Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons.

Nicotinic acetylcholine (ACh) currents were studied in cultured postnatal rat hippocampal neurons, using whole-cell voltage-clamp techniques. In most cells, ACh produces one of two types of response. One class of ACh currents exhibits rapid and profound desensitization and is sensitive to inhibition by alpha-bungarotoxin (alpha BTXN). The second class activates slowly and exhibits no desensitization during prolonged agonist applications. This slow current is insensitive to alpha BTXN. Both the fast and slow responses exhibit inwardly rectifying current-voltage relationships and pass little current at positive membrane potentials. Both currents can be recorded in the presence of 1 microM atropine but are blocked by 0.1-1.0 mM d-tubocurarine and 0.1-1.0 mM mecamylamine. These observations suggest heterogeneity of nicotinic ACh receptors in rat hippocampal neurons and provide support for functional alpha BTXN-sensitive nicotinic receptors in this region.     

盐酸埃他卡林对大鼠海马神经元谷氨酸受体功能及突触活动的影响

目的 :研究盐酸埃他卡林 (Ipt)对脑神经元谷氨酸受体功能及突触活动的影响。方法 :采用原代培养的大鼠海马神经元 ,应用膜片钳全细胞记录技术 ,记录Ipt对培养的海马神经元谷氨酸或天冬氨酸(NMDA)诱发电流及神经元突触后电流的影响。结果 :Ipt(1～ 1 0 0 μmol·L- 1)可浓度依赖性地对抗培养的海马神经元谷氨酸或NMDA诱发电流 ,并为ATP敏感性钾通道拮抗剂格列本脲 30 μmol·L- 1所对抗。Ipt抑制培养的海马神经元之间突触联系形成的自发兴奋性突触后电流 ,降低其发放频率 ,抑制其电流幅度 ;但对微小兴奋性突触后电流无显著性影响。结论 :Ipt可阻断脑神经元谷氨酸受体功能 ,抑制脑神经元谷氨酸的兴奋性突触传递 ,其作用与ATP敏感性钾通道相关     

司帕沙星、联苯乙酸对大鼠海马神经元GABA-A和离子型谷氨酸受体的作用(英文)

目的 :观察司帕沙星单用和与联苯乙酸合用时 ,对γ 氨基丁酸 (GABA) ,NMDA ,AMPA和海人藻酸诱导电流的作用。方法 :应用全细胞式膜片钳技术在急性打散的新生大鼠海马锥体神经元上记录GABA ,NMDA ,AMPA和海人藻酸的诱导电流。结果 :司帕沙星 1mmol·L- 1对GABA诱导的电流有迅速可逆的抑制作用 ,抑制率为 (4 7.6±s2 .9) % ,而联苯乙酸 10 μmol·L- 1无抑制作用。但联苯乙酸 (10 μmol·L- 1)与司帕沙星 1mmol·L- 1合用时 ,稍稍增强抑制作用 ,司帕沙星单用或合用联苯乙酸 (10 μmol·L- 1)对GABA诱导电流的IC50 分别为 (2 38± 2 1) μmol·L- 1和 (89± 5 ) μmol·L- 1。司帕沙星、联苯乙酸或司帕沙星合用联苯乙酸对NM DA ,AMPA和海人藻酸诱导的电流无明显作用。结论 :提示司帕沙星 ,或与联苯乙酸合用时所产生的中枢神经毒性可能与抑制GABA诱导的电流有关 ,而与NMDA ,AMPA和海人藻酸诱导的电流无关。     

Effects of a benz[e]indene on gamma-aminobutyric acid-gated chloride currents in cultured postnatal rat hippocampal neurons.

Benz[e]indenes (BIs) are tricyclic molecules that can be envisioned as steroids without an A-ring. Because certain steroids are known to alter gamma-aminobutyric acid (GABA) responses in central neurons, we examined the effects of a substituted BI resembling 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) on GABA-gated chloride currents in cultured postnatal rat hippocampal neurons. The compound, BI-1, reversibly potentiated GABA currents at concentrations of > 10 nM, with an EC50 value of 0.2 microM. BI-1 increased the apparent affinity of GABA for its receptor, decreasing the GABA EC50 from 9 microM to 3 microM. BI-1 had no effect on the shape of the GABA current-voltage relationship and did not alter the GABA reversal potential. The effects of BI-1 were not altered by benzodiazepine or picrotoxin site antagonists. At concentrations up to 10 microM, where maximal effects on GABA currents were seen, BI-1 did not directly activate a membrane current. This contrasts with the effects of 3 alpha-OH-DHP, which activated chloride currents at concentrations that were subsaturating for GABA potentiation. These results suggest that the BIs may be useful for determining the mechanisms by which steroids potentiate GABA responses and directly gate chloride channels.     

水杨酸钠对大鼠海马神经元电压门控性钙离子通道的影响

目的研究水杨酸钠对大鼠海马神经元上电压门控性钙离子通道的作用,为海马参与耳鸣引起的情绪变化提供电生理学的依据。方法利用脑片膜片钳技术观察水杨酸钠对大鼠海马神经元电压门控性钙离子通道电生理学特性的影响。结果水杨酸钠对电压门控性钙离子通道电流(ICa)有抑制作用,而且具有浓度依赖性(0.1¹0 mmol·L-1)。水杨酸钠对海马神经元ICa的半抑制浓度(IC50)为1.64。1mmol·L-1水杨酸钠对ICa的稳态激活动力学有明显影响,使ICa的稳态激活曲线向超极化方向移动大约9 mV左右,但是对其稳态失活动力学没有明显影响。结论水杨酸钠以浓度依赖的方式抑制ICa,对其稳态激活动力学有明显影响。水杨酸钠对大鼠海马神经元上ICa的影响可能与海马参与耳鸣引起的情绪变化相关。     

Development of GABAB subunits and functional GABAB receptors in rat cultured hippocampal neurons

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) play a critical role in inhibitory synaptic transmission in the hippocampus but the ontogeny of their subunit synthesis and synaptic localisation has not been determined. Here we report the distributions and developmental profiles of GABA(B1) and GABA(B2) subunits in cultured rat embryonic hippocampal neurons. Limited levels of GABA(B1) and GABA(B2) immunoreactivity were present at 3 days in vitro (DIV). At 7 DIV, when baclofen-evoked inwardly rectifying K(+) channel-mediated responses first appear in the cells, there was a more widespread expression within the soma and proximal dendrites. Levels of the K(+) channel GIRK 1 were relatively constant at all time points suggesting channel availability does not limit the appearance of functional GABA(B)Rs. At 14 DIV the staining displayed a punctate dendritic distribution and near maximal GABA(B)R-mediated electrophysiological responses were obtained. About half of the puncta for each GABA(B)R subunit in dendrites co-localised with the synaptic marker SV2a suggesting that these subunits are at or very near to synapses. Interestingly, at all ages strong GABA(B)R immunoreactivity was also present in the nuclei of neurons. These results provide an important developmental baseline for future studies aimed at investigating, for example, the trafficking and functional regulation of these receptors.     

罗哌卡因和布比卡因对大鼠海马神经元谷氨酸电流的影响

目的:观察和比较不同浓度的局麻药罗哌卡因和布比卡因对大鼠海马神经元谷氨酸诱发电流的作用,以探讨罗哌卡因神经毒性低的可能机制。方法:分离大鼠海马神经元并培养,利用全细胞膜片钳技术记录细胞谷氨酸诱发电流,并观察罗哌卡因和布比卡因对谷氨酸电流的影响。结果:谷氨酸(100μmol.L-1)可在海马神经元细胞上诱发出一内向电流,并且此电流可被非N-甲基-天门冬氨酸(NMDA)受体拮抗剂DNQX所阻断;布比卡因(10、50、100μmol.L-1)和罗哌卡因(10、50、100μmol.L-1)均可使海马神经元细胞的谷氨酸诱发电流明显降低;罗哌卡因高浓度(501、00μmol.L-1)时对谷氨酸诱发电流的降低幅度显著高于布比卡因高浓度时。结论:布比卡因和罗哌卡因对大鼠海马神经元细胞的谷氨酸诱发电流都有明显的抑制作用,罗哌卡因的抑制作用明显高于布比卡因。     

异丙酚预处理对大鼠海马神经元凋亡的影响

目的探讨异丙酚预处理对缺氧/复氧后大鼠海马神经元凋亡的影响。方法取离体培养的大鼠海马神经元,随机分成5组:正常组(A)组。CoCl2组(B)组:加入300μmol·L-1(下同)CoCl2处理4h,然后更换正常的培养基培养24h,之后更换无血清的培养基培养。脂肪乳组(C)组:加入10%脂肪乳剂90μl预处理1h后同B组。异丙酚组(D)组:加入100μmol·L-1异丙酚1h后同B组。7-NI组(E)组:如D组,但加入CoCl2的同时加入3·3μl7-NI(25g·L-1)处理4h。MTT法测定细胞增殖,流式细胞技术测定细胞的凋亡。RT-PCR技术检测神经型一氧化氮合酶和凋亡相关基因的表达情况。结果脂肪乳组与CoCl2组比较,各项指标相似(P>0·05);异丙酚可增加神经元的增殖能力,减少凋亡(P<0·05或P<0·01),上调神经型一氧化氮合酶和凋亡抑制基因bcl-2的表达,下调促凋亡基因cyclinD1的表达。而这些调节作用可被神经型一氧化氮合酶抑制剂7-NI抑制(P<0·05或P<0·01)。结论100μmol·L-1异丙酚预处理对缺氧/复氧后大鼠海马神经元有保护作用,神经型一氧化氮合酶和凋亡相关基因在其中起重要作用。     

D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons

1 The amino acid, D-aspartate, exists in the mammalian brain and is an agonist at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D-aspartate on alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D-aspartate inhibited kainate-induced AMPAR current in hippocampal neurons. The inhibitory action of D-aspartate on kainate-induced AMPAR current was concentration-dependent and was voltage-independent in the tested voltage range (-80 to +60 mV). 3 The estimated EC50 of the L-glutamate-induced AMPAR current was increased in the presence of D-aspartate, while the estimated maximum L-glutamate-induced AMPAR current was not changed. D-aspartate concentration-dependently shifted the dose-response curve of kainate to the right. Schild plot analysis indicated that D-aspartate acts competitively to block AMPARs. The K(b) for D-aspartate was estimated to be 0.93 mM. 4 D-aspartate also blocked L-glutamate-induced current in Xenopus laevis oocytes that expressed recombinant homomeric AMPARs. 5 NMDA possessed similar inhibitory action on AMPARs. However, L-aspartate had little inhibitory action on AMPARs. 6 D-Aspartate, but not L-aspartate, was found to reduce the amplitude of miniature excitatory postsynaptic current in cultured hippocampal neurons. 7 Our data are consistent with a model in which D-aspartate directly competes with kainate and L-glutamate in binding to the agonist binding site of AMPARs. The prevalence of D-aspartate in the brain suggests a possible role of D-aspartate in modulating AMPAR-mediated fast excitatory synaptic transmission.     

缺氧复氧诱导的大鼠海马神经元凋亡和一氧化氮合酶表达变化及银杏叶提取物的作用

目的:观察银杏叶提取物(EGb)及其活性成份银杏总内酯(Gin)对缺氧再复氧诱导的大鼠海马神经元凋亡及一氧化氮合酶(nitricoxidesynthase,NOS)表达影响。方法:选用胎龄15d的SD胎鼠的大脑海马细胞进行原代培养,建立缺氧再复氧海马神经元损伤模型。实验分以下几组:正常组、缺氧复氧组、EGb组、Gin组、阳性对照组(MK801组)。应用TUNEL原位末端标记法定量分析细胞凋亡以及NADPHd组织化学染色法观察细胞NOS的表达。结果:(1)TUNEL免疫细胞化学染色结果显示,预先加入EGb和Gin的药物组神经元的凋亡率明显低于缺氧复氧组;(2)NADPHd组织化学染色结果显示,预先加入EGb和Gin的药物组抑制神经元NOS表达,NADPH阳性细胞减少。结论:EGb及其活性成份Gin对缺氧再复氧损伤的海马神经元具有保护作用,可部分拮抗缺氧再复氧条件下体外培养的海马神经元的凋亡,抑制NOS的表达。     

Effects of perfluorooctane sulfonate on ion channels and glutamate-activated current in cultured rat hippocampal neurons

Concern on an emerging persistent contaminant, perfluorooctane sulfonate (PFOS), is increasingly growing. Although the fate, transport, distribution and bioaccumulation of PFOS have been documented, its toxicological effects especially neurotoxicity remain largely unknown. In this study, the effects of PFOS on ion channels including potassium and sodium channels and exogenous glutamate-activated current in cultured rat hippocampal neurons were examined, based on whole-cell patch-clamp recording. PFOS markedly increased two subtypes of potassium currents, including transient outward current and delayed rectifier current, at doses over 10 μM. PFOS did not affect the amplitude of sodium current at all administrated doses (1, 10 or 100 μM) but clearly shifted the activation current–voltage curve toward negatively potential. Further, PFOS significantly altered the glutamate-activated current at all doses. Taken together these findings indicated that PFOS disturbs the neuronal physiological processes, which revealed the damage of this pollutant to nerve system and will be helpful for further exploration to its underlying mechanism.     

Effects of emodin on synaptic transmission in rat hippocampal CA1 pyramidal neurons in vitro

Rhubarb extracts provide neuroprotection after brain injury, but the mechanism of this protective effect is not known. The present study tests the hypothesis that rhubarb extracts interfere with the release of glutamate by brain neurons and, therefore, reduce glutamate excitotoxicity. To this end, the effects of emodin, an anthraquinone derivative extracted from Rheum tanguticum Maxim. Ex. Balf, on the synaptic transmission of CA1 pyramidal neurons in rat hippocampus were studied in vitro. The excitatory postsynaptic potential (EPSP) was depressed by bath-application of emodin (0.3-30 microM). Paired-pulse facilitation (PPF) of the EPSP was significantly increased by emodin. The monosynaptic inhibitory postsynaptic potential (IPSP) recorded in the presence of glutamate receptor antagonists (DNQX and AP5) was not altered by emodin. Emodin decreased the frequency, but not the amplitude, of the miniature EPSP (mEPSP). The inhibition of the EPSP induced by emodin was blocked by either 8-CPT, an adenosine A1 receptor antagonist, or by adenosine deaminase. These results suggest that emodin inhibits the EPSP by decreasing the release of glutamate from Schaffer collateral/commissural terminals via the activation of adenosine A1 receptors in rat hippocampal CA1 area and that the neuroprotective effects of rhubarb extracts may result from decreased glutamate excitotoxicity.     

Effects of Resibufogenin and Cinobufagin on voltage-gated potassium channels in primary cultures of rat hippocampal neurons

Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (I_K) and outward transient potassium current (I_A) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both I_K and I_A, whereas CBG inhibited I_K without noticeable effect on I_A. Moreover, at 1 μM concentration both RBG and CBG could alter some channel kinetics and gating properties of I_K, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that I_K is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides’ pathological effects on central nervous system.     

Effects of hydrogen peroxide on sodium current in acutely isolated rat hippocampal CA1 neurons

The effects of hydrogen peroxide (H2O2) on sodium currents (Na+ currents) in freshly dissociated rat hippocampal neurons were studied using the whole-cell patch-clamp techniques. H2O2 caused a reversible increase of the voltage-activated Na+ currents in a concentration- and voltage-dependent manner. The half-increasing concentration (EC50) of H2O2 on Na+ currents was 10.79 microM. In addition, 10 microM H2O2 shifted the steady-state inactivation curve of Na+ currents toward positive potential (control Vh = -64.58 +/- 1.22 mV, H2O2 Vh = -53.55 +/- 0.94 mV, n = 10, P < 0.01 without changing the slope factor). However, the steady-state activation curve was not affected. These results indicated that H2O2 could increase the amplitudes of Na+ currents and change the inactivation properties of Na+ channels even in very low concentration.     

Electrophysiological effects of phencyclidine on rat hippocampal pyramidal neurons

The effects of local application of phencyclidine to hippocampal CAl pyramidal neurons was investigated using electrophysiological techniques. The predominant response was a decrease in firing rate, although excitation and biphasic responses were also seen. The stereospecificity of these responses was demonstrated using the (+) and (?) isomers of 1-[1-phenylcyclohexy]-3-methylpiperidine (PCMP). The (+) enantiomer was 4–15 times more potent than the (?) enantiomer. Interactions between PCP and catecholaminergic synapses were also investigated. Catecholamine depletion by reserpine markedly increased the amount of phencyclidine needed to elicit effects on cell discharge; in many cases, no response was seen with the largest dose of phencyclidine tested. The α-adrenergic antagonist, phentolamine, reversibly antagonized phencyclidine-induced inhibition in the firing rate of CAl cells but did not seem to affect excitation; in contrast, timolol, a β-adrenergic antagonist, did not affect the depression but seemed to antagonize reversibly the phencyclidine-induced excitations. It is concluded that the mechanism of phencyclidine-induced changes in pyramidal neuron discharge involves adrenergic input to these cells.     

Effects of presenilins and β-amyloid precursor protein on delayed rectifier potassium channels in cultured rat hippocampal neurons1

AIM: To study the effects of presenilin-1 (PS-1), presenilin-2 (PS-2), and amyloid beta-protein precursor (APP695) on delayed rectifier potassium channels (IK) in the cultured rat hippocampal neurons. METHODS: PS-1, PS-2, and APP695 were transfected into the cultured rat hippocampal neurons by transient transfection techniques. The IK current was observed by the whole cell patch-clamp techniques. RESULTS: IK was increased in cultured rat hippocampal neurons, after transient transfection of PS-1, PS-2, and APP695. IK amplitudes and densities were significantly increased from (1689 +/- 412) pA, (48 +/- 18) pA/pF (mock cells, GFP alone, n=17) to (5565 +/- 1403) pA, (252 +/- 107) pA/pF (PS-1/GFP, n=22, P<0.01), (3804 +/- 1651) pA, (120 +/- 58) pA/pF (PS-2/GFP, n=16, P<0.01), and (4978 +/- 904) pA, (218 +/- 70) pA/pF (APP695, n=22, P<0.01). But PS-1, PS-2, and APP695 did not alter the activation curve of IK (P>0.05). CONCLUSION: Overexpression of PS-1, PS-2, and APP695 increased IK in the cultured rat hippocampal neurons. The upregulation of IK may be related to neuronal apoptosis after PS-1, PS-2, and APP695 were transfected.     

Effects of carvedilol on delayed rectifier and transient inactivating potassium currents in rat hippocampal CA1 neurons

1. The aims of the present study were to investigate the mechanism(s) underlying the protective effect of carvedilol against neural damage. 2. The transient inactivating potassium current (I_A) and the delayed rectifier potassium current (I_K) in rat hippocampal CA1 pyramidal neurons were recorded using whole‐cell patch‐clamp techniques. 3. Carvedilol (0.1–3 μmol/L) significantly inhibited I_K with an IC₅₀ of 1.3 μmol/L and the inhibition was voltage independent. Over the same concentration range, carvedilol had no effect on the amplitude of I_A. At 1 μmol/L, carvedilol did not significantly change the steady state activation curves of I_A and I_K, but did negatively shift their steady state inactivation curves. Recovery from inactivation was slowed for both I_A and I_K. The inhibitory effect of carvedilol on I_K was not affected by the adrenoceptor agonists phenylephrine and prazosin or the adrenoceptor antagonist isoproterenol, but propranolol was able to shift the dose–response curve of carvedilol for I_K to the right. 4. Because I_K is the main pathway for loss of intracellular potassium from depolarized neurons, selective obstruction of I_K by carvedilol could be useful for neuroprotection.     

Effects of EGCG on voltage-gated sodium channels in primary cultures of rat hippocampal CA1 neurons

(-)-Epigallocatechin-3-gallate (EGCG), the main active component of green tea, is commonly known for its beneficial properties at low doses. On the other hand, little is known about the adverse effects of EGCG. Voltage-gated sodium channel (VGSC) is responsible for both initiation and propagation of action potentials of the neurons in the hippocampus and throughout the central nervous system (CNS). In this study, the effects of EGCG on voltage-gated sodium channel currents (I(Na)) were investigated in rat primary cultures of hippocampal CA1 neurons via the conventional whole-cell patch-clamp technique. We found that I(Na) was not affected by EGCG at the concentration of 0.1microM, but was completely blocked by EGCG at the concentration of 400microM and higher, and EGCG reduced the amplitudes of I(Na) in a concentration-dependent manner in the range of 0.1-400microM. Furthermore, our results also showed that at the concentration of 100microM, EGCG was known to have the following performances: (1) it decreased the activation threshold and the voltage at which the maximum I(Na) current was evoked, caused negative shifts of I(Na) steady-state activation curve. (2) It enlarged I(Na) tail-currents. (3) It induced a left shift of the steady-state inactivation. (4) It reduced fraction of available sodium channels. (5) It delayed the activation of I(Na) in a voltage-dependent manner. (6) It prolonged the time course of the fast inactivation of sodium channels. (7) It accelerated the activity-dependent attenuation of I(Na). On the basis of these findings, we propose that EGCG could impair certain physiological functions of VGSCs, which may contribute, directly or indirectly, to EGCG's effects in CNS.     

Effects of ohmefentanyl stereoisomers on phosphorylation of cAMP-response element binding protein in cultured rat hippocampal neurons

AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R,4S,2'S) OMF (F9204), and (-)-cis-(3S,4S,2'R) OMF (F9203)] on the phosphorylation of cAMP-response element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. RESULTS: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25μmol/L, 15min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phosphorylation from 2.88 to 3.59 folds over control levels after 30-min exposure. This effect was reversed by naloxone, but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioidsinduced CREB phosphorylation, while H-89 and P     

地昔帕明和氟西汀拮抗5,7—二羟色胺对海马和皮质神经元的损伤

AIM: To assess the protective effect of desipramine (Des) and fluoxetine (Flu) on the neurons against the lesion induced by a selective serotonergic neurotoxin in vitro. METHODS: The 10-day cultured primary neurons of hippocampus and cortex of rat was exposed to 5,7-dihydroxytryptamine (5,7-DHT) to determine the optimal lesion concentration and duration. Before exposing to 5,7-DHT, Des and Flu was added to the medium for 30 min to observe the protective effects. RESULTS: The optimal concentration and duration for 5,7-DHT was 600 micromol.L-1 and 4 h, respectively. Both Des and Flu showed a protective effect in the dose range of 0.8 micromol.L-1 to 10 micromol.L-1 and 0.04 micromol.L-1 to 0.6 micromol.L-1, respectively, when the neurons were injured by 5,7-DHT 600 micromol.L-1 for 4 h. Flu showed a higher efficacy than Des. Both exhibited a more powerful protective effect on the hippocampal neuron than on the cortical neuron. CONCLUSION: The antidepressant effect of Des and Flu was attributed to their protective effect on the injured serotonergic neuron of the hippocampus and the cortex.