Gerbil hippocampal extracellular glutamate and neuronal activity after transient ischemia

In order to elucidate the role of glutamate in the pathogenesis of delayed neuronal death, we analyzed changes in extracellular levels of glutamate induced by transient ischemia in the Mongolian gerbil hippocampus by a new brain microdialysis method combined with an enzymatic cycling technique. We also studied the effect of this change in glutamate on CA1 spontaneous neuronal discharges. The level of glutamate significantly increased during the 5 min of ischemia and during the first 5 min of recirculation. However, neuronal hyperactivity anticipated as a result of the increased extracellular glutamate was not observed. Spike discharges disappeared during and shortly after 5 min of ischemia; CA1 spontaneous spike discharges reappeared about 15 min after the recirculation. The frequency and amplitude of the discharges of CA1 neurons returned to normal by 30 min of the recirculation. However, the pattern of discharges was different from that recorded before the ischemia. CA1 neurons were found dead 4 days after the ischemia. Brief exposure to toxic concentrations of glutamate may cause the delayed neuronal death.     

Delayed neuronal death is induced without postischemic hyperexcitability: continuous multiple-unit recording from ischemic CA1 neurons

It has been proposed that neuronal hyperexcitability during postischemic chronic stage mediates delayed neuronal death in the hippocampal CA1 region. In the present study, multiple-unit spike discharges were continuously recorded from hippocampal CA1 neurons of the awake Mongolian gerbil for 5 days after 5 min of ischemia. Before ischemia, CA1 neurons showed burst-like spike discharges (so-called complex spikes). Spike discharges disappeared 8-40 s after the onset of 5-min ischemia and reappeared 5-30 min after recirculation. The frequency of discharges gradually increased but did not return to the preischemic level. The amplitude of the spike discharges was smaller than that recorded before ischemia and the number of spikes composing the burst-like discharges diminished. CA1 neurons did not show any hyperexcitability for 5 days. However, histological examinations revealed widespread neuronal death in the CA1 region. These results indicate that the delayed neuronal death in the hippocampal CA1 region is induced without postischemic neuronal hyperexcitability.     

预缺血诱导大鼠CA1神经元中蛋白伴侣hsp70的表达并抑制蛋白聚集物的形成

目的 研究预缺血对蛋白伴侣hsp70表达和蛋白聚集物形成的影响,探讨其可能的脑保护机制.方法 采用大鼠双侧颈总动脉暂时夹闭法建立全脑缺血模型.大鼠分为3min缺血组,10min缺血组以及预缺血组.苏木素-伊红染色,光镜下随机计数分析预缺血后海马CA1区死亡神经元数量变化.免疫组织化学及激光扫描共聚焦显微镜法观察蛋白伴侣hsp70在CAI区神经元内的分布.差速离心分离细胞浆、细胞核及蛋白聚集物.蛋白印迹法检测不同缺血状态下海马CA1神经元内蛋白聚集物含量的变化,以及胞浆、胞核及蛋白聚集物内蛋白伴侣hsp70含量的变化.结果 组织学检查显示预缺血能够显著减少海马CA1区神经元死亡数量.预缺血诱导海马CA1区神经元内蛋白伴侣hsp70在再灌注后24h表达.预缺血处理后,海马CA1区神经元内蛋白聚集物显著减少.预缺血诱导的蛋白伴侣hsp70与再缺血形成的异常蛋白结合在一起并防止其聚集.结论 预缺血可能通过诱导蛋白伴侣hsp70的表达和抑制再缺血后蛋白聚集物的形成,减少再缺血引起的神经元死亡.     

Nitric oxide synthase inhibitor reduces delayed neuronal death in gerbil hippocampal CA1 neurons after transient global ischemia without reduction of brain temperature or extracellular glutamate concentration

We planned a study to determine whether or not the mechanism of nitric oxide (NO) neurotoxicity involves the elevation of extracellular glutamate or changes of brain temperature in the pathogenesis of delayed neuronal death of gerbil hippocampal CA1 neurons following 5-min transient forebrain ischemia. Intraventricular injection of 5 μl of 5.0 mg/ml N^ω-nitro-l-arginine (LNNA) significantly preserved neuronal density in the central part of the CAI region examined 7 days after 5-min ischemia [188.5 ± 8.5/mm: 90.0% of the 209.5 ± 11.1 /mm density in the sham-operated controls vs. 16.7 ± 6.4/mm in those injected with artificial cerebrospinal fluid (CSF) only]. There was no difference between these two groups in hippocampal temperature before, during or after 5-min ischemia. The glutamate concentration ([Glu.]) during 5-min ischemia measured by a microdialysis technique was similar in the two groups (peak [Glu.] = 2.76 ± 0.62 pmol/μl dialysate in the artificial CSF group and = 2.93 ± 0.64 pmol/μ1 dialysate in the LNNA group). It was found that the neuronal toxicity of NO does not involve hyperthermia or the increase of extracellular glutamate concentration in the hippocampal CA1 region during 5-min ischemia.     

Potassium attenuates zinc-induced death of cultured cortical astrocytes

Transient global ischemia induces CA1 hippocampal neuronal death without astrocyte death, perhaps mediated in part by the toxic translocation of zinc from presynaptic terminals to postsynaptic neurons. We tested the hypothesis that cellular depolarization, which occurs in the ischemic brain due to increased extracellular potassium and energy failure, might contribute to astrocyte resistance to zinc-induced death. We previously reported that neurons in mixed cortical neuronal-astrocyte cultures were more vulnerable to a 5-15-min exposure to Zn(2+) than astrocytes in the same cultures. In the present report, we show that (1) neurons in isolation or in conjunction with astrocytes were 2-3-fold more sensitive to a 15-min nondepolarizing Zn(2+) exposure than are glia; (2) KCl-induced depolarization attenuated glial vulnerability to zinc toxicity but potentiated neuronal vulnerability to zinc toxicity; (3) Zn(2+)-induced glial death was attenuated by T-type Ca(2+) channel blockade, as well as compounds that increase NAD(+) levels; and (4) both astrocytic (65)Zn(2+) accumulation and the increase in astrocytic [Zn(2+)](i) induced by Zn(2+) exposure were also attenuated by depolarization or T-type Ca(2+) channel blockers. Zn(2+)-induced cell death in astrocytes was at least in part apoptotic, as caspase-3 was activated, and the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone partially attenuated Zn(2+)-induced death. The levels of peak [Zn(2+)](i) achieved in astrocytes during this toxic nondepolarizing Zn(2+) exposure (250 nM) were substantially greater than those achieved in neurons (40 nM). In glia, exposure to 400 microM Zn(2+) induced a 13-mV depolarization, which can activate T-type Ca(2+) channels. This Zn(2+)-induced astrocyte death, like neuronal death, was attenuated by the addition of pyruvate or niacinamide to the exposure medium.     

Calpain induces proteolysis of neuronal cytoskeleton in ischemic gerbil forebrain

We investigated the relationship between the activity of calcium-dependent protease (calpain) and the ischemic neuronal damage. We also investigated the mechanism of ischemic resistance in astrocytes. In gerbil, a 10-min forebrain ischemia was induced by occlusion of both common carotid arteries. The calpain-induced proteolysis of cytoskeleton (fodrin) was examined by immunohistochemistry. Immunolocalization of micro and m-calpain was also examined. Intact fodrin was observed both in neurons and astrocytes, but proteolyzed fodrin was not observed in normal brain. Fifteen minutes after ischemia, proteolysis of fodrin took place in putamen, parietal cortex and hippocampal CA1. The proteolysis extended to thalamus 4 h after ischemia after which the immunoreactivity faded down in all areas except hippocampus. On day 7, the proteolysis was still observed only in hippocampus. Neurons with the proteolysis of soma resulted in neuronal death. Throughout the experiment, the proteolysis was not observed in astrocytes. micro -Calpain was observed only in neurons but m-calpain was observed both in neurons and astrocytes. The ischemia induced only micro -calpain activation, which resulted in fodrin proteolysis of neurons with differential spatial distribution and temporal course. The proteolysis was developed rapidly and was completed within 24 h in all vulnerable regions except hippocampal CA1. The proteolysis preceded the neuronal death. The mechanism of the proteolysis seemed to be involved by Ca(2+) influx via glutamate receptor and rapid neuronal death seemed reasonable. The reason why neuronal death in CA1 evolved slowly was not clarified. In astrocytes, fodrin was not proteolyzed by m-calpain. The low Ca(2+)-sensitivity of m-calpain may be the reason of ischemic resistance in astrocytes.     

Efflux of glutamate produced by short ischemia of varied severity in rat striatum.

BACKGROUND AND PURPOSE: Evidence has accumulated suggesting that ischemia-induced neuronal damage may be linked to an extracellular overflow of glutamate. The purpose of this study was to provide new information about the time course of the increase in extracellular glutamate concentration associated with moderate and severe ischemia, and its relationship with electrical changes including anoxic depolarization. METHODS: Changes in the extracellular concentration of glutamate were continuously monitored in the rat striatum by microdialysis. Ischemia was induced by four-vessel occlusion for 3 or 5 minutes, and in some cases its severity was increased with a neck tourniquet. The severity of ischemia was assessed by electroencephalogram and direct current potential recording to detect anoxic depolarization. RESULTS: In all experiments, the extracellular glutamate concentration began to increase shortly after the onset of ischemia and steadily rose throughout the ischemic period. Increases up to 35.0 mumol/l (2-3 mumol/l baseline; p less than 0.005) were observed when ischemia provoked the rapid occurrence of a large and sustained anoxic depolarization. Relatively smaller but still significant increases (6.9 mumol/l; p less than 0.005) were observed in penumbral conditions (electroencephalogram loss without anoxic depolarization). Glutamate began to be cleared immediately after reperfusion and 90% of released glutamate was cleared within 5 minutes, even when the preceding ischemia had been severe. CONCLUSIONS: We propose that the extracellular glutamate concentration may not reach critical levels during short episodes of penumbral ischemia, but this might happen with a longer ischemic period.     

The role of remaining presynaptic terminals in the hippocampal CA1 after selective neuronal death: ischemia-induced glutamate efflux

Following selective neuronal death, numerous presynaptic terminals maintain their structural integrity in the brain region. The role that these remaining presynaptic terminals play in the brain region showing selective neuronal death is not known. In the present study, we investigated the possibility that brief transient ischemia induces an excessive release of glutamate from the remaining presynaptic terminals, which then spreads by diffusion. The glutamate could act as an excitotoxin and be a pathogenic factor in the local injured brain region. Transient ischemia of 3.5 min duration was used in the gerbil as a pretreatment to obtain hippocampal CA1 in which most of postsynaptic neurons were eliminated but numerous presynaptic terminals remained normal. At 10–14 days after the pretreatment, brain microdialysis experiments were performed in vivo in the CA1 to measure the levels of extracellular glutamate induced by 5 min ischemia. Prior to 5 min ischemia the basal concentration of glutamate in the CA1 was the same as that observed in gerbils that had been subjected to sham pretreatment. During 5 min ischemia, no significant increase in glutamate was induced in the CA1 which showed selective neuronal death. However, a massive increase in glutamate was induced in the CA1 of the sham-pretreated gerbils. These results suggest that the remaining presynaptic terminals are unlikely to play a pathogenic role in the CA1 after selective neuronal death has occurred. Received: 6 June 1995 / Revised, accepted: 4 August 1995     

Effects of Glutamate Application on the Rhythm of Low Magnesium-induced Epileptiform Activity in Hippocampal Slices of Guinea-pigs

The extracellular concentration of glutamate has previously been reported to increase to more than 10-fold the basal level during seizure activity. In the present study, we tested whether localized increases in extracellular glutamate concentration influence the rhythm of epileptiform discharges in the low-magnesium epilepsy model. In hippocampal slices of guinea-pigs, epileptiform activity was induced by omission of magnesium from the bath fluid. Glutamate and its subreceptor agonists N -methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) were ejected into different strata of the CA3 and CA1 regions using microiontophoretic and micropressure application. Glutamate, NMDA and AMPA applied to the CA3 region, but not to the CA1 region, induced a short-lasting increase in epileptiform discharge frequency, often followed by a transient reduction. The effect was most pronounced with application into the stratum lacunosum-moleculare of the CA3 region and could only be evoked in slices exceeding 400 μm in thickness. The effects on the rhythm of epileptiform discharges induced by NMDA and AMPA were blocked by their specific receptor antagonists. They were not influenced by application of GABA_A and GABA_B receptor antagonists. Changes in somatic membrane potential of CA3 pyramidal neurons did not correlate with changes in the rhythm of epileptiform discharges elicited in this region. The transient suppression of epileptiform discharges that followed the increase in discharge frequency was abolished by an adenosine A₁ receptor antagonist. We propose that localized increases in extracellular glutamate concentration modify the rhythm of epileptiform discharges due to changes in neuronal network activity.     

Neuronal damage following non-lethal but repeated cerebral ischemia in the gerbil

Summary Brief, non-lethal transient forebrain ischemia in the gerbil can injure selectively vulnerable neurons when such ischemia is induced repeatedly. The influence of the number and interval of the ischemic insults on neuronal damage, as well as the time course of damage, following repeated 2-min forebrain ischemia were examined. A single 2-min forebrain ischemia were examined. A single 2-min ischemic insult caused no morphological neuronal damage. A moderate number of hippocampal CA1 neurons were destroyed following two ischemic insults with a 1-h interval, and destruction of almost all CA1 neurons resulted from three or five insults at 1-h intervals. Three and five insults also resulted in moderate to severe damage to the striatum and thalamus, depending on the number of episodes. Although three ischemic insults at 1-h intervals caused severe neuronal damage, this number of insults at 5-min and 4-h intervals caused destruction of relatively few neurons, and non neurons were destroyed at 12-h intervals. Following three ischemic insults at 1-h intervals, damage to the striatum, neocortex, hippocampal CA4 subfield and thalamus was observed at 6–24 h of survival, whereas damage to the hippocampal CA1 subfield appeared at 2–4 days. The results indicate that even a brief non-lethal ischemic insult can produce severe neuronal damage in selectively vulnerable regions when it is induced repeatedly at a certain interval. The severity of neuronal damage was dependent on the number and interval of ischemic episodes.     

The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats.

Glial glutamate transporter-1 (GLT-1) plays an essential role in removing glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. To explore whether GLT-1 plays a role in the acquisition of brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP), the present study was undertaken to observe in vivo changes in the expression of GLT-1 and glial fibrillary acidic protein (GFAP) in the CA1 hippocampus during the induction of BIT, and the effect of dihydrokainate (DHK), an inhibitor of GLT-1, on the acquisition of BIT in rats. Immunohistochemistry for GFAP showed that the processes of astrocytes were prolonged after a CIP 2 days before the lethal ischemic insult, which could protect pyramidal neurons in the CA1 hippocampus against delayed neuronal death induced normally by lethal ischemic insult. The prolonged processes extended into the area between the pyramidal neurons and tightly surrounded them. These changes made the pyramidal layer look like a 'shape grid'. Simultaneously, the prolonged and extended processes showed a great deal of GLT-1. Western blotting analysis showed significant upregulation of GLT-1 expression after the CIP, especially when it was administered 2 days before the subsequent lethal ischemic insult. Neuropathological evaluation by thionin staining showed that DHK dose-dependently blocked the protective role of CIP against delayed neuronal death induced normally by lethal brain ischemia. It might be concluded that the surrounding of pyramidal neurons by astrocytes and upregulation of GLT-1 induced by CIP played an important role in the acquisition of the BIT induced by CIP.     

'Ischemic tolerance' phenomenon found in the brain

We investigated the possibility that neuronal cells given a mild ischemic treatment sufficient to perturb the cellular metabolism acquired tolerance to a subsequent, and what would be lethal, ischemic stress in vivo. Cerebral ischemia was produced in the gerbils by occlusion of both common carotids for 5 min, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. Minor 2-min ischemia in this model depletes high-energy phosphate compounds and perturbs the protein synthesis, but never causes neuronal necrosis, and therefore was chosen as mild ischemic treatment. Single 2-min ischemia 1 day or 2 days before 5 min ischemia exhibited only partial protective effects against delayed neuronal death. However, two 2-min ischemic treatments at 1 day intervals 2 days before 5 min ischemia exhibited drastically complete protection against neuronal death. The duration and intervals of ischemic treatment, enough to perturb cellular metabolism and cause protein synthesis, were needed respectively, because neither 1-min ischemia nor 2-min ischemia received twice at short intervals exhibited protective effects. This 'ischemic tolerance' phenomenon induced by ischemic stress--which is unquestionably important--and frequent stress in clinical medicine, is intriguing and may open a new approach to investigate the pathophysiology of ischemic neuronal damage.     

Short-term ischemia usually used for ischemic preconditioning down-regulates central cannabinoid receptors in the gerbil hippocampus

Transient forebrain ischemia of 5-min duration causes delayed neuronal death (DND) of vulnerable CA1 neurons in the gerbil hippocampus, which can be prevented by preconditioning with a short ischemic stimulus of 2.5-min duration. While a key role of excitatory glutamate receptors for both phenomena has been widely accepted, little is known about the postischemic regulation of central cannabinoid (CB1) receptors. The present study was designed to test whether ischemic preconditioning is associated with specific alterations of protein expression and/or ligand binding of these receptors compared to ischemia severe enough to induce DND. Gerbils were subjected to either a 5-min ischemic period resulting in DND of CA1 neurons, or a 2.5-min period of ischemia usually used for preconditioning. Postischemic hippocampal CB1 receptor protein expression was investigated immunohistochemically, while postischemic ligand binding of [³H]CP 55940 to CB1 receptors was analyzed by quantitative receptor autoradiography in both experimental groups after 24, 48, and 96 h (n=4–5 per time point), respectively, and compared to sham-treated gerbils (n=10). Short-term ischemia of 2.5-min duration caused a transient reduction of hippocampal CB1 receptor protein expression, while receptor binding density was permanently decreased. In contrast, 5-min ischemia did not alter protein expression or ligand binding up to 48 h. Based on these data, postischemic down-regulation of hippocampal CB1 receptors, specifically seen after short-term ischemia usually used for preconditioning, may participate in the mechanisms of endogenous postischemic neuroprotection.The first two authors contributed equally     

‘Ischemic tolerance’ phenomenon found in the brain

We investigated the possibility that neuronal cells given a mild ischemic treatment sufficient to perturb the cellular metabolism acquired tolerance to a subsequent, and what would be lethal, ischemic stress in vivo. Cerebral ischemia was produced in the gerbils by occlusion of both common carotids for 5 min, which consistently resulted in delayed neuronal death in the CA1 region of the hippocampus. Minor 2-min ischemia in this model depletes high-energy phosphate compounds and perturbs the protein synthesis, but nerver causes neuronal necrosis, and therefore was chosen as mild ischemic treatment. Single 2-min ischemia 1 day or 2 days before 5 min ischemia exhibited only partial protective effects against delayed neuronal death. However, two 2-min ischemic treatments at 1 day intervals 2 days before 5 min ischemia exhibited drastically complete protection against neuronal death. The duration and intervals of ischemic treatment, enough to perturb cellular metabolism and cause protein syhthesis, were needed respectively, because neither 1-min ischemia nor 2-min ischemia received twice at short intervals exhibited protective effects. This ‘ischemic tolerance’ phenomenon induced by ischemic stress — which is unquestionably important — and frequent stress in clinical medicine, is intriguing and may open a new approach to investigate the pathophysiology of ischemic neuronal damage.     

'Ischemic tolerance' phenomenon detected in various brain regions.

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the 'ischemic tolerance' phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of 'ischemic tolerance'.     

Repeated focal cerebral ischemia in gerbils is associated with development of infarction.

We induced repeated focal cerebral ischemia in gerbils. Single 5-min ischemia produced neuronal damage limited to the ipsilateral CA1 and CA4 hippocampus. Two 5-min ischemic insults spaced at a 1-h interval caused selective neuronal damage to the CA1, CA3 and CA4 hippocampus, striatum, neocortex, and thalamus. Three 5-min ischemic insults at 1-h intervals produced infarction. Thus, repeated focal ischemia produced cumulative brain damage by conversion of sublethal damage into selective neuronal damage and of the neuronal damage into infarction.     

‘Ischemic tolerance’ phenomenon detected in various brain regions

We investigated the effects of mild and non-lethal ischemic insult on neuronal death following subsequent lethal ischemic stress in various brain regions, using a gerbil model of bilateral cerebral ischemia. Single 10-min ischemia consistently caused neuronal damage in the hippocampal CA1, CA2, CA3 and CA4, layer III/IV of the cerebral cortex, dorsolateral part of the caudoputamen and ventrolateral part of the thalamus. On the other hand, in double ischemia groups, 2-min ischemic insult 2 days before 10-min ischemia exhibited significant protection in the CA1 and CA3 of the hippocampus, the cerebral cortex, the caudoputamen and the thalamus. Five-min ischemic insult 2 days before 10-min ischemia also showed protective effect in the same areas as those of 2-min ischemia except for the CA1 region of the hippocampus, while 1-min ischemic insult exhibited no protective effect in any brain regions. In the immunoblot analysis, both 2- and 5-min ischemia caused increased synthesis of heat shock protein 72 (HSP 72) in the hippocampus, but 1-min ischemia did not. The present study demonstrated that the ‘ischemic tolerance’ phenomenon was widely found in the brain and also suggested that ischemic treatment severe enough to cause HSP 72 synthesis might be needed for induction of ‘ischemic tolerance’.     

Calpain inhibitor entrapped in liposome rescues ischemic neuronal damage

Transient forebrain ischemia induces activation of calpain and proteolysis of a neuronal cytoskeleton, fodrin, in gerbil hippocampus. This phenomenon precedes delayed neuronal death in hippocampal CA1 neurons. We examined effects of a calpain inhibitor on delayed neuronal death after transient forebrain ischemia. In gerbils, a selective calpain inhibitor entrapped in liposome was given transvenously and 30 min later, 5-min forebrain ischemia was produced by occlusion of both common carotid arteries. On day 7, CA1 neuronal damage was examined in the hippocampal slices stained with cresyl violet. Calpain-induced proteolysis of fodrin was also examined by immunohistochemistry and immunoblot. Additionally, to assure entrapment of the inhibitor by CA1 neurons, the inhibitor-liposome complex was labeled with FITC and given to gerbils. Fluorescence in the hippocampal slices was examined by confocal laser scanning microscope. Selective CA1 neuronal damage induced by forebrain ischemia was prevented by administration of the inhibitor in a dose-dependent manner. Calpain-induced proteolysis of fodrin was also extinguished by the calpain inhibitor in a dose-dependent manner. Bright fluorescence of the FITC-labeled inhibitor was observed in the CA1 neurons. The data show an important role of calpain in the development of the ischemic delayed neuronal death. Calpain seems to produce neuronal damage by degrading neuronal cytoskeleton. Our data also show a palliative effect of the calpain inhibitor on the neurotoxic damage, which offers a new and potent treatment of transient forebrain cerebral ischemia.     

Metabotropic glutamate receptor subtypes are differentially expressed after transient cerebral ischemia without, during and after tolerance induction in the gerbil hippocampus

Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons can be prevented by a preconditioning sublethal ischemic stimulus. To check for possible participation of metabotropic glutamate receptors (mGluRs) in neuronal death or survival, we analyzed postischemic protein expression of subtypes 1b and 5 of group I mGluRs, which are thought to exert neurotoxic effects after pathological activation due to ischemia, and subtypes 2 and 3 of group II mGluRs, which in contrast are thought to be neuroprotective in this state, respectively. Therefore, three groups of gerbils with reperfusion intervals between 8 h and 4 days (n=5 each) were investigated: one group was subjected to 5 min ischemia, resulting in DND of CA1 neurons, a second group to a tolerance inducing 2.5 min period of ischemia and a third group to 5 min ischemia after prior tolerance induction. The major finding was a transient postischemic reduction of mGluR1b and 5 expression in the ischemic tolerant CA1 subfield at 8 h. This downregulation of neurotoxic mGluRs may indicate a contribution to the survival of highly vulnerable CA1 neurons in the ischemic tolerant state.     

Global ischemia downregulates the function of metabotropic glutamate receptor subtype 5 in hippocampal CA1 pyramidal neurons

Within the hippocampus, electrophysiological and immunohistochemical studies showed that metabotropic glutamate receptor subtype 5 (mGluR5) is the major postsynaptic mGluR expressed in CA1 pyramidal neurons. To better understand the role of mGluR5 in ischemia-induced neuronal death, whole-cell patch-clamp recordings using hippocampal slices were performed to investigate the functional change of mGluR5 in CA1 pyramidal neurons following transient global ischemia. Our results indicated that 6 to 24 h after global ischemia, mGluR5-induced cationic currents and mGluR5-mediated enhancement of NMDA-evoked currents in CA1 pyramidal neurons were significantly reduced. Further TaqMan real-time quantitative RT-PCR assay showed that mGluR5 mRNA expression in hippocampal CA1 region or single CA1 pyramidal neurons was significantly downregulated following ischemic insults. The present study suggests that transient global ischemia downregulates mGluR5 function of CA1 pyramidal neurons by decreasing mGluR5 mRNA and that the resulting reduced mGluR5-mediated excitotoxicity could contribute to the survival of CA1 pyramidal neurons after ischemic insult.