GluN2B subunits of the NMDA receptor contribute to the AMPA receptor internalization during long-term depression in the lateral amygdala of juvenile rats.

The lateral nucleus of the amygdala (LA) is a critical structure involved in fear conditioning. We recently showed that regulated exocytosis and endocytosis of postsynaptic A-amino-3-hydroxy-5-methylisoxazole-4-propionate subtype of glutamate receptors (AMPARs) are involved in the expression of N-methyl-D-aspartate subtype glutamate receptors (NMDARs) dependent long-term potentiation (LTP) and long-term depression (LTD) in coronal slices of the LA. However, the molecular mechanisms of this effect remain unclear. In the present study, we investigated the role of distinct NMDAR subtypes in the endocytosis of AMPARs during LTD expression at the synapses of the thalamic inputs to the LA neurons. Here we show that the NMDARs antagonist DL-2-amino-5-phosphonovalerate (D-APV) blocked the induction of LTD and thus prevented endocytosis of surface AMPARs, indicating that NMDAR activation enhanced the internalization of AMPARs in LTD expression. Furthermore, the selective blocking of GluN2B-containing NMDARs completely abolished the NMDAR-induced AMPAR endocytosis, whereas preferential inhibition of GluN2A-containing NMDARs did not block the NMDAR-induced AMPAR endocytosis during LTD expression. These results suggest that there exist a preferred NMDAR subtype for AMPAR internalization and activation of GluN2B-containing NMDARs represent the predominate pathway triggered during the early stages of this NMDAR-induced endocytosis of AMPARs during LTD in the thalamic inputs to the LA of juvenile rats.

Sublethal cerebral ischemia inhibits caspase-3 activation induced by subsequent prolonged ischemia in the C57Black/Crj6 strain mouse.

Caspase-3 activation has been implicated in ischemic neuronal death. In the present study, we examined if cerebral ischemic tolerance induced by sublethal ischemia is associated with an attenuation of caspase-3 activation in a mouse forebrain ischemia model. Forebrain ischemia in C57Black/Crj6 strain mice was induced by bilateral common carotid artery occlusion (BCCAO) for 18 min. Two episodes of 6-min ischemia were carried out as preconditioning 48 and 72 h before the 18-min BCCAO. Caspase-3-like activity was determined by fluorescently monitoring the release of amino-4-methylcoumarin from N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin in the striatal protein extracts at 4, 24, and 72 h after reperfusion. The results showed that the ischemic preconditioning significantly attenuated caspase-3 activation at 4, 24, and 72 h after reperfusion, and reduced neuronal loss caused by the 18-min ischemia as examined on the 7th day after reperfusion. The present results suggest that the neuroprotection achieved by ischemic preconditioning is related to an attenuation of caspase-3 activation.

A forebrain ischemic preconditioning model established in C57Black/Crj6 mice.

Although many kinds of rat and gerbil cerebral ischemic preconditioning models are available, only a focal ischemic preconditioning model in mice has been reported. As most genetic alterations have been performed in mice, it is urgent to develop mouse ischemic preconditioning models for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice. In the present study, we developed a forebrain ischemic preconditioning model in C57Black/Crj6 (C57BL/6) mice. Forebrain ischemia was induced in C57BL/6 mice (8-10 weeks old) by bilateral common carotid artery occlusion (BCCAO) for 18 min. The conditioning ischemic insult lasting for 6 min was carried out 48 h before the 18-min BCCAO. On the seventh day after BCCAO, neuronal damage was visualized by microtubule-associated protein-2 immunohistochemistry and quantified by cresyl violet staining. Terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) was performed 72 h after reperfusion to detect DNA fragmentation. Ischemia for 18 min resulted in injury to the striatum, cortex and hippocampus. In comparison to the hippocampus, striatal neuronal injury was more severe and reproducible. Although the conditioning ischemia itself caused neither noticeable striatal neuronal damage nor DNA fragmentation, it significantly reduced striatal neuronal damage and DNA fragmentation caused by the subsequent 18-min ischemia. These results indicate that striatal neuronal injury after transient BCCAO can be strongly reduced by a sublethal ischemic episode in C57BL/6 mice. As many kinds of gene-altered C57BL/6 mice are available, this preconditioning model may be useful for investigating the molecular mechanisms of ischemic preconditioning in transgenic mice.

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

Intravenous anesthetics differentially reduce neurotransmission damage caused by oxygen-glucose deprivation in rat hippocampal slices in correlation with N-methyl-D-aspartate receptor inhibition.

OBJECTIVE: To examine the relation between the effect of intravenous anesthetics on ischemic neurotransmission damage and their actions on N-methyl-d-aspartate (NMDA) receptors in an in vitro cerebral ischemic model. DESIGN: Prospective, randomized study in freshly prepared rat hippocampal slices. SETTING: University research laboratory. SUBJECTS: Hippocampal slices were prepared from male Wistar rats (4-5 wks old). INTERVENTIONS AND MEASUREMENTS: In vitro ischemia was induced by exposing slices to glucose-free Krebs solution gassed with 95% N2 /5% CO2 at 37.1-37.3 degrees C. Ischemic neurotransmission damage was indicated by the amplitudes of population spikes (PS) recorded from the CA1 pyramidal layer after stimulation of the Schaffer collaterals. The effect of anesthetics on NMDA receptors was determined by measuring the NMDA-mediated changes in intracellular calcium in the CA1 pyramidal layer with a calcium indicator, fura-2. RESULTS: Following 4, 6, and 7.5 mins ischemia in vitro, the recoveries of PS (% control) were 100%, 17.5 +/- 21.8%, and 5.4 +/- 2.1%, respectively. 3-(R)-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 5 microM), an NMDA receptor antagonist, increased the recovery of PS to 88.3 +/- 24.5% after 6 mins ischemia, and to 42.1 +/- 18.7% after 7.5 mins ischemia. Thiopental (400 microM), thiamylal (400 microM), and ketamine (100 microM), but not propofol (100 microM) and etomidate (10 microM), improved the recovery of PS after 6 and 7.5 mins ischemia; the degrees of their protection were comparable to that of 5 microM CPP. The NMDA-mediated increases in intracellular calcium were almost completely inhibited by thiamylal, reduced to half by ketamine and thiopental, augmented by propofol, and not affected by etomidate. CONCLUSIONS: The results indicate that the efficacy of intravenous anesthetics in attenuating ischemic neuronal damage varies among agents, relating to their effects on NMDA receptors.

Thiopental inhibits increases in [Ca2+]i induced by membrane depolarization, NMDA receptor activation, and ischemia in rat hippocampal and cortical slices.

BACKGROUND: This study examined the effects of thiopental on intracellular calcium ([Ca2+]i) changes induced by membrane depolarization, N-methyl-D-aspartate (NMDA) receptor activation, and ischemia. METHODS: Experiments were performed in brain slices prepared from Wistar rats. [Ca2+]i measurements were taken on the CA1 pyramidal cell layer of the hippocampus or layers II to III of the somatosensory cortex using the fura-2 fluorescence technique. Membrane depolarization and NMDA receptor activation were induced by exposing slices to 60 mM K+ and 100 microM NMDA, respectively. In vitro ischemia was induced by superfusing slices with glucose-free Krebs solution equilibrated with 95% nitrogen and 5% carbon dioxide. Thiopental was applied 5 min before application of high K+ and NMDA, or before in vitro ischemia. RESULTS: Ischemia for 15 min produced a characteristic [Ca2+]i increase in both hippocampal and cortical slices. Thiopental prolonged the latency to the appearance of the [Ca2+]i plateau and reduced the magnitudes of increase in [Ca2+]i 8, 10, and 15 min after the onset of ischemia. Thiopental also suppressed the high K+- and NMDA-induced [Ca2+]i increases. The NMDA-induced [Ca2+]i increases were attenuated to a greater extent in cortical slices than were those in hippocampal slices. The inhibition of thiopental on the 200-microM NMDA-mediated [Ca2+]i response was confirmed in cultured cortical neurons. CONCLUSIONS: The results indicate that thiopental attenuates ischemia-induced [Ca2+]i increases in the hippocampus and cortex in vitro, probably because of its inhibition of both voltage-gated calcium channels and NMDA receptors. The regionally different inhibition of thiopental on NMDA receptors may relate to its region-specific action against ischemia.

Quantitative comparison of apoptosis to cell proliferation and p53 protein in breast carcinomas.

OBJECTIVE: To clarify the correlation between apoptosis and tumor cell proliferative activity in human breast cancer and to investigate their relevance to p53 protein. STUDY DESIGN: Seventy-one breast carcinomas with histologic grading were analyzed, using counting of mitotic activity index (MAI) and apoptotic index (AI) to examine apoptosis and cellular proliferation, which were then compared with the expression of p53 protein by using a semiquantitative immunohistochemical method. RESULTS: Both the mean MAI and AI were significantly higher in the grade 3 groups (18.30 +/- 2.18 SE, 13.58 +/- 1.94) and 2 (11.32 +/- 1.30, 9.96 +/- 1.84) than in the grade 1 groups (8.24 +/- 1.10, 8.30 +/- 2.20) (P < .001). Also, MAI/AI was significantly highest in the grade 3 group (P < .001). A significant correlation was found between MAI and AI (r = .767, P < .01). Positive expression of p53 protein, indicated by distinct nuclear staining, was found in 35 of 71 carcinomas and was related to neither MAI nor AI (P > .05); there was no significant relation between p53-positive scoring and histologic grading (P > .05). CONCLUSION: Apoptosis in breast cancer seems to correlate with proliferative activity assessed by the mitotic index and supports the hypothesis that apoptosis may play a role in the selection of clonal subpopulations with high growth potential but is not regulated by the p53 system. Further research needs to be conducted to elucidate the relation between apoptosis and tumor progression and the significance of p53 in abnormalities in breast cancer.

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca2+ and glycolysis.

To elucidate the mechanism of pHi changes induced by membrane depolarization, the variations in pHi and [Ca2+]i induced by a number of depolarizing agents, including high K+, veratridine, N-methyl-D-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pHi and an elevation of intracellular calcium ([Ca2+]i) in the CA1 pyramidal cell layer. The increases in [Ca2+]i caused by the depolarizing agents almost completely disappeared in the absence of Ca2+ (0 mM Ca2+ with 1 mM EGTA). In Ca2+ free media, pHi acid shifts produced by high K+, veratridine or NMDA were attenuated by 10-25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pHi acid shifts induced by both high K+ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K+, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na+-K+ ATPase activity. A Ca2+-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H+ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.

Differential inhibitory effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels and NMDA receptors in rat hippocampal slices.

Although it is known that there are some pharmacological differences between the structurally similar barbiturates, the underlying mechanism of action remains unclear. We have compared the effects of thiopental, thiamylal and phenobarbital on both voltage-gated calcium channels (VGCC) and N-methyl-D-aspartate (NMDA) receptors in rat hippocampal slices by determining changes in intracellular calcium ([Ca2+]i). Experiments were performed in adult rat hippocampal slices perfused with Krebs solution (37 degrees C). Concentrations of [Ca2+]i in the pyramidal cell layer of the CA1 region were measured using a calcium indicator dye, fura-2. To activate VGCC and NMDA receptors, slices were exposed to K+ 60 mmol litre-1 (< or = 60 s) and NMDA 100 mumol litre-1 (30 s), respectively. Thiopental, thiamylal and phenobarbital were present 5 min before, during and 1 min after high K+ or NMDA application. Both thiamylal and thiopental (50-600 mumol litre-1) attenuated the increases in [Ca2+]i produced by high K+ or NMDA in a concentration-dependent manner, while phenobarbital 50-1000 mumol litre-1 only slightly attenuated the [Ca2+]i increase produced by high K+ at concentrations of more than 200 mumol litre-1 and was ineffective on the [Ca2+]i response produced by NMDA. Although the increases in [Ca2+]i caused by membrane depolarization with high K+ were reduced equally with thiamylal and thiopental, thiamylal was more effective in attenuating the increase in [Ca2+]i produced by NMDA receptor activation than thiopental. We conclude that the depressant effects of barbiturates on both VGCC and NMDA receptors varied between agents. Differential inhibition of both VGCC and NMDA receptors may determine the pharmacological properties of barbiturates and their ability to protect neurones against ischaemia.

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices.

As alterations in intracellular pH (pH(i)) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH(i) in rat hippocampal slices using the BCECF fluorescent technique. The 'resting' pH(i) in the CA1 pyramidal cell layers was 6.93 +/- 0.07 (mean +/- S.D., n = 72 slices) in 25 mM HCO3-/5% CO2-buffered solution at 37 degrees C. Exposure of hippocampal slices to NMDA in the range of 10-1000 microM produced a biphasic change in pH(i): an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 microM) but not CNQX (40 microM) blocked the NMDA-induced changes in pH(i). In 0 Ca medium (0 mM Ca2+ supplemented 1 mM EGTA, referred to as 0 Ca), pH(i) acid shift caused by NMDA (20 microM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca2+ ([Ca2+]i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH(i) acid shift by two-thirds, while the NMDA-induced pH(i) alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH(i) acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH(i). The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca2+]i. The pH(i) acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.

Regionally different elevation of intracellular free calcium in hippocampus of septic rat brain.

The effect of sepsis on cellular calcium homeostasis in the central nervous system (CNS) was investigated using hippocampal slices of rats in which sepsis was induced by cecal ligation and puncture (CLP). Hippocampal slices were prepared from septic or sham-operated rats at 24 h after abdominal surgery. The basal intracellular calcium ([Ca2+]i) and its response to oxygen-glucose deprivation in hippocampal slices were measured for assessing cellular calcium homeostasis using fura-2 fluorescent imaging technique. The levels of [Ca2+]i were estimated by the fluorescence ratio (R340/380). Twenty-four hours after CLP, spontaneous movement was reduced and plasma lactate was increased in the septic rats in comparison with the sham-operated rats in which laparotomy was performed without CLP. Basal level of R340/380 in the CA4 ara (.72 +/- .07) was significantly higher (p < .001) in the septic group than that in the sham-operated group (.55 +/- (.06). The fluorescence ratio of septic vs. sham-operated in other hippocampal regions were .55 +/- .09 vs. .48 +/- .06 in CA1 (not significant) and .65 +/- .10 vs. .59 +/- .08 (not significant) in CA3, respectively. Increase in [Ca2+]i due to oxygen-glucose deprivation was significant in CA1 and CA3 of the septic group and in all hippocampal regions of sham-operated group. However, it was not significantly increased in CA4 of the septic group. These results suggest that regional deregulation of cellular calcium occurs in the CNS following CLP. Cellular calcium deregulation may be one of the pathogeneses occurred in clinically observed septic encephalopathy.