Glutamate transporter type 3 knockout reduces brain tolerance to focal brain ischemia in mice

Excitatory amino-acid transporters (EAATs) transport glutamate into cells under physiologic conditions. Excitatory amino-acid transporter type 3 (EAAT3) is the major neuronal EAAT and also uptakes cysteine, the rate-limiting substrate for synthesis of glutathione. Thus, we hypothesize that EAAT3 contributes to providing brain ischemic tolerance. Male 8-week-old EAAT3 knockout mice on CD-1 mouse gene background and wild-type CD-1 mice were subjected to right middle cerebral artery occlusion for 90 minutes. Their brain infarct volumes, neurologic functions, and brain levels of glutathione, nitrotyrosine, and 4-hydroxy-2-nonenal (HNE) were evaluated. The EAAT3 knockout mice had bigger brain infarct volumes and worse neurologic deficit scores and motor coordination functions than did wild-type mice, no matter whether these neurologic outcome parameters were evaluated at 24 hours or at 4 weeks after brain ischemia. The EAAT3 knockout mice contained higher levels of HNE in the ischemic penumbral cortex and in the nonischemic cerebral cortex than did wild-type mice. Glutathione levels in the ischemic and nonischemic cortices of EAAT3 knockout mice tended to be lower than those of wild-type mice. Our results suggest that EAAT3 is important in limiting ischemic brain injury after focal brain ischemia. This effect may involve attenuating brain oxidative stress.     

Differential expression of glutamate transporters EAAT1 and EAAT2 in mice deficient for PACAP-type I receptor

Summary. Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic neurotransmission and induces the expression of glutamate transporters EAAT1 and EAAT2 in newborn mouse astroglial cell cultures. Since nanomolar concentrations of PACAP exert this effect, signal transduction via the high affinity PACAP-type I-receptor PAC1 was assumed. To test this hypothesis and to assess the importance of PAC1-signalling in vivo, we analyzed glutamate transporter expression in mice with a PAC1 knockout.EAAT1 and EAAT2 expression was investigated in the hippocampus and the cerebral cortex of PAC1 mutant mice and wildtype littermates by semiquantitative in-situ-hybridization.PAC1-knockout mice show a subtle but significant reduction of EAAT1 expression in the dentate gyrus. In contrast, reduced expression levels of EAAT1 in the cerebral cortex did not reach statistical significance and EAAT2 expression was unchanged in CA3 and cerebral cortex of PAC1 mutant mice.Our data confirm the previously reported in-vitro-regulation of EAAT1 in the adult nervous system in vivo. EAAT2 expression, however, is unchanged in PAC1 knockout mice, most likely due to counterbalancing factors.     

Expression of EAAT2 in neurons and protoplasmic astrocytes during human cortical development

The major regulators of synaptic glutamate in the cerebral cortex are the excitatory amino acid transporters 1–3 (EAAT1–3). In this study, we determined the cellular and temporal expression of EAAT1–3 in the developing human cerebral cortex. We applied single‐ and double‐label immunocytochemistry to normative frontal or parietal (associative) cortex samples from 14 cases ranging in age from 23 gestational weeks to 2.5 postnatal years. The most striking finding was the transient expression of EAAT2 in layer V pyramidal neuronal cell bodies up until 8 postnatal months prior to its expression in protoplasmic astrocytes at 41 postconceptional weeks onward. EAAT2 was also expressed in neurons in layer I (presumed Cajal–Retzius cells), and white matter (interstitial) neurons. This expression in neurons in the developing human cortex contrasts with findings by others of transient expression exclusively in axon tracts in the developing sheep and rodent brain. With western blotting, we found that EAAT2 was expressed as a single band until 2 postnatal months, after which it was expressed as two bands. The expression of EAAT2 in pyramidal neurons during human brain development may contribute to cortical vulnerability to excitotoxicity during the critical period for perinatal hypoxic–ischemic encephalopathy. In addition, by studying the expression of EAAT1 and EAAT2 glutamate transporters, it was possible to document the development of protoplasmic astrocytes. J. Comp. Neurol. 520:3912–3932, 2012. © 2012 Wiley Periodicals, Inc.     

Glutamate transporter type 3 mediates isoflurane preconditioning-induced acute phase of neuroprotection in mice

A pre-exposure to isoflurane reduces ischemic brain injury in rodents (isoflurane preconditioning). This neuroprotection has acute and delayed phases. Our previous in vitro studies suggest that the acute phase may involve excitatory amino acid transporters (EAATs). We determine whether this protection involves EAAT3, the major neuronal EAAT. Adult male EAAT3 knockout mice and their wild-type littermates were exposed or were not exposed to 1.5% isoflurane for 30 min. Sixty minutes later, they were subjected to a 90- or 60-min middle cerebral arterial occlusion (MCAO). Their neurological outcomes were evaluated 24 h after the MCAO. In another experiment, cerebral cortex was harvested for Western blotting at 30 min after animals were exposed to 1.5% isoflurane for 30 min. Here, we showed that isoflurane reduced brain infarct volumes and improved neurological functions of wild-type mice after a 90-min MCAO. However, isoflurane pre-exposure did not change the neurological outcome of EAAT3 knockout mice no matter whether the MCAO was for 90 min or 60 min. Isoflurane increased phospho-Akt, a survival-promoting protein, in the wild-type mice but not in the EAAT3 knockout mice. The isoflurane-induced neuroprotection in the wild-type mice was abolished by LY294004, an Akt activation inhibitor. LY294004 alone did not affect the neurological outcome of the wild-type or EAAT3 knockout mice after focal brain ischemia. These results suggest that the isoflurane preconditioning-induced acute phase of neuroprotection involves EAAT3. The downstream event includes Akt activation.     

ARIP1,2 mRNA及其蛋白在小鼠脑组织表达和分布的比较研究

目的比较新发现的激活素受体相互作用蛋白1,2(ARIP1,2)在小鼠脑组织的表达与分布。方法采用Northern杂交检测ARIP1,2 mRNA,Western杂交及免疫组化染色检测ARIP1,2蛋白。结果Northern杂交显示ARIP1,2 mRNA在小鼠组织中的表达形式明显不同,ARIP1主要在脑组织表达,ARIP2组织表达广泛。Western杂交进一步揭示ARIP1在大脑、小脑、海马、下丘脑及垂体表达,而ARIP2在大脑、小脑、海马、下丘脑、垂体及脾脏均有不同程度表达。免疫组化染色显示,ARIP1,2成熟蛋白在大脑、小脑及垂体均有表达,ARIP1在大脑和小脑皮质主要表达在小细胞神经元,而ARIP2主要表达在大细胞神经元。ARIP1在神经垂体和腺垂体表达水平明显高于ARIP2。结论ARIP1,2均表达于脑和垂体,但其表达的细胞类型和强度明显不同,这种差异可能与其在神经细胞中介导激活素生物学活性差异有关。     

GLT-1 expression and Glu uptake in rat cerebral cortex are increased by phencyclidine

Using western blottings, microdialysis, and functional assays we tested the hypothesis that phencyclidine (PCP) modifies the expression and function of glutamate (Glu) transporters in the rat frontal cortex. Western blotting studies revealed that administration of PCP (10 mg/kg/day; 7 days) increased significantly the expression of the astrocytic Glu transporter GLT-1/EAAT2. Functional studies showed that PCP increased significantly Na+-dependent Glu uptake in slices and in neuron/astrocyte co-cultures, and microdialysis studies evidenced that PCP treatment reduced basal Glu output. In our experimental conditions, PCP did not induce toxicity. These studies show that PCP increases the expression of GLT-1 in the cerebral cortex, thereby increasing Glu uptake and reducing extracellular [Glu].     

Neuronal damage and changes in the expression of muscarinic acetylcholine receptor subtypes in the neonatal rat cerebral cortical upon exposure to sparteine, a quinolizidine alkaloid

Sparteine is a quinolizidine alkaloid (QA) produced by Lupine species that has generated much interest due to its anti-hypertensive, anti-pyretic, and anti-inflammatory properties. In the nervous system, sparteine has been shown to display anti-cholinergic and depressive activity, although how sparteine exerts its toxic effects in the brain remains unclear. We have addressed this issue by administering subcutaneous injections of sparteine (25 mg/kg of body weight) to rats on postnatal days 1 and 3, and then examining the expression of the muscarinic acetylcholine receptor (mAChR) subunits m1-m4 in the brains of the neonatal rats 14-60 days later. Administration of sparteine to neonatal rats caused neuronal damage in the cerebral motor cortex accompanied by transient changes in the expression of m1-m4 mAChR subunits as revealed by both RT-PCR and Western blotting. This effect could be prevented by pre-treatment with atropine (10 mg/kg) 1 h prior to the injection of sparteine, suggesting that the cytotoxic activity of sparteine is mediated through mAChRs.     

Distribution of the VIP-like immunoreactive neurons in the cat central nervous system

Immunohistochemical topographic localization of the vasoactive intestinal polypeptide (VIP)-like immunoreactive neurons in the cat brain was investigated using a peroxidase anti-peroxidase technique. VIP-like immunoreactive neurons were mainly localized in the cerebral cortex, limbic cortex, hypothalamic nuclei; suprachiasmatic nucleus, supraoptic nucleus, paraventricular nucleus, periventricular nucleus and arcuate nucleus, and in the midbrain; such as the central grey and the raphe nucleus. It was demonstrated that VIP-like immunoreactive neurons were widely distributed in the cat brain, particularly in the hypothalamus, compared with those of the rat and mouse; though whether these differences were species-related or due to differences in the physiological conditions remains to be determined. This is the first report of VIP neuronal perikarya in the arcuate nucleus of mammalian species, although these cells are present in the arcuate nucleus of birds.     

Increased expression of glutamate transporters in subcortical white matter after transient focal cerebral ischemia

Transient focal cerebral ischemia leads to extensive excitotoxic glial damage in the subcortical white matter. Efficient reuptake of released glutamate is essential for preventing glutamate receptor overstimulation and neuronal and glial death. The present study evaluates the expression of the main glutamate transporters (EAAT1, EAAT2, and EAAT3) in subcortical white matter of the rat after transient middle cerebral artery occlusion. Western blot analysis and immunohistochemistry show an increase in the expression of EAAT1 and EAAT2 in subcortical white matter early after ischemia which subsequently decreases at longer reperfusion periods. However, expression of both EAAT1 and EAAT2 remains higher in astrocytes forming the gliotic scar and in microglial/macrophage cells at the border of or within the infarct area, respectively. Taken together, these results indicate that there is a transient enhanced expression of EAATs in the subcortical white matter early after ischemia. Our findings reveal an adaptive response of subcortical white matter to increased levels of glutamate during focal cerebral ischemia which may limit excitotoxic damage.     

Glutamate transporter EAAT2 expression is up-regulated in reactive astrocytes in human periventricular leukomalacia

The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% +/- 5.6%) compared with control white matter (21.4% +/- 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation.     

Decrease of aquaporin‐4 and excitatory amino acid transporter‐2 indicate astrocyte dysfunction for pathogenesis of cortical degeneration in HIV‐associated neurocognitive disorders

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV‐associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter‐2 (EAAT‐2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian‐human immunodeficiency virus (SHIV)‐infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543‐3‐infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV‐infected macaques, three SIVsm543‐3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte‐specific protein aquaporin‐4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV‐infected macaques, which was associated with EAAT‐2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT‐2. Some astrocytes express EAAT‐2 but not AQP4, and decrease of EAAT‐2 expression tended to be less than the decrease of AQP4. Active‐caspase‐3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT‐2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT‐2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT‐2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.     

Differences in routing of pelvic visceral afferent fibers in the dog and cat

Averaged evoked potentials were recorded after stimulation of nerve fibers to the pelvic viscera in nine dogs and six cats. Bladder visceral afferent fibers reach the spinal cord via pelvic nerves and hypogastric nerves in both dog and cat. However, only hypogastric neural activity is relayed by the spinal cord to the cerebral cortex in the dog, whereas both hypogastric and pelvic neural activity are relayed by the spinal cord to the cerebral cortex in the cat. Neural activity from the urethra, rectum, and genitalia ascends to the cord by the pelvic nerve and then is relayed by the spinal cord to the cerebral cortex in both dog and cat.     

Loss of astrocytic glutamate transporters in Wernicke encephalopathy

Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age‐matched controls, concomitant with decreases in α‐internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte‐specific proteins. Treatment of TD rats with N‐acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE. © 2009 Wiley‐Liss, Inc.     

The glutamate transporter EAAT2 is transiently expressed in developing human cerebral white matter

The major brain abnormality underlying cerebral palsy in premature infants is periventricular leukomalacia (PVL), a lesion of the immature cerebral white matter. Oligodendrocyte precursors (pre-OLs; O4(+)O1(-)) predominate in human cerebral white matter during the peak time frame for PVL (24-32 gestational weeks) and are vulnerable to excitotoxicity. We hypothesize that PVL reflects, in part, excitotoxicity to pre-OLs resulting from cerebral ischemia/reperfusion. Reversal of glutamate transport in the setting of energy failure is a major source of pathologic accumulation of extracellular glutamate. Here, we identify and localize the glutamate transporters in human cerebral white matter during the age range of PVL. In situ hybridization was performed with digoxigenin-labeled probes directed against the full-length coding regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA was abundant in human fetal white matter during the period of peak incidence of PVL and virtually disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting demonstrated that EAAT2 protein was highly expressed in early development relative to adult values. Double-label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the window of peak vulnerability for PVL, suggesting that this developmentally up-regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant.