Enhancement in activities of large conductance calcium-activated potassium channels in CA1 pyramidal neurons of rat hippocampus after transient forebrain ischemia

It has been reported previously that the neuronal excitability persistently suppresses and the amplitude of fast afterhyperpolarization (fAHP) increases in CA1 pyramidal cells of rat hippocampus following transient forebrain ischemia. To understand the conductance mechanisms underlying these post-ischemic electrophysiological alterations, we compared differences in activities of large conductance Ca(2+)-activated potassium (BK(Ca)) channels in CA1 pyramidal cells acutely dissociated from hippocampus before and after ischemia by using inside-out configuration of patch clamp techniques. (1) The unitary conductance of BK(Ca) channels in post-ischemic neurons (295 pS) was higher than that in control neurons (245 pS) in symmetrical 140/140 mM K(+) in inside-out patch; (2) the membrane depolarization for an e-fold increase in open probability (P(o)) showed no significant differences between two groups while the membrane potential required to produce one-half of the maximum P(o) was more negative after ischemia, indicating no obvious changes in channel voltage dependence; (3) the [Ca(2+)](i) required to half activate BK(Ca) channels was only 1 microM in post-ischemic whereas 2 microM in control neurons, indicating an increase in [Ca(2+)](i) sensitivity after ischemia; and (4) BK(Ca) channels had a longer open time and a shorter closed time after ischemia without significant differences in open frequency as compared to control. The present results indicate that enhanced activity of BK(Ca) channels in CA1 pyramidal neurons after ischemia may partially contribute to the post-ischemic decrease in neuronal excitability and increase in fAHP.     

ATP modulation of large conductance Ca-activated K channels via a functionally associated protein kinase A in CA1 pyramidal neurons from rat hippocampus

Using inside-out configuration of patch clamp techniques, ATP modulation of BK(Ca) channels was studied in hippocampal CA1 pyramidal neurons of adult rat. Intracellular ATP application markedly increased BK(Ca) channel activity, and this ATP-produced increase in BK(Ca) channel activity was characterized by a higher opening frequency with no changes in channel open times. In the presence of specific inhibitor against protein kinase A, H-89, ATP did not induce any increase in the channel activity. Furthermore, adding H-89 after addition of ATP reversed the modulation produced by ATP. In contrast, protein kinase C inhibitor chelerythrine exerted no apparent effects on ATP-induced channel activation. The present study suggests that BK(Ca) channels from hippocampal CA1 pyramidal neurons could be modulated by ATP via a functionally associated protein kinase A-like protein.     

Tetrahydroberberine blocks membrane K channels underlying its inhibition of intracellular message-mediated outward currents in acutely dissociated CA1 neurons from rat hippocampus

In the patch-clamp perforated whole-cell recording mode, tetrahydroberberine (THB), a novel dopamine (DA) receptor antagonist, inhibits not only DA-induced outward K⁺ currents, but also acetylcholine-, caffeine- or strychnine-induced outward current. However, THB does not affect either GABA- or glycine-induced Cl⁻ currents, or non-NMDA receptor agonist-induced cation currents. As expected for a K⁺ channel blocker, THB evokes a downward current deflection accompanied by a decrease of conductance. It is concluded that the direct blockade of membrane K⁺ channels by THB underlies its inhibition of intracellular message-mediated outward currents.     

Elevation of Na+-K+ ATPase immunoreactivity in GABAergic neurons in gerbil CA1 region following transient forebrain ischemia

In a previous study, we suggested that GABAergic neurons might be resistant to ischemic insult, because of the maintenance of the GABA shunt, which is one of the ATP synthetic pathways in neurons. In the present study, we identified Na(+)-K(+) ATPase immunoreactivity in the gerbil hippocampus in order to determine whether changes in Na(+)-K(+) ATPase immunoreactivity correlate with GABA shunt following ischemic insult. At 12 h after ischemia-reperfusion, Na(+)-K(+) ATPase immunoreactivity accumulated in some neurons in the CA1 region. However, the protein content of Na(+)-K(+) ATPase was not altered. Interestingly, the density of Na(+)-K(+) ATPase immunoreactivity in neurons and the protein content in the CA1 region was intensified in the 24 h post-ischemic group. As a result of double immunofluorescence study, Na(+)-K(+) ATPase immunoreactive neurons were identified with GABAergic neurons. Therefore, our findings suggest that the increase of Na(+)-K(+) ATPase in GABAergic neurons may be able to explain the resistance of these cells to ischemic insult, and support our previous hypothesis that GABA may play an important role as a metabolite in the survival of GABAergic neurons after ischemic insult.     

Melatonin administration protects CA1 hippocampal neurons after transient forebrain ischemia in rats

Melatonin administered at the beginning of cerebral reperfusion protected CA1 neurons against 10, 20 and 30 min of transient forebrain ischemia. Intraperitoneal injections of saline or melatonin (10 mg/kg) were given after 0, 2 and 6 h, or 1, 2 and 6 h of cerebral reperfusion, or 30 min prior to ischemia. One week later, quantitative histological analysis demonstrated that CA1 neuronal density was significantly increased in the melatonin groups that were treated at 0, 2, 6 h compared to the saline-treated controls. Ischemic protection of CA1 was lost in the animals in which the melatonin treatment was delayed by 1 h, or given 30 min prior to the ischemia.     

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.     

Agonist-specific maturation of GIRK current responses in acutely isolated pyramidal neurons of rat neocortex

We performed whole-cell recordings from acutely isolated pyramidal cell somata of rat neocortex to measure and compare G protein-activated, inwardly rectifying K+ (GIRK) currents induced by adenosine, serotonin and baclofen at different postnatal stages (postnatal days 3-19). In about two thirds of neurons, baclofen-induced GIRK currents were already detected at postnatal days 3 and 4 (P3-P4) and almost all neurons between P5 and P19 were responsive. This robust response suggests that postsynaptic effects of baclofen occur much earlier than previously thought. Sensitivity to adenosine was around 70% during the first two postnatal weeks. Given the late maturation of functional synaptic inhibition in neocortex, we propose that phasic and/or tonic activation of GIRK current by baclofen and adenosine might serve as a mechanism to control neuronal excitability during early postnatal development. In marked contrast to the pronounced early sensitivity to baclofen and adenosine, only 20% of the neurons displayed a GIRK current response to serotonin during the first postnatal week. After that, about half of the neurons tested positive for serotonin. GIRK current densities for baclofen and adenosine attained a maximum at the end of the second postnatal week, whereas the serotonin-induced current showed a linear increase during the second and third week of life. Set in relationship with previous data on the postnatal expression of receptor protein and GIRK channel mRNA, our findings suggest that the maturation of GIRK current responses is determined predominantly by the different postnatal patterns of receptor expression.     

Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia

Summary We show a differential up-regulation of immunomolecules in the rat dorsal hippocampus accompanying neuronal cell death as a consequence of transient forebrain ischemia (four-vessel occlusion model). Using a panel of monoclonal antibodies (mAbs), we have examined the time course of expression of major histocompatibility complex (MHC) antigens class I (OX-18) and class II (OX-6), leukocyte common antigen (OX-1), CD4 (W3/25) and CD8 (OX-8) antigens, CR3 complement receptor (OX-42), as well as brain macrophage antigen (ED2). The study was performed at time intervals ranging from 1 to 28 days after reperfusion. Throughout all post-ischemic time periods, strongly enhanced immunoreactivity on microglial cells in the CA1 region and dentate hilus and, to a lesser extent, in CA3 was demonstrated with mAb OX-42. MHC class I-positive cells (OX-18) appeared on day 2, whereas cells immunoreactive with OX-1 and W3/25 became evident in the CA1 and hilar regions on post-ischemic day 6. In contrast, MHC class II (Ia) antigen was first detected on indigenous microglia by day 13. In some animals, the OX-8 antibody resulted in the labelling of scattered CD8-positive lymphocytes, but perivascular inflammatory infiltrates were absent. No changes in the expression of ED2 immunoreactivity on perivascular cells could be observed. The results show that following ischemic injury, microglial cells demonstrate a timedependent up-regulation and de novo expression of certain immunomolecules, indicative of their immunocompetence. The findings are compared with those obtained in other models of brain injury.Supported in part by NIH/NINCDS PO 1 NS27511     

Gabapentin potentiates the conductance increase induced by nipecotic acid in CA1 pyramidal neurons in vitro

The anticonvulsant gabapentin (1-(aminomethyl)cyclohexane acetic acid) has been found to be effective for treatment of partial seizures, but the mechanism of action is unknown. Recent evidence from the rat optic nerve suggests that gabapentin may enhance promoted release of GABA, which is thought to be due to reverse operation of the GABA transporter. We have used whole-cell patch clamp recordings from CA1 pyramidal neurons in hippocampal slices to directly measure currents induced by nipecotic acid (NPA) during exposure to gabapentin. Under control conditions, pressure microejection of NPA increased whole-cell conductance with a reversal potential equal to the chloride equilibrium potential. This response was mimicked by GABA application, and blocked by bicuculline. The response to NPA was also present after blockade of synaptic transmission in the presence of calcium-free solution. These results are consistent with NPA promoting nonvesicular release of GABA from neighboring neurons or glia via reverse operation of the GABA uptake system, which then activated GABA_A receptors on the recorded neurons. In control solution, the response to NPA slowly decreased over 45 min to approximately 50% of the initial response, consistent with GABAA receptor ‘rundown’. However, in the presence of gabapentin there was a ;slow increase in the response, reaching approximately 170% of the control level after 45 min of gabapentin exposure. These results demonstrate that gabapentin enhances the promoted release of GABA by more than three-fold. The potentiation of the NPA response may be due to gabapentin increasing cytosolic GABA in neighboring cells via a delayed metabolic effect, and would have the functional effect of increasing neuronal inhibition during periods of hyperexcitability.     

Beta-agkistrodotoxin inhibits large-conductance calcium-activated potassium channels in rat hippocampal CA1 pyramidal neurons

Effect of β-agkistrodotoxin (β-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK_Ca) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K⁺ (150 mM) and 1 μM intracellular Ca²⁺ conditions, internal application of β-AgTx inhibited the activity of BK_Ca by reducing open probability (P_o) of the channels in a concentration-dependent manner. High concentration (74 nM) of β-AgTx completely eliminated opening of the channels. However, 37 nM β-AgTx (at −40 mV) decreased P_o from 0.49±0.07 to 0.03±0.03, switched two open time constants (0.51±0.32 and 8.77±1.63 ms) to be a single time constant of 0.46±0.40 ms. The results indicate that inhibition of BK_Ca by β-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.     

Rapid decrease of high affinity ouabain binding sites in hippocampal CA1 region following short-term global cerebral ischemia in rat

High affinity [3H]ouabain binding was examined in the hippocampal CA1 region and frontal cortex of rats subjected to 5 min complete cerebral ischemia in a clinical death model, and to subsequent resuscitation. A decrease of Bmax directly after ischemia and its further gradual decrease during 120 min of reperfusion were noted in the ischemia-vulnerable CA1 region, whereas no change of Bmax was observed in frontal cortex. The apparent Kd constant showed insignificant fluctuations in either of the two brain regions. Since ouabain binds with high affinity to the neuronal (alpha +)-form of Na+/K+-ATPase, the results indicate a rapid enzyme loss in CA1 neurons. The high affinity ouabain binding test proved to be a sensitive detector of premorphological changes in nerve cell membranes in ischemia.     

Postischemic alterations of spontaneous activities in rat hippocampal CA1 neurons

Changes of spontaneous impulse discharges in rat hippocampal neurons during and after transient forebrain ischemia were investigated electrophysiologically. Spontaneous impulse frequencies of CA1 neurons before ischemia were varied from 0.4 to 20.0 impulses/s and its average was5.8 ± 1.2 (means±S.E., n = 36). These spontaneous discharges were completely suppressed during forebrain ischemia exept for the transient hyperactivity observed just after the beginning of ischemia. Recovery of spontaneous discharges of CA1 neurons from suppression induced by 5 min ischemia started at 5 min, and neuronal activities were restored to pre-ischemic levels approximately 30 min after reperfusion. On the other hand, spontaneous impulse frequencies at all time points recorded after 20 min ischemia were less than 40% of the pre-ischemic levels. These continuous suppression of spontaneous activity after 20 min ischemia may suggest that neuronal function is impaired during and/or in the early stages of reperfusion, and functional disorders precede morphological degeneration.     

Acetylcholine responses in snail neurons: Increase and decrease in potassium conductance succeeding inward currents

In the identified neurons B1 and B3 of the buccal ganglion of Helix pomatia, the initial acetylcholine (ACh) inward current was succeeded by two types of secondary responses. The secondary responses consisted either in an outward current or in a long-lasting inward current or in a combination of both. The secondary outward current was decreased with membrane hyperpolarization, associated with a decrease of membrane resistance and abolished in Ca2+-free Co2+ solution. It is assumed to be a K+ current activated by an influx of Ca2+. The secondary inward current also decreased with membrane hyperpolarization, but was associated with an increase of the membrane resistance and could be mimicked by an injection of Na+ into the cells. It is suggested to be due to a block of K+ channels by intracellular Na+. When the secondary responses appeared combined, the outward current preceded the inward current.     

Temporal changes in extracellular acetylcholine and CA1 pyramidal cells in gerbil hippocampus following transient cerebral ischemia

Temporal changes in cholinergic functions following transient cerebral ischemia (10 min) were studied in the hippocampus of awake unrestrained gerbils using in vivo microdialysis. These data were compared with the results for temporal change in the area of each CA1 cell soma, measured with a microcomputer imaging device. KCl-induced release of acetylcholine (ACh) tended to be lower within 1 day after recirculation, and was significantly lower on the 4th, 7th and 14th days. Atropine-induced release of ACh gradually decreased over the test period. In histological estimation, no differences were observed within the 1st day, but a significant decrease of the area of CA1 cell soma was observed from the 4th to 14th days. Moreover, ischemia over 2 min decreased KCl- and atropine-induced ACh release on the 14th day without significant changes of hippocampal CA1 pyramidal cell. From these results, it is clear that ischemia produced dysfunction of hippocampal cholinergic neurons, and that dysfunction of the hippocampal cholinergic system following transient ischemia precedes pyramidal cell damage in the hippocampal CA1 subfield.     

Delayed neuronal death in the rat hippocampus following transient forebrain ischemia

Summary An unusual, slowly progressing neuronal damage has been reported to occur in the gerbil hippocampus following ischemia (Kirino 1982). Delayed neuronal death following ischemia has also been noticed in the rat four-vessel occlusion model (Pulsinelli et al. 1982). By light microscopy this slow neuronal injury in the rat was not different from the previously known neuronal ischemic cell change. This report lead us to the question as to whether neurons in the rat hippocampus are damaged rapidly following an initial latent period or deteriorate slowly and progressively until they display overt changes. To clarify this point, observation was done on the hippocampal CA1 sector of the rat following ischemia. Rats were subjected to four-vessel occlusion, and those which developed ischemic symptoms were perfusion-fixed. Although the change appeared very slowly and lacked microvacuolation of the cytoplasm, neuronal alteration was practically not different from classical ischemic cell change. By electron microscopy, however, the change was detectable when the neurons still appeared intact by light microscopy. An increase in the membranous organelles and deposition of dark substances were the initial manifestations. It seemed that the CA1 neurons deteriorated very slowly and progressively, and that they retained partial viability in the initial phase of the change. In spite of the difference in light-microscopic findings, the mechanisms underlying delayed neuronal death in the rat and gerbil hippocampus seemed to be identical.     

Voltage-gated K channels in chemoreceptor sensory neurons of rat petrosal ganglion

A subpopulation of sensory neurons in the petrosal ganglion transmits information between peripheral chemoreceptors (glomus cells) in the carotid body and relay neurons in the nucleus of the solitary tract. Expression of voltage-gated K⁺ channels in these neurons was characterized by immunohistochemical localization. Five members of the Kv1 family, Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 and members of two other families, Kv2.1 and Kv4.3, were identified in over 90% of the chemoreceptor neurons. Although the presence of these channel proteins was consistent throughout the population, individual neurons showed considerable variation in K⁺ current profiles.     

Sevoflurane-induced ionic current in acutely dissociated CA1 pyramidal neurons of the rat hippocampus

The electrophysiological properties of sevoflurane (Sev)-induced current (I_Sev) were investigated in CA1 pyramidal neurons freshly dissociated from the rat hippocampus by using the nystatin perforated patch recording configuration under voltag-clamp condition. Within the range of Sev concentrations from 3 · 10⁻⁴ to 2 · 10⁻³ M, I_sev was an inward current which consisted of an initial transient peak component and a successive steady-state plateau component. The peak current component increased in a concentration-dependent manner with a conductance increase. The application of Sev over 2 · 10⁻³ M, however, suppressed the peak and steady-state current components with a concomitant decrease in conductance and elicited a transient inward current (‘hump’ current) immediately after wash out. The current-voltage relationship for I_Sev showed some outward rectification suggesting a slight voltage-dependency of the I_sev. The reversal potential of I_Sev (E_Sev) was close to the E_Cl and shifted by 52 mV for a 10-fold change in extracellular Cl⁻ concentrations, indicating that I_Sev is passing through Cl⁻ channels. The single channel conductance obtained from the analysis of the variance of I_Sev fluctuations was 15.3 ± 1.3pS.     

Methionine alters Na,K-ATPase activity, lipid peroxidation and nonenzymatic antioxidant defenses in rat hippocampus

In the present study we investigated the effect of methionine exposure of hippocampus homogenates on Na+,K+-ATPase activity from synaptic plasma membrane of rats. Results showed that methionine significantly decreased this enzyme activity. We also evaluated the effect of incubating glutathione (GSH) and trolox (alpha-tocopherol) alone or combined with methionine on Na+,K+-ATPase activity. The tested antioxidants per se did not alter the enzymatic activity, but prevented the inhibitory action of methionine on Na+,K+-ATPase activity, indicating that Met inhibitory effect was probably mediated by free radical formation. Besides, we tested the in vitro effect of methionine on some parameters of oxidative stress, namely chemiluminescence, thiobarbituric acid reactive substances (TBARS), total radical-trapping antioxidant potential (TRAP), as well as on the antioxidant enzyme activities catalase, glutathione peroxidase and superoxide dismutase in rat hippocampus. We observed that methionine significantly increased chemiluminescence and TBARS, decreased TRAP, but did not change the activity of the antioxidant enzymes. These findings suggest that reduction of Na+,K+-ATPase activity and induction of oxidative stress may be involved in the brain damage observed in human hypermethioninemia.     

缺氧对大鼠大脑皮层神经元钙激活性钾通道的影响

目的　研究缺氧对大鼠大脑皮层神经元钙激活性钾 (Kca)通道的影响 ,以揭示神经元抗缺血损伤的电生理机制。方法　在不同缺氧条件下 ,应用膜片钳技术记录大脑皮层神经元上Kca通道电流活动。电流信号经放大、滤波及A/D、D/A转换后输入微机进行采样和储存。实验数据应用PClamp(6 .0 .2 )软件进行分析处理。结果　缺氧对通道的开放概率 (Po)及平均开放时间 (To)有明显影响 ,在缺氧实验早期通道Po明显增加 ,其中 10 μmol·L-1NaCN缺氧组其增加程度大于 2 0 μmol·L-1和 30 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。而在缺氧实验后期通道Po和To明显降低 ,其中 30 μmol·L-1NaCN缺氧组其降低程度大于 2 0 μmol·L-1和 10 μmol·L-1NaCN缺氧组 (P <0 .0 5 )。结论　缺氧早期大脑皮层神经元Kca通道激活 ,产生超极化电位 ,从而稳定细胞膜 ,降低细胞兴奋性 ,延缓缺氧除极的发生 ,这可能是神经元自身的一种代偿作用     

Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus

The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94+/-4.96 microM and 10.86+/-1.06 microM, Emax of 67.47+/-4.00% and 67.14+/-1.79%, n of 0.77+/-0.08 and 0.96+/-0.07, respectively. Berberine 30 microM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 microM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage.