN2) gene encodes the β-III spectrin protein with high expression in Purkinje cells that is involved in excitatory glutamate signaling through stabilization of the glutamate transporter, and its mutation is known to cause spinocerebellar ataxia type 5. Three years and 5 months old boy with delayed development showed leukodystrophy and cerebellar atrophy in brain magnetic resonance imaging (MRI). Diagnostic exome sequencing revealed that the patient has heterozygous mutation in SPTBN2 (p.Glu1251Gln) which is a causative genetic mutation for spinocerebellar ataxia type 5. With the patient's clinical findings, it seems reasonable to conclude that p.Glu1251Gln mutation of SPTBN2 gene caused spinocerebellar ataxia type 5 in this patient.

In Silico Model-driven Assessment of the Effects of Brain-derived Neurotrophic Factor Deficiency on Glutamate and Gamma-Aminobutyric Acid: Implications for Understanding Schizophrenia Pathophysiology

OBJECTIVE: Deficient brain-derived neurotrophic factor (BDNF) is one of the important mechanisms underlying the neuroplasticity abnormalities in schizophrenia. Aberration in BDNF signaling pathways directly or circuitously influences neurotransmitters like glutamate and gamma-aminobutyric acid (GABA). For the first time, this study attempts to construct and simulate the BDNF-neurotransmitter network in order to assess the effects of BDNF deficiency on glutamate and GABA. METHODS: Using CellDesigner, we modeled BDNF interactions with calcium influx via N-methyl-D-aspartate receptor (NMDAR)-Calmodulin activation; synthesis of GABA via cell cycle regulators protein kinase B, glycogen synthase kinase and β-catenin; transportation of glutamate and GABA. Steady state stability, perturbation time-course simulation and sensitivity analysis were performed in COPASI after assigning the kinetic functions, optimizing the unknown parameters using random search and genetic algorithm. RESULTS: Study observations suggest that increased glutamate in hippocampus, similar to that seen in schizophrenia, could potentially be contributed by indirect pathway originated from BDNF. Deficient BDNF could suppress Glutamate decarboxylase 67-mediated GABA synthesis. Further, deficient BDNF corresponded to impaired transport via vesicular glutamate transporter, thereby further increasing the intracellular glutamate in GABAergic and glutamatergic cells. BDNF also altered calcium dependent neuroplasticity via NMDAR modulation. Sensitivity analysis showed that Calmodulin, cAMP response element-binding protein (CREB) and CREB regulated transcription coactivator-1 played significant role in this network. CONCLUSION: The study presents in silico quantitative model of biochemical network constituting the key signaling molecules implicated in schizophrenia pathogenesis. It provides mechanistic insights into putative contribution of deficient BNDF towards alterations in neurotransmitters and neuroplasticity that are consistent with current understanding of the disorder.

Blockade of Trigeminal Glutamate Recycling Produces Anti-allodynic Effects in Rats with Inflammatory and Neuropathic Pain

The present study investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain or trigeminal neuropathic pain. Experiments were carried out on male Sprague-Dawley rats weighing between 230 and 280 g. Under anesthesia, a polyethylene tube was implanted in the atlanto-occipital membrane for intracisternal administration. IL-1β-induced inflammation was employed as an orofacial acute inflammatory pain model. IL-1β (10 ng) was injected subcutaneously into one vibrissal pad. We used the trigeminal neuropathic pain animal model produced by chronic constriction injury of the infraorbital nerve. DL-threo-β -benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block the spinal glutamate transporter and the glutamine synthetase activity in astroglia. Intracisternal administration of TBOA produced mechanical allodynia in naïve rats, but it significantly attenuated mechanical allodynia in rats with interleukin (IL)-1 β-induced inflammatory pain or trigeminal neuropathic pain. In contrast, intracisternal injection of MSO produced anti-allodynic effects in rats treated with IL-1β or with infraorbital nerve injury. Intracisternal administration of MSO did not produce mechanical allodynia in naive rats. These results suggest that blockade of glutamate recycling induced pro-nociception in naïve rats, but it paradoxically resulted in anti-nociception in rats experiencing inflammatory or neuropathic pain. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic pain conditions.

Reduction in hypoxia-derived neuroinflammation and dysfunctional glutamate transporters by minocycline may restore hypoxia-injured cognition of neonatal rat / 生理学报

The aim of the present study was to investigate the effects of minocycline on cognitive functions in neonatal rat after hypoxia exposure and the underlying mechanism. A model of hypoxic brain damage (HBD) was developed by exposing postnatal 1 day (P1) rats to systemic hypoxia. The rats were intraperitoneally injected with normal saline (Hy group) or minocycline (Hy + M group) 2 h after hypoxia exposure. Some other P1 rats that were not subjected to systemic hypoxia were used as normal control (NG group). The Y-maze test was used to evaluate learning and memory ability on postnatal day 30. Inflammatory mediators (Iba-1, IL-1β, TNF-α and TGF-β1), glutamate transporters (EAAT1 and EAAT2), total Tau and phosphorylated Tau (phosphorylation sites: Tyr18, Thr205, Thr231, Ser396 and Ser404) protein expressions in the hippocampus were detected by Western blot 7 d after hypoxic exposure. The results showed that hypoxia induced learning and memory impairments of the neonatal rats, and minocycline administration could reverse the effects of hypoxia. The protein expression levels of Iba-1, IL-1β, TNF-α, EAAT2 and Tau phosphorylated at T231 were increased, but the total Tau expression was decreased in the hippocampus of the rats from Hy group 7 d after hypoxia exposure. In the hypoxia-treated rats, minocycline down-regulated Iba-1, IL-1β, TNF-α and EAAT2 protein expressions significantly, but did not affect total Tau and phosphorylated Tau protein expressions. Our results suggest that minocycline can prevent cognitive deficits of rats with hypoxia exposure, and the underlying mechanism may involve the inhibition of neuroinflammation and dysfunctional glutamate transporters but not the regulation of the Tau hyperphosphorylation.

Absenteísmo-doença no serviço público municipal de Goiânia / Sickness absence in a municipal public service of Goiânia, Brazil

INTRODUÇÃO: O absenteísmo-doença, enquanto falta ao trabalho justificada por licença médica, é um importante indicador das condições de saúde dos trabalhadores. Em geral, características sociodemográficas e ocupacionais situam-se entre os principais fatores associados ao absenteísmo-doença. A administração pública é responsável por 21,8% dos empregos formais no Brasil. Esta população permite o estudo de uma grande variedade de categorias profissionais. OBJETIVO: Analisar o perfil e os indicadores de absenteísmo-doença entre servidores municipais de Goiânia, no Estado de Goiás, Brasil. Métodos: Estudo transversal das licenças certificadas para tratamento de saúde superiores a três dias, de todos os servidores, desde janeiro de 2005 a dezembro de 2010. Foram calculadas as prevalências, utilizando como critérios o número de indivíduos, os episódios e os dias de afastamento. RESULTADOS: Foram concedidas 40.578 licenças certificadas para tratamento de saúde a 13.408 servidores numa população média anual de 17.270 pessoas, o que resultou em 944.722 dias de absenteísmo. A prevalência acumulada de licença no período foi de 143,7%, com média anual de 39,2% e duração de 23 dias por episódio. A prevalência acumulada de absenteísmo-doença foi maior entre mulheres (52,0%) com idade superior a 40 anos (55,9%), com companheiro (49,9%), de baixa escolaridade (54,4%), profissionais de educação (54,7%), > 10 anos de serviço (61,9%) e múltiplos vínculos profissionais (53,7%). Os grupos de diagnósticos (CID-10) com as maiores prevalências acumuladas de licenças foram os do capítulo de transtornos mentais (26,5%), doenças osteomusculares (25,1%) e lesões (23,6%). CONCLUSÕES: Os indicadores de absenteísmo-doença expressam a magnitude desse fenômeno no serviço público e podem auxiliar no planejamento das ações de saúde do trabalhador, priorizando os grupos ocupacionais mais vulneráveis. .

BACKGROUND: Sickness absence, as work absenteeism justified by medical certificate, is an important health status indicator of the employees and, overall, sociodemographic and occupational characteristics are among the main factors associated with sickness absence. Public administration accounts for 21.8% of the formal job positions in Brazil. This population allows the study of a wide range of professional categories. OBJECTIVE: To assess the profile and indicators of sickness absence among public workers from the municipality of Goiania, in the State of Goiás, Brazil. METHODS: A cross-sectional study on certified sick leaves, lasting longer than three days, of all civil servants from January 2005 to December 2010. Prevalence rates were calculated using as main criteria the number of individuals, episodes and sick days. RESULTS: 40,578 certified sick leaves were granted for health treatment among 13,408 public workers, in an annual average population of 17,270 people, which resulted in 944,722 days of absenteeism. The cumulative prevalence of sick leave for the period was of 143.7%, with annual average of 39.2% and duration of 23 days per episode. The cumulative prevalence of sickness absence was higher among women (52.0%), older than 40 years old (55.9%), with a partner (49.9%), low schooling (54.4%), education professionals (54.7%), > 10 years of service (61.9%), and with multiple work contracts (53.7%). Diagnoses groups (ICD-10) with higher cumulative prevalence of sick leaves were those with mental disorders (26.5%), musculoskeletal diseases (25.1%), and injuries (23.6%). CONCLUSIONS: Indicators of sickness absence express the magnitude of this phenomenon in the public sector and can assist in planning health actions for the worker, prioritizing the most vulnerable occupational groups. .

Dietary Glutamate: Interactions With the Enteric Nervous System

BACKGROUND/AIMS: Digestion of dietary protein elevates intraluminal concentrations of glutamate in the small intestine, some of which gain access to the enteric nervous system (ENS). Glutamate, in the central nervous system (CNS), is an excitatory neurotransmitter. A dogma that glutamatergic neurophysiology in the ENS recapitulates CNS glutamatergic function persists. We reassessed the premise that glutamatergic signaling in the ENS recapitulates its neurotransmitter role in the CNS. METHODS: Pharmacological analysis of actions of receptor agonists and antagonists in concert with immunohistochemical localization of glutamate transporters and receptors was used. Analysis focused on intracellularly-recorded electrical and synaptic behavior of ENS neurons, on stimulation of mucosal secretion by secretomotor neurons in the submucosal plexus and on muscle contractile behavior mediated by musculomotor neurons in the myenteric plexus. RESULTS: Immunoreactivity for glutamate was expressed in ENS neurons. ENS neurons expressed immunoreactivity for the EAAC-1 glutamate transporter. Neither L-glutamate nor glutamatergic receptor agonists had excitatory actions on ENS neurons. Metabotropic glutamatergic receptor agonists did not directly stimulate neurogenic mucosal chloride secretion. Neither L-glutamate nor the metabotropic glutamatergic receptor agonist, aminocyclopentane-1,3-dicarboxylic acid (ACPD), changed the mean amplitude of spontaneously occurring contractions in circular or longitudinal strips of intestinal wall from either guinea pig or human small intestinal preparations. CONCLUSIONS: Early discoveries, for excitatory glutamatergic neurotransmission in the CNS, inspired enthusiasm that investigation in the ENS would yield discoveries recapitulating the CNS glutamatergic story. We found this not to be the case.

MrgC receptor activation reverses chronic morphine-evoked alterations of glutamate transporters and nNOS in rats / 生理学报

This study was aimed to investigate the mechanisms underlying the modulation effect of Mas-related gene (Mrg) C receptors (MrgC) on morphine tolerance. Saline, morphine (20 μg), morphine plus bovine adrenal medulla 8-22 (BAM8-22, 1 nmol) or (Tyr(6))-2-MSH-6-12 (MSH, 5 nmol) were administered intrathecally in rats for 6 days. Pain-related molecules in the spinal cord and dorsal root ganglion (DRG) were examined using Western blot, immunocytochemistry and RT-PCR techniques. The results showed that intrathecal administration of the selective MrgC receptor agonists (BAM8-22 or MSH) remarkably attenuated or abolished chronic morphine-evoked reduction in glutamate transporters (GLAST, GLT-1 and EAAC1) in the spinal cord and increase in neuronal nitric oxide synthase (nNOS) in the spinal cord as well as DRG. In addition, MrgC receptor-like immunoreactivity (IR) was detected in superficial laminae of the spinal cord. Chronic morphine induced significant increases in MrgC receptor-IR in the spinal cord and MrgC receptor mRNA levels in DRG. These results suggest that the modulation of pro-nociceptive mediators in the spinal cord and DRG underlies the inhibition of morphine tolerance by MrgC receptor activation.

Inibição do receptor de glutamato do tipo NMDA em um modelo de hipóxia-isquemia prenatal: avaliação morfofuncional do cerebelo / Inhibition of glutamate NMDA receptor in a prenatal hipoxia-ischemia model: morphofunctional analises of cerebellum

Lesões sistêmicas peri e pré-natais alteram o desenvolvimento do SNC, levando a problemas cognitivos e motores em crianças que podem perdurar por toda a vida. Um tipo particular de lesão é a hipóxia-isquemia (HI), caracterizada pela interrupção momentânea ou permanente do fluxo sanguíneo. Um dos mecanismos propostos para as lesões decorrentes da HI é a excitotoxicidade glutamatérgica. O uso de inibidores da neurotransmissão glutamatérgica tem sido estudados em diversos modelos de HI. Neste trabalho, avaliamos os efeitos morfofuncionais da administração de um antagonista não-competitivo do receptor de glutamato NMDA sobre o desenvolvimento do cerebelo. Ratas no 18º dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo H). Animais controle tiveram os úteros expostos, sem a obstrução (Grupo S). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um dia após o nascimento, metade de cada ninhada foi designada para receber MK801, 0,3mg/kg/dia, (grupos SM e HM) e a outra metade recebeu solução salina (grupos SS e HS), por 5 dias. Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23, 30 e 60 dias pós-natais, cortes parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para calbindina, GFAP, GLAST, PDGFRα e MBP. A partir de 45 dias de vida, os animais foram testados em vários de testes comportamentais: labirinto em cruz elevado (LCE), campo vazado (CV), ROTAROD, teste de caminhada sobre barras (ladder test) e teste do comprimento da passada (stride length). Aos 9 dias, a espessura da árvore dendrítica era menor nos animais SM, HS/HM, demonstrando efeitos deletérios tanto do MK801 quanto da HI. Menor número de células PDGFRα+ foi observado nos animais HS/HM, sem efeitos da administração de MK801. Aos 23 dias, maior número de células PDGFRα+ foi observado nos animais HM comparado aos outros 3 grupos, indicando efeito ...

Peri and prenatal systemic lesions alter CNS development leading to motor and cognitive problems in children that might persist throughout life. A particular kind of injury, the hypoxic ischemic (HI), is characterized by a permanent or temporary blockage of blood flow. One of the proposed mechanisms downstream from a HI event is called glutamatergic excitotoxicity. The administration of glutamate inhibitors has been studied in HI models for several years. In this work, we evaluated the effects of administration of a non-competitive antagonist of glutamate receptor, NMDA, on cerebellar development and behavioral tests of HI animals. Pregnant rats in the 18th gestational day were anesthetized, the uterine horns were exposed and the four uterine arteries were clamped for 45 minutes (group H). Sham controls had the uterine horns exposed, but no arteries were clamped (group S). Gestation proceeded after surgery. Only full term animals were used. One day after birth half the animals was assigned to receive either SALINE (groups SS and HS) or MK801 (groups SM and HM). Animals were anesthetized and perfused with 4% paraformaldehyde at 9, 23, 30 and 60 days of age. Parasagittal cerebellar sections were submitted to Calbindin, GFAP, GLAST, PDGFRα and MBP immunohistochemistry. Beginning at P45 animals were subjected to a battery of behavioral tests: elevated plus maze (EPM), hole board (HB), ROTAROD, ladder test and stride length. At P9 the dendritic tree of Purkinje cells were thinner in SM, HS/HM animals, indicating that both HI and MK801 are deleterious regarding this Purkinje cell differentiation. A lower number of PDGFRα+ cells was observed in HS/HM animals, with no effects of MK801 administration. At P23 a greater number of PDGFRα+ cells was found in HM animals when compared to the other 3 groups, demonstrating a neuroprotector effect of MK801. A lower number of myelinated fibers (MBP+) was observed in HS animals at P9, and MK801 administration reverse this ...

Suppressing SNAP-25 and reversing glial glutamate transporters relieves neuropathic pain in rats by ameliorating imbalanced neurotransmission / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neuropathic pain results from a lesion or disease affecting the somatosensory system at either the peripheral or central level. The transmission of nociception within the central nervous system is subject to modulation by release and reuptake of neurotransmitters, which maintain a dynamic balance through the assembly and disassembly of the SNARE complex as well as a series of neurotransmitter transporters (inhibitory GABA transporters GAT and excitatory glutamate transporters GT). Neuronal hyper-excitability or defected inhibition involved in neuropathic pain is one of the outcomes caused by imbalanced neurotransmission. SNAP-25, which is one of the SNARE complexes, can modulate the release of neurotransmitters. Glia glutamate transporter (GLT) is one of the two glutamate transporters which account for most synaptic glutamate uptake in the CNS. The role of SNAP-25 and GLT as well as GAT is not clearly understood.</p><p><b>METHODS</b>We used the rat chronic constriction injury (CCI) model for research, and degraded SNAP-25 by a single intrathecal administration of BoNT/A. The mechanical (MWT) and thermal withdrawal latency (TWL) were tested. The level of SNAP-25, GLT, and GAT-1 were assayed using RT-PCR and Western blotting.</p><p><b>RESULTS</b>SNAP-25 was suppressed by a single intrathecal administration of 0.01U BoNT/A and the reduction of SNAP- 25 was correlated with the relief of nociceptive responses in CCI rats. MWT and TWL returned to normal from the 5th to 14th day (P < 0.05) after the administration. On the 14th day after surgery, compared to the sham group, the upregulation of SNAP-25 in CCI rats was reversed after BoNT/A treatment (P < 0.05). The decreased GLT was reversed after BoNT/A treatment but increased GAT-1 was not influenced by BoNT/A treatment.</p><p><b>CONCLUSIONS</b>SNAP-25 and GLT play important roles in the development of neuropathic pain, and the mechanism may involve the imbalance of neurotransmission after peripheral nerve injury. Intrathecal administration of BoNT/A reversed the upregulation of SNAP-25 and downregulation of GLT after CCI, but had no significant effect on the expression of GAT-1.</p>

Alexander Disease

Alexander disease (ALXD) is a rare demyelinating disease of the white matter of the brain that is caused by a mutation in the glial fibrillary acidic protein (GFAP) gene. The overexpression of GFAP in astrocytes induces a failure in the developmental growth of the myelin sheath. The neurodegenerative destruction of the myelin sheath of the white matter is accompanied by an accumulation of abnormal deposits of Rosenthal fibers in astrocytes, which is the hallmark of ALXD. The disease can be divided into four groups based on the onset age of the patients: neonatal, infantile, juvenile, or adult. Early-onset disease is more severe, progresses rapidly, and results in a shorter life span than late-onset cases. Magnetic resonance imaging and genetic tests are mostly used for diagnostic purposes. Pathological tests of brain tissue for Rosenthal fibers are definitive diagnostic methods. Therapeutic strategies are being investigated. Ceftriaxone, which is an enhancer of glial glutamate transporter (GLT-1) expression, is currently in clinical trials for the treatment of patients with ALXD. To date, there are no clinically available treatments. The cause, pathology, pathophysiology, inheritance, clinical features, diagnosis, and treatment of ALXD will be reviewed comprehensively.

Effects of intrathecal injection of ginsenoside Rg1 on the level of glutamate transporter in the arthritis rats with chronic morphine tolerance / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effects of intrathecal injection of ginsenoside Rg1 at different doses on the changes of the behavior and the expressions of excitatory amino-acid transporter 1 (EAAT1), i. e., glutamate-aspartate transporter (GLAST) in the spinal dorsal horn of the arthritis rats with chronic morphine tolerance, and further to explore its mechanisms for morphine tolerance.</p><p><b>METHODS</b>After successful intrathecal injection, an adjuvant arthritis model was established in 36 healthy male SD rats. They were randomly divided into 6 groups, 6 in each group. They were intrathecally injected with 10 microL normal saline (Group NS), 10 microg morphine (Group M), 10 microg morphine + 50 microg ginsenoside Rg1 (Group MG50), 10 microg morphine +100 microg ginsenoside Rg1 (Group MG100), 10 microg morphine + 200 microg ginsenoside Rg1 (Group MG200), and 100 microg ginsenoside Rg1 (Group G100), respectively. The normal saline and morphine were intrathecally injected twice daily, while ginsenoside Rg1 at different doses was intrathecally injected once daily, for 7 successive days. Fifty percent mechanical paw withdrawal threshold (PWT) was dynamically detected to evaluate their behaviors. The rats were sacrificed on day 7 after medication. The L3-L5 segment of the spinal cord was isolated for determining the expression of GLAST in the spinal dorsal horn using immunofluorescence staining.</p><p><b>RESULTS</b>The PWT of Group M was significantly higher than that of Group NS on the 1st and 3rd day after medication (P < 0.05). But it was gradually shortened along with the increasing days of medication. There was no statistical difference between Group M and Group NS on the 7th day (P > 0.05), indicating the formation of morphine tolerance. The PWT of Group MG100 also showed a decreasing tendency, but obviously slower than that of Group M (P < 0.05). The PWT of Group G100 was higher than that of Group NS (P < 0.05). Compared with Group NS, the expression of GLAST in the spinal dorsal horn of rats in Group M was down-regulated (P < 0.01). Compared with Group M, the expression of GLAST in the spinal dorsal horn of rats in Group MG100 and Group G100 was up-regulated (P < 0.05).</p><p><b>CONCLUSIONS</b>Single application of ginsenoside Rg1 showed mild antinociceptive effect in adjuvant-induced arthritis rats. Intrathecal injection of 100 microg ginsenoside Rg1 could attenuate the formation of morphine tolerance. Its mechanisms might be correlated with up-regulating of the expression of GLAST.</p>

Effects of clonidine on the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes / 대한마취과학회지

BACKGROUND: Clonidine has been shown to be a potent neuroprotectant by acting at alpha2 receptors on glutamatergic neurons to inhibit the release of glutamate. The aim of this study is to investigate the effects of clonidine on the activity of EAAT3 that can regulate extracellular glutamate. METHODS: EAAT3 was expressed in the Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate both in the presence and absence of various concentrations of clonidine. To determine the effects of clonidine on the Km and Vmax of EAAT3 and the reversibility of clonidine effects, membrane currents were recorded after the application of various concentrations of L-glutamate both in the presence and absence of 1.50 x 10(-7) M clonidine. RESULTS: Clonidine reduced the EAAT3 responses to L-glutamate in a concentration-dependent manner. This inhibition was statistically significant at higher concentrations than at the clinically relevant range. Clonidine at 1.50 x 10(-7) M reduced the Vmax, but did not affect the Km of EAAT3 for L-glutamate. CONCLUSIONS: These results suggest that the direct inhibition of EAAT3 activity is not related to the sedation effect of clonidine and that the clonidine-induced reduction of EAAT3 activity provides additional data for the possible involvement of glutamatergic hyperactivity in the proconvulsant effect of clonidine.

Polymorphism of the Glutamate Transporter Protein EAAT2 and Migraine Transformation into Chronic Daily Headache

BACKGROUND AND PURPOSE: The progression of migraine into chronic daily headache involves multiple risk factors, but the main contributor is not known. Glutamate is the major excitatory neurotransmitter in central sensitization, which is an important process in the pathogenesis of migraine transformation. The glutamate transporter protein excitatory amino acid transporter 2 (EAAT2) is the primary modulator of glutamatergic neurotransmission, and genetic polymorphisms of its gene, EEAT2, have been identified. The aim of this study was to determine the effect of EAAT2 polymorphisms on migraine transformation into chronic daily headache. METHODS: We included 74 migraine patients with episodic attack (M-E) and 59 migraine patients with chronic daily headache (M-CDH). After amplifying EAAT2 by polymerase chain reaction, we assessed its genotype frequencies based on restriction fragment length polymorphisms. We reclassified all migraine patients into two groups according to their EAAT2 genotype, either with the A allele (n=62) or without it (n=71), and compared the clinical variables between the two groups. RESULTS: The genotype frequencies of EAAT2 polymorphisms did not differ between the M-E and M-CDH groups. Comparison between EEAT2 genotypes revealed that the frequency of analgesic usage was significantly higher among migraine patients with the A allele (12.9+/-1.6 days/month) than in those without the A allele (8.1+/-1.2 days/month; p=0.019). The other clinical variables of migraine did not differ between the two groups. CONCLUSIONS: The results suggest that EEAT2 polymorphism contributes to the tendency toward frequent analgesic usage in migraine patients. This implies a potential genetic influence on the progression of migraine into chronic daily headache through the development of medication-overuse headache.

Effect of propofol on oxidative stress-attenuated glutamate transporter EAAT4 activity

BACKGROUND: Propofol (2, 6-diisopropylphenol) has been known to have neuroprotective effects. Excitatory amino acid transporter 4 (EAAT4) is a glutamate transporter predominantly expressed in the cerebellar Purkinje cells, which is vulnerable to ischemic injury. Thus, we hypothesized that propofol reverses reduced EAAT4 activity which was induced by oxidative stress and investigated the effects of propofol on EAAT4 under oxidative stress induced by tert-butyl hydroperoside (t-BHP). METHODS: EAAT4 was expressed in Xenopus oocytes by injection of its mRNA. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-aspartate (3 microM) in the presence or absence of t-BHP and propofol. RESULTS: L-aspartate induced an inward current in EAAT4 expressing oocytes. Exposure of these oocytes to t-BHP (1-20 mM) for 10 min dose-dependently decreased EAAT4 activity (1 +/- 0.01 microC for control; 0.88 +/- 0.05 microC for 1 mM; 0.83 +/- 0.03 microC for 2mM; 0.65 +/- 0.04 microC for 3 mM; 0.51 +/- 0.07 microC for 5 mM; 0.45 +/- 0.03 f microC for 10 mM and 0.24 +/- 0.06 microC for 20 mM). IC50 for t-BTH was 6.05 mM and further study was performed with 10 mM t-BTH. Propofol (3-10 microM) dose-dependently reversed this t-BHP-attenuated EAAT4 activity. CONCLUSIONS: Oxidative stress by t-BHP decreased EAAT4 activity and 3-10 microM propofol restored oxidative stress-reduced EAAT4 activity.

The effects of hypoxic-ischemia on glutamate transporters / 生物医学工程学杂志

Glutamate is a kind of excitatory transmitter in the central nervous system of mammals; at the same time, being with excitatory toxicity, its extracellular concentration is mainly modulated by the glutamate transporters. In the case of hypoxic-ischemia, the energy failure would lead to the generation of lactate and free radical, and to the crash of [Na+] gradient, thus exerting influence on the activity and the expression of glutamate transporters. As a result, the glutamate accumulates in the synaptic cleft, activates the glutamate receptors, and causes the death of neurons.

Remifentanil increases the activity of the glutamate transporter, EAAC1, expressed in Xenopus oocytes

BACKGROUND: Remifentanil has gained wide clinical acceptance during anesthesia due to its short context-sensitive half time and organ-independent metabolism. However, its mechanism as an anesthetic remains unclear. Glutamate transporters may be important targets for anesthetic action in the central nervous system, and we tested whether remifentanil affected the activity of the primary neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1). METHODS: EAAC1 was expressed in Xenopus oocytes by mRNA injection. By using two-electrode voltage clamping, membrane currents were recorded before, during, and after application of L-glutamate (30microM) in the presence or absence of remifentanil. Oocytes were exposed to a protein kinase C (PKC) activator and inhibitor to study the role of PKC on EAAC1 activity. RESULTS: L-Glutamate induced an inward current in EAAC1-expressing oocytes. This response increased in a bell-shaped manner in the presence of 0.1microM to 1 mM remifentanil. Remifentanil significantly increased Vmax (3.1 +/- 0.2microC for controls vs. 4.9 +/- 0.3 microC for remifentanil treatment; n = 12-15; P < 0.05). However, remifentanil did not significantly change Km. Treatment of the oocytes with phorbol-12-myristate-13-acetate (PMA), a PKC activator, caused a significant increase in transporter current (1.00 +/- 0.03 to 1.35 +/- 0.03microC; P < 0.05). Oocytes pretreated with the PKC inhibitor alone (staurosporine) abolished remifentanilenhanced EAAC1 activity. CONCLUSIONS: Our data suggests that remifentanil enhances EAAC1 activity and that PKC is involved in mediating this effect.

study of glutamate transporter 3 [EAAT3] expression, serum TNF-alpha level, and NF-KB activity in ischemic tolerance induced by prolonged and intermittent normobaric hyperoxia

Recent studies suggest that intermittent and prolonged normobaric hyperoxia [HO] results in ischemic tolerance to preventing ischemia brain injury. In this research attempts were made to see the changes in excitatory amino-acid transporter 3 [EAAT3], TNF- alpha levels, and NF- kappa B activity following prolonged and intermittent NBHO preconditioning. Rats were divided into four experimental groups, each with 21 animals. The first two groups were exposed to 95% inspired HO for 4h/day for 6 consecutive days [intermittent HO; InHO] or for 24 continuous hours [prolonged HO; PrHO]. The second two groups acted as controls, and were exposed to 21% oxygen in the same chamber [nomobaric normoxia, RA; room air] continuously for six days [intermittent RA, InRA] or for 24 hours [prolonged RA; PrRA]. Each main group was subdivided to MCAO- operated [middle cerebral artery occlusion], sham-operated [without MCAO], and intact [without any surgery] subgroups. After 24 h, MCAO-operated subgroups were subjected to 60min of right MCAO. After 24h reperfusion, neurologic deficit score [NDS] were assessed in MCAO-operated subgroups. Immediately and 48 h after pretreatment, blood sampling for assessment of serum TNF- alpha levels were performed. Then, the effect of InHO and PrHO on serum TNF - alpha levels, NF - kappa B activity and EAAT3 expression were measured. Reconditioning with InHO and PrHO decreased NDS and upregulate EAAT3 and increase serum TNF- alpha level and NF- kappa B activity significantly. Although further studies are needed to clarify the mechanisms of ischemic tolerance, InHO and PrHo seems to partly exert their effects via increase in serum TNF- alpha levels, NF- kappa B activity and upregulation of glutamate transporters

Altered Expression of Parvalbumin in Neural Retina of the Insulin-dependent Diabetic Rat / 대한해부학회지

Calcium-binding proteins in the nervous system are functioned in Ca2+ buffering and Ca2+ transport and regulation of various enzyme systems. They potentially have a number of different effects on cells includingaltering the duration of action potentials, promoting neuronal bursting activity and protecting cells against the damaging effects of excessive calcium influx. The present study has been designed to clarify the differential responding patterns of parvalbumin immunoreactive neurons in the rat retina following diabetic injury, for better understandings of role of parvalbumin in the retinal circuitry and in calcium homeostasis. Experimental diabetes was induced by a single intravenous injection of streptozotocin in a dose of 60 mg/kg body weight. Diabetic rats showing high blood glucose levels (above 300 mg/dL) were cared for 1, 4, 8, 12 and 24 weeks, respectively. The retinas at each time point were processed for immunohistochemistry and Western blotting using antiparvalbumin antibody. In the rat retina at normal, parvalbumin immunoreactivity appeared in AII amacrine cells, amacrine cells of a widefield type and displaced amacrine cells. A few bipolar cells are also showed the reactivity. During diabetes, the intensity of parvalbumin immunoreactivity is decreased especially in the AII amacrine cells. The cell number of parvalbumin immunoreactive neurons has showed no large changes throughout the diabetes, except that of bipolar cells. That population of parv immunoreactive of bipolar cells has increased remarkably at later diabetic periods. The protein levels of parvalbumin have showed transiently a slight increase at earlier diabetic periods, and then decreased to lower than that of normal. Parvalbumin immunoreactive bipolar cells at diabetes are co-localized not with PKC-alpha or recoverin, but with glutamate transporter Glt-1b. AII amacrine cell processes were joined with each other and with axon terminals of presumed cone bipolar cells by gap junction. These results suggest that the calcium buffering activity of parvalbumin is shifted from AII amacrine cells to a certain type of cone bipolar cells, in response to diabetes. This event may be occurred through electrically coupled gap junction in between these cell processes.