灵芝多糖对癫痫大鼠脑中谷氨酸转运体的影响

目的：观察和探讨灵芝多糖干预后戊四氮（ PTZ）致痫大鼠皮质和海马区兴奋性谷氨酸转运体（EAAT）GLAST（EAAT1）、GLT1（EAAT2）、EAAC1（EAAT3）的变化,进一步研究癫痫的发病机制及灵芝多糖的作用机制。方法 SD 大鼠32只,随机分为正常对照组、癫痫模型组、灵芝多糖组和卡马西平组,每组8只。癫痫组、灵芝多糖组和卡马西平组采用 PTZ 腹腔注射制作慢性癫痫点燃模型。实验结束后断头迅速取脑,采用免疫组化法检测皮质和海马区各指标的变化。结果癫痫模型组大鼠脑中 GLAST、GLT1、EAAC1的表达较正常对照组降低：皮质（31.87±4.76）、（48.00±5.34）,（42.87±4.01）、（52.12±3.75）,（40.25±2.81）、（46.87±3.04）;海马：（29.87±4.32）、（44.51±4.81）,（36.50±3.02）、（47.00±3.20）,（35.62±3.42）、（42.12±3.56）;灵芝多糖和卡马西平组大鼠脑中 GLAST1、GLT1、EAAC1的表达较癫痫模型组升高：皮质（40.50±4.47）、（31.87±4.76）,（48.87±3.48）、（42.87±4.01）,（43.87±2.53）、（40.25±2.81）;海马：（37.75±3.61）、（29.87±4.32）,（41.25±2.60）、（36.50±3.02）,（39.50±2.61）、（35.62±3.42）。灵芝多糖组和卡马西平组之间 GLAST1、GLT1、EAAC1三者的表达无统计学意义。结论灵芝多糖能够升高癫痫大鼠脑中 GLAST、GLT1、EAAC1的表达,加速了兴奋性氨基酸 Glu（谷氨酸）的清除。降低神经元兴奋性、减少神经系统损伤而抑制癫痫的发作。     

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Prefrontal cortical network activity: Opposite effects of psychedelic hallucinogens and D1/D5 dopamine receptor activation

The fine-tuning of network activity provides a modulating influence on how information is processed and interpreted in the brain. Here, we use brain slices of rat prefrontal cortex to study how recurrent network activity is affected by neuromodulators known to alter normal cortical function. We previously determined that glutamate spillover and stimulation of extrasynaptic N-methyl-d-aspartic acid (NMDA) receptors are required to support hallucinogen-induced cortical network activity. Since microdialysis studies suggest that psychedelic hallucinogens and dopamine D1/D5 receptor agonists have opposite effects on extracellular glutamate in prefrontal cortex, we hypothesized that these two families of psychoactive drugs would have opposite effects on cortical network activity. We found that network activity can be enhanced by 2,5-dimethoxy-4-iodoamphetamine (DOI) (a psychedelic hallucinogen that is a partial agonist of 5-HT(2A/2C) receptors) and suppressed by the selective D1/D5 agonist SKF 38393. This suppression could be mimicked by direct activation of adenylyl cyclase with forskolin or by addition of a cAMP analog. These findings are consistent with previous work showing that activation of adenylyl cyclase can upregulate neuronal glutamate transporters, thereby decreasing synaptic spillover of glutamate. Consistent with this hypothesis, a low concentration of the glutamate transporter inhibitor threo-beta-benzoylaspartic acid (TBOA) restored electrically-evoked recurrent activity in the presence of a selective D1/D5 agonist, whereas recurrent activity in the presence of a low level of the GABA(A) antagonist bicuculline was not resistant to suppression by the D1/D5 agonist. The tempering of network UP states by D1/D5 receptor activation may have implications for the proposed use of D1/D5 agonists in the treatment of schizophrenia.     

Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement

Abstract : Background: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure. Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24–96 hr. Using two‐electrode voltage clamp, membrane currents were recorded after the application of L‐glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (μC). Results: Ethanol (10–100 mM) reduced EAAT3 activity in a time‐dependent and reversible manner. After a 96 hr‐incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr‐exposure to 50 mM ethanol significantly decreased Vmax (3.6 ± 0.3 for control versus 2.6 ± 0.3 μC for ethanol, n = 20, p < 0.05) but had no effect on Km (57.6 ± 12.8 for control versus 51.8 ± 16.3 μM for ethanol, n = 20, p > 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)‐treated oocytes were incubated with phorbol‐12‐myrisate‐13‐acetate (50 or 100 nM for 10 min), ethanol‐induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 μM chelerythrine significantly decreased EAAT3 activity (1.00 ± 0.08 for control versus 0.51 ± 0.09 μC for chelerythrine group, n = 18–20, p < 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 μM for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups. Conclusions: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C‐dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.     

Selective changes in the shapes of parasagittal bands of Aldoc (Zebrin) mRNA in the rat vermis of the cerebellum after repeated methamphetamine injections

In the cerebellum the mossy and climbing projections, which excite Purkinje cells, display a parasagittal and striped organization. These projections also excite Zebrin (aldolase C: Aldoc) parasagittally. To evaluate the possibility that external stimuli can change the organization of the bands of Aldoc mRNA, we compared the effects of repeated methamphetamine administration on the Aldoc mRNA stripes in the four transverse (anterior, central, posterior and nodular) regions of the vermis with the effects on the glutamate transporter EAAT4 (SCL1A 6) mRNA stripes. In the posterior region the injections four times daily increased the fragmentation of the Aldoc mRNA stripes. The presence of a large amount of fragmentation (forty/cerebellum slice), was accompanied with large lateral dislocations of the Aldoc mRNA stripes. In the central and nodular regions, where the size of the stripe areas decreased significantly the stripes were dislocated laterally. The dislocations of the Aldoc mRNA bands did not occur after a single methamphetamine injection and thus repeated injections were necessary to change the distributions of the lateral bands. In contrast, the distributions of the SCL1A 6 mRNA stripes did not change, even though there was mild fragmentation (six/slice) of the SLC1A 6 mRNA stripes in the anterior region and decreases in the numbers (twelve/slice) in the nodular region. We concluded that excess dopamine selectively changes the location of the Aldoc mRNA compartments in the vermis while the SLC1A 6 mRNA stripes could be changed by other inputs and thus the specific transmitter system might change the specific compartment of the cerebellum.     

Differential regulation of 5' splice variants of the glutamate transporter EAAT2 in an in vivo model of chemical hypoxia induced by 3-nitropropionic acid

Defective glutamate uptake has been implicated as a pathogenic event of neuronal damage related to cerebral ischemia and hypoxia. In several models of ischemia-hypoxia, a reduced immunoreactivity and altered RNA expression of excitatory amino acid transporter 2 (EAAT2), the major excitatory amino acid transporter, have been reported. However, the gene regulation of EAAT2 under these conditions is incompletely understood. In this study, we investigated alternative splicing of EAAT2 in an in vivo mouse model of chemical hypoxia as induced by 3-nitropropionic acid (3-NP). The neurotoxin 3-NP is an inhibitor of mitochondrial energy production. Furthermore, it is known to inhibit glutamate reuptake directly, representing at least one of the mechanisms responsible for 3-NP-induced neurodegeneration. Here we report an expression analysis of five known (mEAAT2/5UT1-5) and two novel (mEAAT2/5UT6, -7) 5' splice variants of EAAT2 using semiquantitative PCR. The RNA expression was studied at 2, 12, 24, 48, and 72 hr and 7 days after 3-NP administration. mEAAT2/5UT4 and mEAAT2/5UT5 were up-regulated in the frontal cortex and down-regulated in the hippocampus 12-72 hr after chemical hypoxia. In the cerebellum, there was an increased expression of mEAAT2/5UT4 and a down-regulation of mEAAT2/5UT5. mEAAT2/5UT3 show a different regional expression pattern, being regulated in the cerebellum only. mEAAT2/5UT1-7 encoded distinct 5' regulatory sequences, including conserved elements of translational control. It is easily conceivable that expression alterations of 5' splice variants of EAAT2 are related to glutamate transporter malfunction after chemical hypoxia. Our findings contribute to the hypothesis that RNA splicing events can serve as a molecular mechanism of posthypoxic gene regulation.     

Expression of glutamate transporter,GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knock-out mouse for Canavan disease

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the brain. The clinical features of CD are hypotonia, megalencephaly, and mental retardation leading to early death. While aspartoacylase (ASPA) activity increases with age in the wild type mouse brain, there is no ASPA activity in the CD mouse brain. So far ASPA deficiency and elevated NAA have been ascribed with the CD. Other factors affecting the brain that result from ASPA deficiency may lead pathophysiology of CD. The NMR spectra and amino acid analysis showed lower levels of glutamate and gamma-aminobutyric acid in the CD mouse brain compared to the wild type. Microarray gene expression on CD mouse brain showed glutamate transporter-EAAT4 and gamma-aminobutyric acid-A receptor, subunit alpha6 (GABRA6) were lower 9.7- and 119.1-fold, respectively. Serine proteinase inhibitor 2 (Spi2) was 29.9-fold higher in the CD mouse brain compared to the wild type. The decrease of GABRA6 and high expression of Spi2 in CD mouse brain were also confirmed by real-time RT-PCR. This first report showing abnormal expression of EAAT4, GABRA6, Spi2 combined with lower levels of glutamate and GABA are likely to be associated with the pathophysiology of CD.     

Analysis of cerebellar Purkinje cells using EAAT4 glutamate transporter promoter reporter in mice generated via bacterial artificial chromosome-mediated transgenesis

The EAAT4 glutamate transporter helps regulate excitatory neurotransmission and prevents glutamate-mediated excitotoxicity in the cerebellum. Immunohistochemistry and in situ hybridization have previously defined a cerebellar cell population expressing this protein. These methods, however, are not well suited for evaluating the dynamic regulation of the transporter and its gene-especially in living tissues. To better study EAAT4 expression and regulation, we generated bacterial artificial chromosome (BAC) promoter eGFP reporter transgenic mice. Histological analysis of the transgenic mice revealed that the EAAT4 promoter is active predominantly in Purkinje cells, but can also be modestly detected in other neurons early postnatally. EAAT4 promoter activity was not present in non-neuronal cells. Cerebellar organotypic slice cultures prepared from BAC transgenic mice provided a unique reagent to study transporter and Purkinje cell expression and regulation in living tissue. The correlation of promoter activity to protein expression makes the EAAT4 BAC promoter reporter a valuable tool to study regulation of EAAT4 expression.     

Enhanced expression of the high affinity glutamate transporter GLT-1 in C6 glioma cells delays tumour progression in rat

High grade gliomas are known to release excitotoxic concentrations of glutamate, a process thought to contribute to their malignant phenotype through enhanced autocrine stimulation of their proliferation and destruction of the surrounding nervous tissue. A model of C6 glioma cells in which expression of the high affinity glutamate transporter GLT-1 can be manipulated both in vivo and in vitro was used in order to investigate the consequences of increasing glutamate clearance on tumour progression. These cells were grafted in the striatum of Wistar rats and doxycycline was administered after validation of tumour development by magnetic resonance imaging. Both GLT-1 expression examined by immunohistochemistry and glutamate transport activity measured on synaptosomes appeared robustly increased in samples from doxycycline-treated animals. Moreover, these rats showed extended survival times as compared to vehicle-treated animals, an effect that was consistent with volumetric data revealing delayed tumour growth. As constitutive deficiency in glutamate clearance at the vicinity of brain tumours is well established, these data illustrate the potential benefit that could be obtained by enhancing glutamate transport by glioma cells in order to reduce their invasive behaviour.