Differential molecular regulation of glutamate in kindling resistant rats.

Using pentylenetetrazol (PTZ) kindling, we collected hippocampal tissue from standard response and kindling resistant animals, measuring hippocampal mRNA with real-time PCR of glutamate transporters GLAST, GLT-1, and EAAC1 and the sodium-coupled neutral amino acid transporter (SNAT) 1, SNAT2, and SNAT3. In addition, we measured mRNA of glutamine synthetase (GS), phosphate-activated glutaminase (PAG), glutamic acid decarboxylase (GAD) 1, GAD2, and vesicular inhibitory amino acid transporter (VIAAT). Fully kindled animals had decreased expression of mRNA in the hippocampus for GLAST and GAD2 compared with saline injected control. mRNA for SNAT1, SNAT2, SNAT3, GS, and VIAAT was increased. After induction of generalized tonic-clonic seizures by PTZ there were no differences in mRNA at 24h after seizures, equaling baseline quantities except for GAD1, which was decreased. When levels were measured at 30days after a PTZ induced convulsive seizure, we found increased levels of GLT-1, SNAT1 and GS, but decreased levels of GAD1. When these animals, serving as control for the 30day interval between the last convulsive seizure in the kindled experimental group, were analyzed, we found that GLT-1, SNAT3, GAD1 and VIAAT differed in that GLT-1 was decreased and the others increased. Animals found resistant to kindling had strikingly different mRNA patterns, with markedly up-regulated mRNA of proteins that transport glutamate into neurons and glia; SNAT1 was up regulated as well. Up-regulation of genes in kindling resistant animals supports the hypothesis that clearance of glutamate, conversion to glutamine and transport of glutamine into neurons, has the effect of raising the threshold for convulsive seizures and attenuating kindling.

Increased lipid peroxidation in Down's syndrome mouse models.

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

Levetiracetam enhances endogenous antioxidant in the hippocampus of rats: in vivo evaluation by brain microdialysis combined with ESR spectroscopy.

We have attempted to explore the neuroprotective effectiveness of levetiracetam (LEV) by measuring its in vivo antioxidant effect in the hippocampus of rats in a freely moving state. Male Wistar rats were used for the estimation of the in vivo antioxidant effect of LEV through microdialysis combined with electron spin resonance spectroscopy. The antioxidant effect was examined using the principle by which a systemically administered blood-brain barrier-permeable nitroxide radical (PCAM) decreases in an exponential decay manner that is correlated with the amount of antioxidant in the brain. The PCAM decay ratio during perfusion with normal Ringer's solution was compared with that during 32 microM and 100 microM LEV co-perfusion. The in vivo antioxidant effect was examined. In addition, the expressions of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS) protein related to redox regulation were measured in the hippocampus of rats after 14 days of administration of LEV at a dose of 54 mg/day i.p. The half-life of PCAM was statistically shortened after LEV perfusion compared with the results of the control experiment. While the expression of the pro-oxidant protein iNOS was decreased, that of the antioxidant protein xCT was statistically increased by the administration of LEV. The role of xCT is to transport cystine, the internal material of glutathione, into the cell. The shortened half-life of the nitroxide radical by co-perfusion of LEV with increased xCT and decreased iNOS expression revealed the enhancement of the endogenous antioxidant effect or free-radical scavenging activity. The results of this study suggest that LEV synergistically enhances the basal endogenous antioxidant effect in the hippocampus with ascorbic acid and alpha-tocopherol. Our findings further suggest that LEV exerts a neuroprotective role by 1) modifying the expression of xCT and iNOS in connection with lipid peroxidation, 2) synergistically enhancing the increased basal endogenous antioxidant ability in the hippocampus, and 3) decreasing the basal concentration of glutamate followed by up-regulation of the intake of cystine, an internal material of GSH.

Molecular regulation of antioxidant ability in the hippocampus of EL mice.

We recently found that the antioxidant ability was remarkably decreased in the hippocampus (Hipp) of EL at 8 weeks of age utilizing ESR spectroscopy. In this study, in addition to evaluating the extracellular glutamate concentration, we tried to determine whether or not changes in the expression of cystine/glutamate exchanger (xCT) and glutamate transporter take place in the Hipp of EL. EL mice and DDY mice at 5, 10, and 20 weeks of age were used for Exp. I and II, respectively. Exp. I: During the interictal state, dialysate was collected from the ventral Hipp using a microdialysis technique, and an extracellular concentration of glutamate ([Glu](o)) was measured with HPLC-ECD. Exp. II: The hippocampal expression of the glutamate transporter and xCT was estimated by Western blots. Exp. I: The level of [Glu](o) at 10 weeks of age was remarkably higher at other ages of EL mice, while [Glu](o) of DDY was unchanged as a result of age. Exp. II: The excitatory amino acid carrier-1 (EAAC-1) and xCT of EL mice at 10 weeks of age decreased more than those of DDY. GLAST and GLT-1 of EL mice at 5 weeks of age decreased more than those of DDY at the same age. No differences were found between EL and DDY for GLAST and GLT-1 at other ages. According to previous studies, the decreased endogenous antioxidant potential observed at 10 weeks of age is a very likely explanation for ictogenesis. The decreased xCT expression at 10 weeks of age could provide the molecular mechanism to explain the depletion of the endogenous antioxidant ability of EL mice during ictogenesis. In addition to the depletion of antioxidant ability, decreased EAAC-1 at this period could be one reason for the collapse of the molecular action of inhibition. These molecular findings support the idea that the elevation of [Glu](o) at 10 weeks of age triggers ictogenesis.

In vivo EPR estimation of bilateral hippocampal antioxidant ability of rats with epileptogenesis induced by amygdalar FeCl3 microinjection.

PURPOSE: To measure the neural antioxidant function in the hippocampus of rats with epileptogenesis induced by microinjection of FeCl3 into the amygdala using the decay rate of the nitroxide radical as estimated by L-band electron paramagnetic resonance (EPR) spectroscopy. MATERIALS AND METHODS: Region-selected intensity determination (RSID) was used for the estimation of the nitroxide decay ratio. It is possible to estimate the in vivo hippocampal antioxidant ability using the half-life of the EPR signal of the blood-brain barrier-permeable nitroxide radical. Rats were microinjected with aqueous FeCl3 into the right amygdaloid body. Recording from chronically implanted depth electrodes showed the development of spike discharges with recurrent seizures arising from amygdalar regions with propagation into both hippocampi. Rats with unilateral aqueous FeCl3 lesions were injected systemically with the nitroxide radical and then had EPR for RSID estimation at 5, 15, and 30 days after the iron salt injection. RESULTS: The in vivo antioxidant ability of the dorsal hippocampus was significantly decreased bilaterally in animals with FeCl3-induced seizures when compared to the control. CONCLUSION: Neural antioxidant function in the hippocampi of rats with chronic seizures induced by amygdalar FeCl3 was decreased early and both ipsilaterally and bilaterally.

Age-dependent changes in the hippocampal antioxidant ability of EL mice.

Electron spin resonance (ESR) spectroscopy combined with in vivo microdialysis was used to analyze the antioxidant ability in the hippocampus of mice in an interictal state of EL mice utilizing decay ratio of an exogenously applied nitroxide radical (3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM)). In EL mice with a history of frequent seizures, the half-life of the electron paramagnetism of PCAM in the hippocampus was prolonged. These results revealed decreased antioxidant ability, suggesting vulnerability against oxidative stress. Our data suggest that epileptogenesis in EL mice with chronic seizures is associated with functional failure due to the oxidized redox state and revealed that the decreased hippocampal antioxidant ability is related to the regional vulnerability to oxidative stress in the limbic system of EL mice during epileptogenesis.

Effect of levetiracetam on molecular regulation of hippocampal glutamate and GABA transporters in rats with chronic seizures induced by amygdalar FeCl3 injection.

Enhancement of the glutamatergic excitatory synaptic transmission efficacy in the FeCl3 induced epilepsy model is associated with changes in the levels of glutamate and GABA transporter proteins. This study examined the effect of levetiracetam (LEV) on glutamate overflow and glutamate/GABA transporters expression in rats with epileptogenesis induced by the amygdalar injection of 1.0 microl of 100 mM FeCl3 (epileptic rat) and in control rats receiving amygdalar acidic saline injection (non-epileptic rat). In amygdalar acidic saline injected rats, 40 mM KCl-evoked glutamate overflow was significantly suppressed by both 32 and 100 microM LEV co-perfusion. In unilateral amygdalar FeCl3 injected rats, 32 microM LEV was ineffective, but the 100 microM LEV statistically suppressed glutamate overflow. Western blotting was employed to determine the hippocampal expression of glutamate/GABA transporters in epileptic or non-epileptic rats. The rats were treated for 14 days with 54 mg/kg LEV or vehicle intraperitoneally injection. Following 14 days of treatment, the ipsilateral hippocampus was removed for a Western blot analysis. In non-epileptic rats, the expression increased for all of the glutamate and GABA transporters (GLAST, GLT-1, EAAC-1, GAT-1 and GAT-3) while the glutamate transporter regulating protein (GTRAP3-18) decreased in comparison to those of normal rats that were treated with the vehicle. In epileptic rats receiving LEV, the EAAC-1 and GAT-3 levels increased while GTRAP3-18 (89%) decreased in comparison to those of the epileptic rats treated with the vehicle. GTRAP3-18 inhibitor regulates glutamate-binding affinity to EAAC-1. The anti-epileptic action of LEV may be partially due to a reduction of glutamate-induced excitotoxicity and an enhancement of the GABAergic inhibition as observed with the inhibitory effect on the 40 mM KCl-evoked glutamate overflow. These conclusions are supported by the increase in the expression of glial glutamate transporters (GLAST and GLT-1), and the increase in the expression of EAAC-1 and GAT-3 associated with a decrease in GTRAP3-18. The increased expression of EAAC-1 and the decreased expression of GTRAP3-18 in association with the up-regulation of GAT-3 due to such continual LEV administration was thus found to enhance GABA synthesis and reverse the transport of GABA both in non-epileptic and epileptic rats. The suppression of glutamate excitation and the enhancement of GABA inhibition in the rats with continual LEV administration is a result of the up-regulation of glutamate and GABA transporters with the down-regulation of GTRAP3-18. These observations together demonstrated the critical molecular mechanism of the anti-epileptic activity of LEV.

Functional role of GABA transporters for kindling development in GLAST KO mice.

Kindling-induced after discharge in electroencephalograms depends on the protein associated with glutamatergic and/or GABAergic neuronal transmission. In glutamate transporter knockout (GLAST KO) mice, the kindling phenomena in GLAST KO developed more slowly while the after discharge duration (ADD) was briefer than that of the control C57BL-6J mice. These findings indicate that either the excitatory function was suppressed or the inhibitory function was enhanced in GLAST KO kindling. To explain these phenomena, we used Western blotting to evaluate the alterations in the expression of hippocampal GABA transporter proteins, and the estimation of the effect on the process of epileptogenesis. Although no alterations were observed in the GAT-3 expression, the hippocampal GAT-1 expression was significantly suppressed in comparison to that of C57BL-6J mice. A decreased GAT-1 level in the hippocampus, which might be associated with the increased extracellular GABA level, may therefore inhibit both ADD and seizure propagation as shown by the amygdaloid kindling phenomenon observed in GLAST KO mice.