Postischemic CA1 Neurons Research Articles

Emerging Role for KCNQ1 in Ischemia-Induced Neuronal Death

The slowly activating component of the delayed rectifier potassium current, IKs, composed of KCNQ1-KCNE1 channel subunits, is an important contributor to normal cardiac repolarization. Emerging evidence indicates that KCNQ1 subunits are also expressed in brain, but its precise physiological role in neuronal function has not been investigated. Potassium channels are key players in ischemia-induced death of hippocampal CA1 pyramidal neurons which, in turn, leads to impaired cognition. REST, a gene silencing transcription factor, is up-regulated in global ischemia and contributes to neuronal death. Recently we showed that REST is enriched at the promoter of the KCNQ1 channel and that KCNQ1 mRNA and protein expression are increased in post-ischemic CA1 neurons. Additionally, functional KCNQ1 channels were identified in cultured neurons using KCNQ1-specific inhibitors Chromanol 293B and JNJ-303 and induction of oxygen-glucose deprivation (OGD) induced a marked increase in KCNQ1 currents (∼34%, p > 0.02), evident at 48 h post-ischemia. To assess a possible role for KCNQ1 in ischemia-induced neuronal death, we induced OGD in dissociated hippocampal neurons in the absence and presence of Chromanol 293B (100 µM) and treated with propidium iodine. Inhibition of KCNQ1 markedly diminished cell death, assessed at 24 h (21%, p > 0.002) and 48 h (28%, p > 0.001) suggesting that an up-regulation of KCNQ1 channels may contribute to ischemia-induced neuronal death. To examine a possible role for REST in ischemia-induced up-regulation of KCNQ1, we performed single-locus ChIP which revealed enrichment for REST by ∼2-fold and a decrease in trimethylation of lysine 27 on histone 3 (H3K27me3), an epigenetic mark of gene repression at the KCNQ1 promoter, consistent with the increase in KCNQ1 expression after ischemia. Our findings reveal, for the first time, a role for REST in KCNQ1 expression in response to ischemic insults.

Calpain-Mediated Hsp70.1 Cleavage in Monkey CA1 after Ischemia Induces Similar ‘Lysosomal Vesiculosis’ to Alzheimer Neurons

For the past decade evidence has gathered for the implication of lysosomes in the development of programmed cell death. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) not only as a molecular chaperone for damaged/aged proteins but also as a guardian of lysosomal integrity. Hsp70.1-mediated lysosomal stabilization is tightly regulated, but its disorder under extreme conditions results in lysosomal rupture to cause cell death. Comparing with the pathological hallmark ‘granulo-vacuolar degenerations’ in Alzheimer’s disease, the postischemic monkey neurons were carefully observed with microscope. Intriguingly, we found very similar change, and identified it as ‘lysosomal vesiculosis’ by electron microscopy. However, the exact molecular and structural impacts of the Hsp70.1 disorder upon the lysosomal membrane are hardly elucidated in the human brain because of the practical and ethical problems. Accordingly, using the monkey brain tissues after in-vivo and in-vitro oxidative stresses, we studied molecular modifications of Hsp70.1 and its counterpart bis(monoacylglycero) phosphate (BMP), because these molecules are closely related to the lysosomal membrane stability by regulating acid sphingomyelinase. It still remains unelucidated whether ischemia/reperfusion can alter composition or amount of BMP in the brain. Regardless of the brain regions studied, the normal monkey brain tissues showed calpain-mediated cleavage of Hsp70.1 after in-vitro oxidative stress. In the CA1 after in-vivo ischemia/reperfusion, we first found that docosahexaenoic and oleic acids in BMP showed a significant decrease on days 1 and 3, compared to non-ischemic controls. Since Hsp70.1- BMP binding is indispensable for activating acid sphingomyelinase and producing ceramide to stabilize lysosomal membrane, dysfunction of Hsp70.1 and BMP presumably causes storage of sphingomyelin and deficiency of ceramide at the lysosomal membrane, leading to its destabilization in the postischemic CA1 neurons. These data, combined together, suggest that lysosomal destabilization induced by calpain-mediated cleavage of carbonylated Hsp70.1 and the concomitant BMP breakdown causes ‘lysosomal vesiculosis’.

Calpain-mediated Hsp70.1 cleavage in hippocampal CA1 neuronal death

Necrotic neuronal death is recently known to be mediated by the calpain–cathepsin cascade from simpler organisms to primates. The main event of this cascade is calpain-mediated lysosomal rupture and the resultant release of lysosomal cathepsins into the cytoplasm. However, the in-vivo substrate of calpain for inducing lysosomal destabilization still remains completely unknown. The recent proteomics data using the post-ischemic hippocampal CA1 tissues and glaucoma-suffered retina from the primates suggested that heat shock protein (Hsp) 70.1 might be the in-vivo substrate of activated μ-calpain at the lysosomal membrane of neurons. Hsp70.1 is known to stabilize lysosomal membrane by recycling damaged proteins and protect cells from oxidative stresses. Here, we studied the molecular interaction between activated μ-calpain and the lysosomal Hsp70.1 in the monkey hippocampal CA1 neurons after the ischemia–reperfusion insult. Immunofluorescence histochemistry showed a colocalization of the activated μ-calpain and upregulated Hsp70.1 at the lysosomal membrane of the post-ischemic CA1 neurons. In-vitro cleavage assay of hippocampal Hsp70.1 by Western blotting demonstrated that Hsp70.1 in the CA1 tissue is an in-vivo substrate of activated μ-calpain, and that carbonylated Hsp70.1 in the CA1 tissue by artificial oxidative stressors such as hydroxynonenal (HNE) or hydrogen peroxide is much more vulnerable to the calpain cleavage. These data altogether suggested that Hsp70.1 can become a target of the carbonylation by HNE, and Hsp70.1 is a modulator of calpain-mediated lysosomal rupture/permeabilization after the ischemia–reperfusion injury.

Hippocampal CA1 cell loss in a non-human primate model of transient global ischemia: A pilot study

We exposed adult Rhesus (Macaca mulatta) to a transient global ischemia, which was induced by clipping the innominate and subclavian arteries that originated from the aortic arch. NHP1 received 20-min, while NHP2 and NHP3, were exposed to a 15-min transient global ischemia and were euthanized at day 1 (NHP1), day 5 (NHP2) or day 30 (NHP3) after ischemia, respectively. NHP1 displayed severe paralysis and rigidity, and intermittent convulsions over the next 24 h. Although histological examination of the brain revealed no detectable gross brain damage (i.e., swelling) and only minimal cell loss in the hippocampus, the acute survival time after surgery likely prevented the cerebral ischemia to fully develop and to be morphologically manifested. Nonetheless, the 20-min ischemia might have been too severe and caused a systemic multiple organ collapse that produced the abnormal behavioral symptoms. On the other hand, NHP2 and NHP3 which received 15-min ischemia only exhibited minor hindlimb paralysis. Indeed, by 48 h after ischemia, both animals appeared fully recovered with only fine motor deficits. Immunohistochemical examination revealed that NHP2 and 3, but not NHP1, had a marked neuronal cell loss in the hippocampal region, specifically the cornu Ammonis (CA1) region. The cell loss in these two ischemic NHP hippocampi was further confirmed by direct comparison with a normal Rhesus brain. These findings replicate the brain pathology seen in Japanese macaques exposed to the same ischemia model [T. Tsukada, M. Watanabe, T. Yamashima, Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus, J. Neurochem. 79 (2001) 1196–1206; T. Yamashima, Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates, Prog. Neurobiol. 62 (2000) 273–295; T. Yamashima, Y. Kohda, K. Tsuchiya, T. Ueno, J. Yamashita, T. Yoshioka, E. Kominami, Inhibition of ischemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on calpain-cathepsin hypothesis, Eur. J. Neurosci. 10 (1998) 1723–1733; T. Yamashima, T.C. Saido, M. Takita, A. Miyazawa, J. Yamano, A. Miyakawa, H. Nishijyo, J. Yamashita, S. Kawashima, T. Ono, T. Yoshioka, Transient brain ischemia provokes Ca2+, PIP2 and calpain responses prior to delayed neuronal death in monkeys, Eur. J. Neurosci. 8 (1996) 1932–1944; T. Yamashima, A.B. Tonchey, T. Tsukada, T.C. Saido, S. Imajoh-Ohmi, T. Momoi, E. Kominami, Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates, Hippocampus 13 (2003) 791–800]. The present minimally invasive transient global ischemia model using Rhesus shows many histopathological symptoms seen in human patients who experienced global ischemia, and should allow translational validation of experimental therapeutics for ischemic injury. Additional studies are warranted to reveal behavioral deficits associated with this ischemia model.

Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death

Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn(2+) in CA1 neurons just before the onset of histologically detectable cell death. Here we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca(2+)/Zn(2+)-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn(2+) and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn(2+) in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn(2+) and death of CA1 neurons, although a direct action at the time of the rise in Zn(2+) is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans.

Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates.

Because of the paucity of primate experimental models, the precise molecular mechanism of ischemic neuronal death remains unknown in humans. This study focused on nonhuman primates to determine which cascade necrosis or apoptosis is predominantly involved in the development of delayed (day 5) neuronal death in the hippocampal CA1 sector undergoing 20 min ischemia. We investigated expression, activation, and/or translocation of micro-calpain, lysosome-associated membrane protein-1 (LAMP-1), caspase-3, and caspase-activated DNase (CAD), as well as morphology of the postischemic CA1 neurons and DNA electrophoresis pattern. Immunoblotting showed sustained (immediately after ischemia until day 5) and maximal (day 3) activation of micro-calpain. The immunoreactivity of activated micro-calpain became remarkable as coarse granules at lysosomes on day 2, while it translocated throughout the perikarya on day 3. The immunoreactivity of LAMP-1 also showed a dynamic and concomitant translocation that was maximal on days 2-3, indicating calpain-mediated disruption of the lysosomal membrane after ischemia. In contrast, immunoblotting demonstrated essentially no increase in the activated caspase-3 at any time points after ischemia, despite upregulation of pro-caspase-3. Although expression of CAD was slightly upregulated on day 1 or 2, or both, it was much less compared with lymph node or intestine tissues. Furthermore, light and electron microscopy showed eosinophilic coagulation necrosis and membrane disruption without apoptotic body formation, while DNA electrophoresis did not show a ladder pattern, but rather a smear pattern. Sustained calpain activation and the resultant lysosomal rupture, rather than CAD-mediated apoptosis, may cause ischemic neuronal necrosis in primates.

Estrogen Protects against Global Ischemia-Induced Neuronal Death and Prevents Activation of Apoptotic Signaling Cascades in the Hippocampal CA1

The importance of postmenopausal estrogen replacement therapy in affording protection against the selective and delayed neuronal death associated with cardiac arrest or cardiac surgery in women remains controversial. Here we report that exogenous estrogen at levels that are physiological for hormone replacement in postmenopausal women affords protection against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1 of male gerbils. Global ischemia induced a marked increase in activated caspase-3 in CA1, evident at 6 hr after ischemia. Global ischemia induced a marked upregulation of the proapoptotic neurotrophin receptor p75(NTR) in CA1, evident at 48 hr. p75(NTR) expression was induced primarily in terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling-positive cells, indicating expression in neurons undergoing apoptosis. Global ischemia also induced a marked downregulation of mRNA encoding the AMPA receptor GluR2 subunit in CA1. Caspase-3, p75(NTR), and GluR2 were not significantly changed in CA3 and dentate gyrus, indicating that the ischemia-induced changes in gene expression were cell specific. Exogenous estrogen attenuated the ischemia-induced increases in activated caspase-3 and blocked the increase in p75(NTR) in post-ischemic CA1 neurons but did not prevent ischemia-induced downregulation of GluR2. These findings demonstrate that long-term estrogen at physiological levels ameliorates ischemia-induced hippocampal injury and indicate that estrogen intervenes at the level of apoptotic signaling cascades to prevent onset of death in neurons otherwise "destined to die."

Transient forebrain ischemia induces persistent hyperactivity of large conductance Ca2+-activated potassium channels via oxidation modulation in rat hippocampal CA1 pyramidal neurons.

The present study examined temporal changes in activity of large conductance, Ca2+-activated potassium (BKCa) channels in postischemic CA1 pyramidal neurons at 2, 6, 24 and 48 h after reperfusion. These changes in activity and possible cellular mechanisms were examined using the inside--out configuration of patch clamp. The unitary conductance of postischemic BKCa channels increased transiently to 119% of the control at 2 h after reperfusion, and recovered to the control level thereafter. A persistent increase in [Ca2+]i sensitivity of BKCa channels was observed in postischemic CA1 neurons with the maximal sensitivity to [Ca2+]i at 6 h after reperfusion while channel voltage- dependence showed no obvious changes. Kinetic analyses showed that the postischemic enhancement of BKCa channel activity was due to longer open times and shorter closed times as there was no significant changes in opening frequency after ischemia. Glutathione disulphide markedly increased BKCa channel activity in normal CA1 neurons, while reducing glutathione caused a decrease in BKCa channel activity by reducing the sensitivity of this channel to [Ca2+]i in postischemic CA1 neurons. Similar modulatory effects on postischemic BKCa channels were also observed with another redox couple, DTNB and DTT, suggesting an oxidation modulation of BKCa channel function after ischemia. The present results indicate that a persistent enhancement in activity of BKCa channels, probably via oxidation of channels, in postischemic CA1 pyramidal neurons may account for the decrease in neuronal excitability and increase in fAHP after ischemia. The ischemia-induced augmentation in BKCa channel activity may be also associated with the postischemic neuronal injury.

PET imaging of ischemic neuronal death in the hippocampus of living monkeys.

The aim of the present study was to visualize postischemic hippocampal neuronal death in the living monkey brain, using a high-resolution positron emission tomography (PET) and novel radioligands. In preceding papers, we reported on postischemic hippocampal neuronal death in a model of Japanese monkeys (Macaca fuscata) undergoing a 20-min complete whole-brain ischemia. Using the same model here, we investigated the in vivo bindings of two radiotracers, [11C]Ro15-4513 (a type II benzodiazepine receptor ligand) and [11C](+)3-MPB (a muscarinic cholinergic receptor ligand), in the hippocampus on day 7 after ischemia, as compared to the normal hippocampus. A significant decrease in the in vivo binding of [11C]Ro154513 and [11C(+)3-MPB was observed in the postischemic monkey hippocampus on day 7 after ischemia compared to controls. Light and electron microscopic analyses of postischemic CA1 neurons showed typical features of coagulation necrosis, as associated with a marked reduction of postsynaptic densities and presynaptic vesicles. These results suggest that semiquantification of hippocampal neuronal death is possible in the living primate brain using PET, and that the same procedures can be applied for evaluating neuronal cell loss in patients with ischemic injuries and/or dementia.

Differential expression of neurexin mRNA in CA1 and CA3 hippocampal neurons in response to ischemic insult

A short period of cerebral ischemia will trigger a cascade of events leading to neuronal death. In an effort to elucidate molecular mechanisms underlying differential vulnerability of CA1 and CA3 hippocampal neurons to neurodegeneration, we performed a transcriptional analysis of rat hippocampal neurons following transient global ischemia. In response to 15-min ischemia, the mRNA level of neurexins IIα and IIIα was elevated in CA1 neurons and CA3 neurons, respectively. Interestingly, the up-regulated neurexin IIIα mRNA in postischemic CA3 consisted of the insert corresponding to the fourth splicing site, while the transcripts in postischemic CA1 neurons and control CA3 neurons lacked the insert. The observed tissue specific expression and the splicing pattern suggest functional importance of neurexins in postischemic degeneration of hippocampal neurons.

Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia.

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca(2+) permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca(2+) influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca(2+)-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Age-related changes of cornu ammonis 1 pyramidal neurons in gerbil transient ischemia.

This study reports that postischemic apoptotic cell death of the hippocampal cornu ammonis (CA) 1 neurons is delayed in aged gerbils. Age-related changes in the process of CA1 neuronal death following transient ischemia was studied. Two groups of Mongolian gerbils were used in this study, which compared adult (4-month-old) and aged (24-month-old) animals by hematoxylin-eosin stain, in situ nick-end labeling (TUNEL method) and electron microscopy. In the process of neuronal death, neuronal loss of the aged group was histologically less severe than that of the adult group. TUNEL-positive cells were found on days 3-5 after ischemia in the adult group, while they were still found on day 7 in the aged group. The apoptotic process of the aged group was delayed compared to the adult group. Furthermore, lipofuscin was ultrastructurally observed inside the apoptotic body 5 days after ischemia in CA1 pyramidal neurons of the aged group. It is likely that colocalization of lysosomal enzyme cathepsin D with lipofuscin might be associated with the age-related alteration of lysosomal system in the neurons. Altogether these data suggest that age-related lysosomal changes might affect the apoptotic cascade process in postischemic CA1 neurons.

Expression of cell death-associated phospho-c-Jun and p53-activated gene 608 in hippocampal CA1 neurons following global ischemia

Persistent activation of c-Jun N-terminal kinases (JNKs) and phosphorylation of c-Jun has been shown in various cell death paradigms. Inhibition of the JNK signal transduction pathway prevented neuronal cell death both in vitro and in vivo. In the present study, nuclear phospho-c-Jun immunoreactivity became apparent selectively in vulnerable hippocampal CA1 neurons at 24 h after transient global cerebral ischemia. A high constitutive expression of phospho-JNK1 was detected by immunoblot analysis of hippocampal extracts. Expression of JNK interacting protein-1 (JIP-1), which facilitates JNK signaling, remained unchanged in post-ischemic hippocampal neurons. By contrast, p53-activated gene 608 (PAG608), which promotes cell death in vitro, was strongly induced in post-ischemic CA1 neurons. Our data suggest that transcription factors p53 and phospho-c-Jun may contribute to programmed CA1 cell death following ischemia.

Postictal Blockade of Ischemic Hippocampal Neuronal Death in Primates Using Selective Cathepsin Inhibitors

This paper is to study the participation of cathepsin in ischemic neuronal death of the monkey hippocampal cornu ammonis (CA) 1 sector and also to clarify whether its selective inhibitor epoxysuccinyl peptides such as CA-074 and E-64c can inhibit the neuronal death or not. In the preceding reports, we demonstrated μ-calpain activation and subsequent rupturing of the lysosomal membrane of postischemic CA1 neurons and also increase of enzyme activity of cathepsins B and L in monkeys undergoing a complete 20-min whole brain ischemia. Here, morphological, immunohistochemical and enzymatical analyses were performed to examine the efficacy of two selective cathepsin inhibitors in the postictal blockade of delayed neuronal death in the monkey hippocampus. Both inhibitors could significantly decrease enzyme activities of cathepsins B and L in all hippocampal sectors. When CA-074 was intravenously administered immediately after the ischemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 after ischemia. In contrast, when E-64c was similarly administered, approximately 84% of CA1 neurons were saved from delayed neuronal death on day 5. The surviving neurons showed mild central chromatolysis and negligible immunoreactivity for cathepsins B and L. These observations indicate that the use of cathepsin inhibitors may become novel strategy for prevention of ischemic delayed neuronal death in the primate hippocampus.

Prolonged enhancement and depression of synaptic transmission in CA1 pyramidal neurons induced by transient forebrain ischemia in vivo

Evoked postsynaptic potentials of CA1 pyramidal neurons in rat hippocampus were studied during 48 h after severe ischemic insult using in vivo intracellular recording and staining techniques. Postischemic CA1 neurons displayed one of three distinct response patterns following contralateral commissural stimulation. At early recirculation times (0–12 h) approximately 50% of neurons exhibited, in addition to the initial excitatory postsynaptic potential, a late depolarizing postsynaptic potential lasting for more than 100 ms. Application of dizocilpine maleate reduced the amplitude of late depolarizing postsynaptic potential by 60%. Other CA1 neurons recorded in this interval failed to develop late depolarizing postsynaptic potentials but showed a modest blunting of initial excitatory postsynaptic potentials (non-late depolarizing postsynaptic potential neuron). The proportion of recorded neurons with late depolarizing postsynaptic potential characteristics increased to more than 70% during 13–24 h after reperfusion. Beyond 24 h reperfusion, ∼20% of CA1 neurons exhibited very small excitatory postsynaptic potentials even with maximal stimulus intensity. The slope of the initial excitatory postsynaptic potentials in late depolarizing postsynaptic potential neurons increased to ∼150% of control values up to 12 h after reperfusion indicating a prolonged enhancement of synaptic transmission. In contrast, the slope of the initial excitatory postsynaptic potentials in non-late depolarizing postsynaptic potential neurons decreased to less than 50% of preischemic values up to 24 h after reperfusion indicating a prolonged depression of synaptic transmission. More late depolarizing postsynaptic potential neurons were located in the medial portion of CA1 zone where neurons are more vulnerable to ischemia whereas more non-late depolarizing postsynaptic potential neurons were located in the lateral portion of CA1 zone where neurons are more resistant to ischemia. The result from the present study suggests that late depolarizing postsynaptic potential and small excitatory postsynaptic potential neurons may be irreversibly injured while non-late depolarizing postsynaptic potential neurons may be those that survive the ischemic insult. Alterations of synaptic transmission may be associated with the pathogenesis of postischemic neuronal injury.

Cyclic changes in NMDA receptor activation in hippocampal CA1 neurons after ischemia

We studied N-methyl- d-aspartate (NMDA) receptor-mediated synaptic potentials in CA1 pyramidal neurons using hippocampal slices of gerbils after transient forebrain ischemia. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and bicuculline, stimulation of Schaffer collateral/commissural fibers induced field excitatory postsynaptic potentials (fEPSP) activated by NMDA receptors. We found that in many slices after ischemia, prolonged low-frequency stimulation (0.1–10 Hz) caused repeated depression and potentiation of the NMDA-mediated fEPSP. Changes in fEPSP amplitude were dependent on stimulus frequency and the cycle frequency ranged from 0.08 to 2.5 cycles/min. These cyclic changes were blocked by application of BAPTA-AM, a membrane-permeable Ca 2+ chelator, but were little affected by application of verapamil or by lowering the Ca 2+ in bathing solution. Intracellular recordings from CA1 neurons revealed that low-frequency stimulation caused periodic depolarizations of membrane potential accompanied by depression of the excitatory postsynaptic potentials. The cyclic changes of fEPSPs were blocked by inhibitors of protein kinase C (PKC) but were unaffected by inhibitors of Ca 2+/calmodulin-dependent protein kinase II (CaMKII) or myosin light-chain kinase (MLCK). These results suggest that stimulus-dependent NMDA-receptor activation, mediated by PKC, takes place in the postischemic CA1 neurons and that the cyclic change may reflect abnormal intracellular Ca 2+ signaling processes leading to neuronal degeneration.

Transient ischemia induces an early decrease of synaptic transmission in CA1 neurons of rat hippocampus: electrophysiologic study in brain slices.

We examined the functionality of hippocampal CA1 neurons at early times after transient global ischemia, by electrophysiologic recordings in brain slices. Transient ischemia was conducted on rats using the method of 15-minute four-vessel occlusion, and brain slices were obtained from these animals at different times after ischemia. Within 24 hours after insult, CA1 neurons showed no substantial damage as identified by morphologic means, but exhibited dramatic decreases in synaptic activities by 12 hours after insult, which became further decreased at more extended times after recovery. Blocking gamma-aminobutyric acid A (GABAA) receptors with bicuculline produced a reversible augmentation of the diminished synaptic responses in slices prepared from 12-hour postinsult animals, but failed to do so in slices obtained from rats 24 hours after insult. Recorded in whole-cell mode, the minimum depolarizing current required to elicit an action potential was about twofold larger in the ischemic CA1 neurons than in sham controls, suggesting that an elevated spiking threshold exists in these neurons. We suggest that decreases in electrophysiologic activities precede the morphologic deterioration in postischemic CA1 neurons. The early decrease in CA1 synaptic activities may be associated with an imbalance between glutamate-mediated synaptic excitation and GABAA-mediated synaptic inhibition, whereas substantial impairments in synaptic transmission likely take place after prolonged post-ischemic recovery.