Proliferating cells differentiate into neurons in the hippocampal CA1 region of gerbils after global cerebral ischemia

Maternal separation in early life can increase vulnerability to neuropsychiatric disorders over the lifespan. To investigate the effect of acupuncture on cell proliferation in the dentate gyrus (DG), 5-bromo-2'-deoxyuridine (BrdU)-immunohistochemistry was performed in maternally-separated rat pups. Maternal separation, for 7 days from postnatal day 14, induced a significant decrease of BrdU-immunoreactive cells in DG, while acupuncture treatment at acupoint Shenmen (HT7), at the end of the transverse crease of the ulnar wrist, resulted in the significant increase in the number of BrdU-positive cells in DG. However, acupuncture at acupoint ST36, near the knee joint, produced no increase in the number of BrdU-positive cells. These findings indicate that acupuncture at acupoint HT7 appears to stimulate cell proliferation, and we suggested that acupuncture may be useful in the treatment of diseases related to maternal separation.     

PBN fails to suppress in delayed neuronal death of hippocampal CA1 injury following transient forebrain ischemia in gerbils

Free radicals have been suggested to be involved in the genesis of ischemic brain damage, as shown by the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, in ischemic cerebral injury. However, the involvement of free radicals in transient ischemic-induced delayed neuronal death is not fully understood. To clarify this, in the present study, we evaluated the effect of PBN on delayed neuronal death and on the levels of free radicals in hippocampal CA1 region in the gerbil. The administration of PBN (10 mg/kg, i.v.) failed to show any preventive effect on the delayed neuronal death, examined by hematoxylin and eosin staining and the TUNEL method. Furthermore, we observed no free radical formation in delayed neuronal death, determined immunohistochemically using a specific 8-OHdG antibody, after transient ischemic insult. These results suggest that free radical formation may not contribute to the formation of delayed neuronal death.     

Differential effects and changes of ceruloplasmin in the hippocampal CA1 region between adult and aged gerbils after transient cerebral ischemia

In this study, we examined the differential effects and changes of ceruloplasmin between adult and aged gerbil hippocampus after transient forebrain ischemia. Ceruloplasmin in the hippocampal CA1 region of adult and aged gerbils was significantly changed after ischemia/reperfusion. Whereas, it was not significantly changed in the CA2/3 region compared to the CA1 region after ischemia. Ceruloplasmin immunoreactivity and its protein level in aged gerbil CA1 region were higher than those in adult gerbil CA1 region. Ceruloplasmin in the CA1 region was highest in adult gerbils and aged gerbils at 24h and 12h after transient ischemia, respectively. At these time points, strong ceruloplasmin immunoreactivity was observed in CA1 pyramidal cells. Thereafter, ceruloplasmin was decreased with time after ischemia. Four days after ischemia/reperfusion, ceruloplasmin immunoreactivity in both adult and aged gerbils was expressed in astrocytes in the CA1 region. Ceruloplasmin treatment in adult ischemic gerbils showed strong protective effect against ischemic damage in CA1 pyramidal cells compared to that in aged ischemic gerbils. We conclude that ceruloplasmin early increases in the aged gerbil CA1 region compared to that of the adult gerbil CA1 region may be associated with the earlier induction of reactive oxygen species, and ceruloplasmin shows strong neuroprotective effects in adults compared to those in aged gerbils.     

Extrusion of intracellular calcium ion after in vitro ischemia in the rat hippocampal CA1 region

Simultaneous recordings of intracellular Ca(2+) ([Ca(2+)](i)) signal and extracellular DC potential were obtained from the CA1 region in 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid penta-acetoxymethyl ester (Fura-2/AM)-loaded rat hippocampal slices. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) for 5-6 min produced a rapid rise of the [Ca(2+)](i) level in the stratum radiatum (rising phase of the [Ca(2+)](i) signal), which occurred simultaneously with a rapid negative DC potential (rapid negative potential). When oxygen and glucose were reintroduced, the increased [Ca(2+)](i) signal diminished rapidly (falling phase of the [Ca(2+)](i) signal) during the generation of a slow negative DC potential (slow negative potential), which occurred within 1 min from the onset of the reintroduction. Thereafter, the [Ca(2+)](i) signal partially and the slow negative potential completely returned to the preexposure level approximately 6 min after the reintroduction. The changes in [Ca(2+)](i) signal during and after in vitro ischemia were very similar to the changes in the membrane potential of glial cells. The rising and falling phases of [Ca(2+)](i) signal corresponded to the rapid depolarization and a depolarizing hump, respectively, in the repolarizing phase of glial cells. A prolonged application of in vitro ischemia or a reintroduction of either glucose or oxygen suppressed the falling phase after ischemic exposure. The application of ouabain (30 microM) generated both a rapid negative potential and a rapid elevation of [Ca(2+)](i), but no slow negative potential or rapid reduction in [Ca(2+)](i) were observed. When oxygen and glucose were reintroduced to slices in the Na(+)-free or ouabain- or Ni(2+)-containing medium, the falling phase was suppressed. The falling phase was significantly accelerated in Ca(2+)- and Mg(2+)-free with EGTA-containing medium. In contrast, the falling phase was significantly slower in the Ca(2+)-free with high Mg(2+)- and EGTA-containing medium. The falling phase of the [Ca(2+)](i) signal after ischemic exposure is thus considered to be primarily dependent on the reactivation of Na(+), K(+)-ATPases, while the extrusion of cytosolic Ca(2+) via the forward-mode operation of Na(+)/Ca(2+) exchangers in glial cells is thought to be directly involved in the rapid reduction of [Ca(2+)](i) after ischemic exposure.     

Estrogen prevents the loss of CA1 hippocampal neurons in gerbils after ischemic injury

Estrogen replacement therapy is thought to attenuate the incidence of Alzheimer's disease in women and enhance cognitive functions. In rodents, estrogen protects cerebral cortical neurons from ischemic injury and cultured neurons from a variety of perturbations. Because few nuclear estrogen receptors have been detected in the dorsal hippocampus, the present studies used a global ischemia model to evaluate the neuroprotective actions of estrogen in this region. Ovariectomized gerbils were treated with placebo, 0.5 mg or 1 mg pellets of estradiol for 13 days. On day 7, the common carotid arteries were occluded for 5 min and on day 13 the animals were killed. Analysis of neurogranin mRNA, a marker of hippocampal neurons, with in situ hybridization revealed a dramatic and selective loss of CA1 neurons in the placebo-treated ovariectomized gerbils, whereas both 0.5 mg and 1 mg pellets of 17beta-estradiol prevented cell loss. Subsequent studies showed that a variety of estrogens, including diethylstilbestrol, estrone and 17alpha-estradiol as well as vitamin E, also protected CA1 neurons from ischemic injury in ovariectomized gerbils, whereas treatment with the estrogen antagonist tamoxifen was ineffective. The results of in vivo binding studies with 17alpha-iodovinyl-11beta-methoxyestradiol revealed a concentration of nuclear estrogen binding sites in the CA1 region of the ovariectomized gerbil brain, whereas binding in other hippocampal regions was limited. Moreover, the binding studies revealed that the regional pattern of binding was not altered after ischemic injury, with the exception of the hippocampus, where the binding sites were attenuated in ovariectomized animals with time after ischemic injury. Together, these data demonstrate that a variety of steroidal and non-steroidal estrogens are potent neuroprotective agents in an animal model of global ischemia, agents that protect neurons critical for learning and memory and susceptible to neurodegenerative diseases.     

Expression of nerve growth factor in astrocytes of the hippocampal CA1 area following transient forebrain ischemia.

We have examined by immunoassay and immunohistochemistry, the expression of nerve growth factor in the rat hippocampus, one to 28 days after transient forebrain ischemia. In the CA1 area, the overall level of nerve growth factor expression remained constant over the first three days of reperfusion while it increased by about 45% of control levels after longer postischemic periods. In contrast, a slight decrease in nerve growth factor levels, which was most prominent at three days postlesion, was observed in the other hippocampal regions. Immunohistochemical analysis of the distribution of nerve growth factor showed that its expression was up-regulated in astrocytes but not in microglia of the postischemic CA1 region and that the intensity and temporal profile of the changes in nerve growth factor immunostaining in these cells, was consistent with that observed in the immunoassay. Interestingly, the regulation of the nerve growth factor expression in reactive astrocytes of the postischemic CA1 area closely parallels that of kainate receptor subunits GluR5-7, raising the possibility of a cause-effect relationship. These results indicate that after ischemia nerve growth factor expression is up-regulated in reactive astrocytes suggesting that these cells may contribute to rescuing damaged neurons by means of increasing nerve growth factor production.     

Differential changes of potassium currents in CA1 pyramidal neurons after transient forebrain ischemia

CA1 pyramidal neurons are highly vulnerable to transient cerebral ischemia. In vivo studies have shown that the excitability of CA1 neurons progressively decreased following reperfusion. To reveal the mechanisms underlying the postischemic excitability change, total potassium current, transient potassium current, and delayed rectifier potassium current in CA1 neurons were studied in hippocampal slices prepared before ischemia and at different time points following reperfusion. Consistent with previous in vivo studies, the excitability of CA1 neurons decreased in brain slices prepared at 14 h following transient forebrain ischemia. The amplitude of total potassium current in CA1 neurons increased approximately 30% following reperfusion. The steady-state activation curve of total potassium current progressively shifted in the hyperpolarizing direction with a transient recovery at 18 h after ischemia. For transient potassium current, the amplitude was transiently increased approximately 30% at approximately 12 h after reperfusion and returned to control levels at later time points. The steady-state activation curve also shifted approximately 20 mV in the hyperpolarizing direction, and the time constant of removal of inactivation markedly increased at 12 h after reperfusion. For delayed rectifier potassium current, the amplitude significantly increased and the steady-state activation curve shifted in the hyperpolarizing direction at 36 h after reperfusion. No significant change in inactivation kinetics was observed in the above potassium currents following reperfusion. The present study demonstrates the differential changes of potassium currents in CA1 neurons after reperfusion. The increase of transient potassium current in the early phase of reperfusion may be responsible for the decrease of excitability, while the increase of delayed rectifier potassium current in the late phase of reperfusion may be associated with the postischemic cell death.     

Behavior-dependent paired-pulse responses in the hippocampal CA1 region

Summary Paired-pulses of 20–100 ms interpulse interval (IPI) were delivered to the Schaffer collaterals/commissural fibers in order to excite the apical dendrites of the hippocampal CA1 region in freely behaving rats. Significant differences were observed for the paired-pulse responses during different behavioral states. The responses recorded during awake immobility (IMM), and slow-wave sleep (SWS) were similar, but as a group, were different from those during walking (WLK) and rapid-eye-movement sleep (REM). During WLK and REM, the population spike evoked by the second pulse (P2) at IPI of 30 and 50 ms, was greatly facilitated as compared to the population spike evoked by the first pulse (P1), i.e. P2 > P1. During IMM and SWS and using moderate stimulus intensities, P2 was generally smaller than P1 (paired-pulse suppression) at IPI of 30 and 50 ms. The P2/P1 relation with behavior was not caused by the slight variations of P1 with behavior. In addition, paired-pulse facilitation of the population excitatory postsynaptic potentials (EPSP) was relatively small and not significantly dependent on behavior. Behavioral dependence of the paired-pulse responses was not generally found for IPI of 20 or 100 ms. It is concluded that paired-pulse facilitation at 30–50 ms IPI can best be explained by EPSP facilitation combined with a behaviorally dependent disinhibition.     

Changes in membrane properties of CA1 pyramidal neurons after transient forebrain ischemia in vivo

We have previously identified three distinct populations of CA1 pyramidal neurons after reperfusion based on differences in synaptic response, and named these late depolarizing postsynaptic potential neurons (enhanced synaptic transmission), non-late depolarizing postsynaptic potential and small excitatory postsynaptic neurons (depressed synaptic transmission). In the present study, spontaneous activity and membrane properties of CA1 neurons were examined up to 48 h following approximately 14 min ischemic depolarization using intracellular recording and staining techniques in vivo. In comparison with preischemic properties, the spontaneous firing rate and the spontaneous synaptic activity of CA1 neurons decreased significantly during reperfusion; spontaneous synaptic activity ceased completely 36-48 h after reperfusion, except for a low level of activity which persisted in non-late depolarizing postsynaptic potential neurons. Neuronal hyperactivity as indicated by increasing firing rate was never observed in the present study. The membrane input resistance and time constant decreased significantly in late depolarizing postsynaptic potential neurons at 24-48 h reperfusion. In contrast, similar changes were not observed in non-late depolarizing postsynaptic potential neurons. The rheobase, spike threshold and spike frequency adaptation in late depolarizing postsynaptic potential neurons increased progressively following reperfusion. Only a transient increase in rheobase and spike threshold was detected in non-late depolarizing postsynaptic potential neurons and spike frequency adaptation remained unchanged in these neurons. The amplitude of fast afterhyperpolarization increased in all neurons after reperfusion, with the smallest increment in non-late depolarizing postsynaptic potential neurons. Small excitatory postsynaptic potential neurons shared similar changes to those of late depolarizing postsynaptic potential neurons. These results suggest that the enhancement and depression of synaptic transmission following ischemia are probably due to changes in synaptic efficacy rather than changes in intrinsic membrane properties. The neurons with enhanced synaptic transmission following ischemia are probably the degenerating neurons, while the neurons with depressed synaptic transmission may survive the ischemic insult.     

NR2B反义寡核苷酸对脑缺血海马CA1区NR2B蛋白质表达的影响

目的：观察前脑缺血后NR2B反义寡核苷酸（ANR2B）对海马CA1区NR2B蛋白质表达的影响,为临床脑血管疾病的防治以及研制特异性新药提供理论基础。方法：正常SD大鼠,脑缺血手术48h前海马CA1区分别立体定位预注射ANR2B、NR2B正义寡核苷酸（SNR2B）。后行四动脉阻断全脑缺血手术,免疫组织化学反应,观察NR2B蛋白质在ANR2B的作用下的表达变化。结果：海马CA1区立体定位注射ANR2B后,注射区及其周围NR2B免疫组织化学染色强度明显下降,仅有少量锥体细胞散在分布。结论：ANR2B可以在体局部抑制缺血早期NR2B亚单位蛋白表达上升趋势。     

SDF-1α/CXCR7促进BMSCs向缺血/再灌注大鼠海马CA1区迁移

目的:研究基质细胞衍生因子-1α(stromal cell derived-factor-1α,SDF-1α)及其受体CXCR7在介导骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)向大鼠脑缺血区迁移的作用。方法:BMSCs的原代培养、四血管阻断脑缺血模型制备大鼠脑缺血模型、侧脑室移植组(培养基干预组、BMSCs组和CXCR7抗体预处理BMSCs组)、免疫组织化学和免疫荧光组织化学染色方法。结果:免疫组织化学染色结果显示:在各组大鼠海马CA1区细胞内可见不同程度的呈黄色或棕黄色颗粒的SDF-1α免疫阳性产物,主要表达在细胞胞质中,CXCR7蛋白则主要表达在胞核和胞膜中;与假手术组比较,BMSCs组和CXCR7抗体预处理BMSCs组SDF-1α和CXCR7的表达均升高(P<0.05)。免疫荧光组织化学结果显示:侧脑室注射BMSCs并移植48 h后,BMSCs组、CXCR7抗体预处理BMSCs组海马CA1区均有不同程度荧光标记的阳性细胞;与BMSCs组比较,CXCR7抗体预处理BMSCs组阳性细胞数降低(P<0.05)。结论:缺血/再灌注损伤后,促进了SDF-1α及CXCR7阳性产物的表达;同时SDF-1α/CXCR7能够促进BMSCs向损伤区域的迁移。     

Hyperexcitability of hippocampal CA1 region in brain slices after GABA withdrawal.

The interruption of GABA infusion in the cerebral cortex and in the hippocampus produces electrographic seizures in rats. Here, we have used the hippocampal slice preparation to induce a 'GABA withdrawal syndrome (GWS)'. With the stimulation parameters used (0.2 Hz, 200 microseconds), activation of the Schaffer afferents produced one population spike in the CA1 subfield, while multiple population spikes were observed in the slices previously incubated in GABA. Also, we recorded an increase in the amplitude of the population spike when compared to its control value. Paired pulse test showed absence of recurrent inhibition in these slices. These results suggest a dysfunction in GABAergic neurotransmission.     

Protective effect of topiramate against hippocampal neuronal damage after global ischemia in the gerbils

The purpose of this study was to examine whether topiramate would reduce neuronal damage after transient global ischemia in the gerbils because topiramate blocks voltage sensitive sodium channels and non-N-methyl-D-aspartate receptors and enhances gamma-aminobutyric acid-mediated inhibitory transmission. Both common carotid arteries were occluded for 3 min with microaneurysmal clips. The gerbils were treated with topiramate (50, 100, or 200 mg/kg, i.p.) immediately after ischemia. Neuronal cell damage in the hippocampal CA1 region was evaluated quantitatively 7 days after ischemia. Topiramate at the dose of 50 mg/kg failed to reduce hippocampal neuronal damage. However, topiramate when administered at the dose of 100 or 200 mg/kg significantly reduced hippocampal neuronal damage in dose-dependent manner (P<0.001 and P<0.0005, respectively). These results suggest that topiramate has a neuroprotective effect against neuronal damage following global ischemia in the gerbils.     

Alterations of single potassium channel activity in CA1 pyramidal neurons after transient forebrain ischemia.

Selective neuronal injury in the CA1 zone of hippocampus following transient cerebral ischemia has been well documented. Extracellular potassium concentration markedly increases during ischemia/hypoxia. Accumulating evidence has indicated that the outward potassium currents, including delayed rectifier potassium current, not only influence membrane excitability but also mediate apoptosis. It has been shown that the amplitude of delayed rectifier potassium current in CA1 neurons significantly increased after cerebral ischemia. To elucidate the mechanisms underlying the changes of potassium currents following ischemia, single potassium channel activities of rat CA1 neurons were compared before and after transient forebrain ischemia. Using cell-attached configuration, depolarizing voltage steps activated outward single channel events. The channel properties, the kinetics and pharmacology of these events resemble the delayed rectifier potassium current. After ischemia, the unitary amplitude of single channels significantly increased, the open probability, mean open time and open time constant also significantly increased while the conductance remained unchanged. These data indicate that the increase of single channel activity is responsible, at least in part, for the increase of delayed rectifier potassium current in CA1 neurons after cerebral ischemia.     

Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence against programmed cell death

Summary The ultrastructural changes in the pyramidal neurons of the CA1 region of the hippocampus were studied 6 h, 24 h, 48 h, and 72 h following a transient 10 min period of cerebral ischemia induced by common carotid occlusion combined with hypotension. The pyramidal neurons showed delayed neuronal death (DND), i.e. at 24 h and 48 h postischemia few structural alterations were noted in the light microscope, while at 72 h extensive neuronal degeneration was apparent. The most prominent early ultrastructural changes were polysome disaggregation, and the appearance of electron-dense fluffy dark material associated with tubular saccules. Mitochondria and nuclear elements appeared intact until frank neuronal degeneration. The dark material accumulated with extended periods of recirculation in soma and in the main trunks of proximal dendrites, often beneath the plasma membrane, less frequently in the distal dendrites and seldom in spines. Protein synthesis inhibitors (anisomycin, cycloheximide) and an RNA synthesis inhibitor (actinomycin D), administered by intrahippocampal injections or subcutanously, did not mitigate neuronal damage. Therefore, DND is probably not apoptosis or a form of programmed cell death. We propose that the dark material accumulating in the postischemic period represents protein complexes, possibly aggregates of proteins or internalized plasma membrane fragments, which may disrupt vital cellular structure and functions, leading to cell death.     

Transient enhancement of inhibitory synaptic transmission in hippocampal CA1 pyramidal neurons after cerebral ischemia

Pyramidal neurons in hippocampal CA1 regions are highly sensitive to cerebral ischemia. Alterations of excitatory and inhibitory synaptic transmission may contribute to the ischemia-induced neuronal degeneration. However, little is known about the changes of GABAergic synaptic transmission in the hippocampus following reperfusion. We examined the GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in CA1 pyramidal neurons 12 and 24 h after transient forebrain ischemia in rats. The amplitudes of evoked inhibitory postsynaptic currents (eIPSCs) were increased significantly 12 h after ischemia and returned to control levels 24 h following reperfusion. The potentiation of eIPSCs was accompanied by an increase of miniature inhibitory postsynaptic current (mIPSC) amplitude, and an enhanced response to exogenous application of GABA, indicating the involvement of postsynaptic mechanisms. Furthermore, there was no obvious change of the paired-pulse ratio (PPR) of eIPSCs and the frequency of mIPSCs, suggesting that the potentiation of eIPSCs might not be due to the increased presynaptic release. Blockade of adenosine A1 receptors led to a decrease of eIPSCs amplitude in post-ischemic neurons but not in control neurons, without affecting the frequency of mIPSCs and the PPR of eIPSCs. Thus, tonic activation of adenosine A1 receptors might, at least in part, contribute to the enhancement of inhibitory synaptic transmission in CA1 neurons after forebrain ischemia. The transient enhancement of inhibitory neurotransmission might temporarily protect CA1 pyramidal neurons, and delay the process of neuronal death after cerebral ischemia.     

Na+/Ca2+ exchanger activity induces a slow DC potential after in vitro ischemia in rat hippocampal CA1 region

In rat hippocampal CA1 neurons recorded intracellularly from tissue slices, a rapid depolarization occurred approximately 5 min after application of ischemia-simulating medium. In extracellular recordings obtained from CA1 region, a rapid negative-going DC potential (rapid DC potential) was recorded, corresponding to a rapid depolarization. When oxygen and glucose were reintroduced after generating the rapid depolarization, the membrane further depolarized and the potential became 0 mV after 5 min. Contrary, the DC potential began to repolarize slowly and subsequently a slow negative-going DC potential (slow DC potential) occurred within 1 min. A prolonged application of ischemia-simulating medium suppressed the slow DC potential. Addition of a high concentration of ouabain in normoxic medium reproduced a rapid but not a slow DC potential. The slow DC potential was reduced in low Na+- or Co2+-containing medium, but was not affected in low Cl-, high K+ or K+-free medium, suggesting that the slow DC potential is Na+-and Ca2+-dependent. Ni2+ (Ca2+ channel blocker as well as the Na+/Ca2+ exchanger blocker) and benzamil hydrochloride (Na+/Ca2+ exchanger blocker) reduced the slow DC potential dose-dependently. These results suggest that the slow DC potential is mediated by forward mode operation of Na+/Ca2+ exchangers in non-neuronal cells, and that reactivation of Na+, K+-ATPase is necessary to the Na+/Ca2 +exchanger activity.     

Morphine modulates glutamate release in the hippocampal CA1 area in mice

Opiate abuse is associated with long-lasting neural adaptative changes in the brain. Increasing evidence demonstrates that opiates significantly alter the function of the glutamatergic system, while how the system is regulated still remains elusive. In the present study, we studied the effect of morphine on extracellular glutamate concentration in the hippocampus, a nucleus rich of the glutamatergic neurons. The results showed that glutamate concentration in the extracellular fluid of the hippocampus was decreased following either acute or chronic treatment of morphine. However, naloxone-induced withdrawal increased glutamate concentration significantly. These results suggest an adaptation of the glutamatergic neuronal transmission in the hippocampus after morphine treatment.     

Auguementations in activity of calcium-activated large conductance potassium channel in pyramidal neurons acutely isolated from adult rat hippocampal CAI region after transient forebrain ischemia

<正> Recent studies have demonstrated that many electrophysiological characteristics of phramidal neurons in hippocanmpalCAl region are changed after transient forebrain ischemia.Large conductance calcium-adtivated potassium(BK_(Ca))channel plays critical roles insetting neuronal electrophysiological properties.Here we examined whether activity of BK_(Ca) channel in CA1 pyramidal neurons of adult rat hip-pocampus changed after transient forebrain ischemia using inside-out configuration of patch clamp techocniques.The results were as follows:(a)Alternation in sensitvity to [Ca~(2+)],was found when the [Ca~(2+)]_i.required to half activate(P_0=0.5)BK_(Ca) channel increased from 2×10~(-6)M to1×10~(-6)M and voltage dependence of BK_(Ca)channel showed no obvious change after ischewia;(b)The unitary conductance of BK_(Ca) channel in-creased by about 20% (from 245 pS to 295 pS in symmetrical 14O/140mM K~+ in inside-out patch)after ischemia;(c)Mean open time of BK_(Ca)channel increased from 12.20 ms to 17.32ms,and