过氧亚硝酸阴离子通过鸟苷酸环化酶途径抑制钠电流影响海马神经元兴奋性

过氧亚硝酸阴离子（ONOO-）是一种性质活泼的自由基,可引起强的氧化性损伤,介导了一氧化氮（NO）的大部分毒性作用．应用全细胞膜片钳技术,探讨ONOO-对脑片海马神经元电压门控钠通道电流（INa）和神经元兴奋性的影响．结果表明,ONOO-供体SIN-1（10,500,2000μmol／L）可浓度依赖性抑制INa电流峰值．SIN-1与ONOO-清除剂尿酸共处理,并不影响INa．500μmol／L的SIN-1可使INa的I-V曲线上移,并可抑制其失活后恢复过程,但对INa的激活和失活过程无影响．SIN-1还可抑制动作电位发放频率和幅值．脑片预处理腺苷酸环化酶（adenylate cyclase,AC）抑制剂MDL-12,330A（25μmol／L）和NEM（50μmol／L）对SIN-1的作用无影响．然而,预处理鸟苷酸环化酶（CG）抑制剂ODQ可抑制SIN-1对INa的作用．以上结果提示,ONOO-通过cGMP-INa-AP信号级联系统作用于海马神经元,与PKA和蛋白巯基亚硝化途径无关,这可能是ONOO-神经毒性的机制之一．     

一氧化氮通过巯基亚硝化途径抑制海马神经元的延迟整流型钾电流

应用膜片钳全细胞记录模式研究了内源性一氧化氮(NO)对培养海马神经元延迟整流型钾电流的调控作用及其机制.给予NO合成酶的底物L-精氨酸(L-Arg,2mmol/L)可显著抑制海马神经元上的延迟整流型钾电流,但其同分异构体D-精氨酸(2mmol/L)对钾电流则无明显影响.并且,经一氧化氮合成酶抑制剂L-NAME(nomega-nitro-L-argininemethylester,0.5mmol/L)预处理后,L-Arg对钾电流的抑制作用消失,表明L-Arg抑制钾电流是通过产生NO而不是精氨酸本身.特异性鸟苷酸环化酶抑制剂ODQ(1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one,10!mol/L)预处理不影响L-Arg对钾电流的抑制作用,但巯基烷化剂NEM(N-ethylmaleimide,1mmol/L)预处理可完全阻断L-Arg的抑制效应.以上结果表明,内源性NO主要通过巯基亚硝化途径抑制海马神经元的延迟整流型钾电流.     

二氧化硫衍生物对铅引起的大鼠海马神经元钠电流变化的影响

运用全细胞膜片钳技术研究慢性铅暴露和急性给二氧化硫衍生物对大鼠海马神经元钠电流的影响,结果发现,慢性铅暴露组钠电流在-70mV激活,-30mV达到峰值;对照组钠电流在-70mV激活,-40mV达到峰值．两组峰值不具有显著性差异．急性给二氧化硫衍生物于慢性铅暴露组,钠电流在-80mV开始激活,-40mV达到峰值,I-V曲线显著下移．慢性铅暴露使穿越钠通道离子的绝对数量稍微有些减少,但不具有统计学差异;二氧化硫可使慢性铅暴露的海马神经元的INa显著增大．慢性铅暴露推迟了INa达到峰值的时间,但不影响失活时间常数;急性加入二氧化硫衍生物不改变慢性铅暴露达到峰值的时间,却使失活时间常数显著延长．慢性铅暴露使INa的激活曲线右移,失活曲线左移;二氧化硫衍生物使慢性铅暴露的海马神经元上的INa的激活和失活曲线都往超极化方向移动．这些结果表明,铅和二氧化硫改变了细胞膜钠通道对于电压的感应,延长了钠通道的开放时程,这些可能是这两种大气污染物联合损伤海马神经元的作用机制之一．     

Hypoxic Excitability Changes and Sodium Currents in Hippocampus CA1 Neurons

1. The objective of the present study was to distinguish if inhibition of neuronal activity by hypoxia is related to a block of voltage-gated Na+ channels. 2. The effect of chemical hypoxia induced by cyanide (0.5 mM, 10 min perfusion) was studied with patch-clamp technique in visualized intact CA1 pyramidal neurons in rat brain slices. Action potentials were elicited in whole cell current-clamp recordings and the threshold was estimated by current pulses of 50-ms duration and incremental amplitudes (n = 31). The effect of cyanide on the Na+ current and conductance was studied in voltage clamp recordings from cell-attached patches (n = 13). 3. Cyanide perfusion during 10 min increased the threshold for excitation by 73 +/- 79 pA (p = 0.001), which differed from the effect in control cells (11 +/- 41 pA, ns). The change in current threshold was correlated to a change in membrane potential (r = -0.88, p < 0.0001). Cyanide had no significant effect on the peak amplitude, duration, or rate of rise of the action potential. 4. Cyanide perfusion did not change the Na+ current size, but caused a small decrease in ENa (-17 +/- 22 mV, ns) and a slight increase in Na+ conductance (+14 +/- 26%, ns), which differed (p = 0.045) from controls (-19 +/- 23 %, ns). 5. In conclusion, chemical hypoxia does not cause a decrease in Na+ conductance. The decreased excitability during hypoxia can be explained by an increase in the current threshold, which is correlated with the effect on the membrane potential.     

Ablation of NMDA Receptors Enhances the Excitability of Hippocampal CA3 Neurons

Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons.     

Blockade of Excitability of Hippocampal Neurons after Coagulation of the Dorsal Amygdalofugal Pathway

In chronic experiments on rabbits, we demonstrated that electrolytic destruction of the stria terminalis results in the intense depression of the integral electrical activity of the hippocampus. Characteristics of the responses induced by electrical and chemostimulation of the hippocampus under the above conditions allowed us to conclude that a significant drop in the excitability of hippocampal neurons themselves, but not changes in the structure and intensity of afferent impulsation from the extrahippocampal structures, underlies such a depression. Morphological changes in neurons and satellite glial cells, such as lysis of the Nissl substance, hyperchromatosis, and wrinkling of the cells after destruction of the stria terminalis, were proof of the correctness of our conclusion. It is postulated that one of the main factors underlying the generalized effects observed in our experiments is probably the disturbance of functional characteristics of the hypothalamo-hypophyseal neurosecretory system induced by destruction of the amygdalofugal connections; this disturbance results in significant pathological shifts of metabolism in the hippocampus.     

琥珀酸对大鼠海马CA1区神经元电压依赖性钙电流的影响

目的：观察不同浓度的琥珀酸对大鼠海马CA1区神经元电压依赖性钙通道（voltage—dependent calcium channels,VDCC）电流的作用,初步探讨琥珀酸对神经元保护的电生理学基础。方法：采用传统全细胞膜片钳技术和制霉菌素（nystatin）穿孔膜片钳技术观察琥珀酸对海马CA1区神经元VDCC电流的影响。结果：不同浓度的琥珀酸（10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1）在海马CA1区对低电压激活（low—voltage activated,LVA）钙通道电流未见任何影响,而对高电压激活（high—voltage activated,HVA）钙通道电流的抑制呈浓度依赖性。对照组HVA钙电流为580．05±17．32pA,分别给予10^-6、10^-5、10^-4、10^-3、10^-2和10^-1mol·L^-1。的琥珀酸后,HVA钙电流依次为563．74±16．65,517．99±15．24,444．66±13．26,405．32±19．11,269．03±9．96和86．41±3．25pA,同对照组相比差异有统计学意义（n=8,P〈0．01）。结论：琥珀酸能浓度依赖性地抑制HVA钙电流,而对LVA钙电流无影响。由此推测琥珀酸可能通过抑制HVA钙电流减少Ca^2＋内流而影响海马CA1区神经元的兴奋性,从而抑制癫痫的形成,其脑保护作用可能与此有关。     

Enhancement of Lanthanum (III) on Sodium Currents in Acutely Isolated Hippocampal CA1 Neurons of Rat

The effects of lanthanum (III) (La³⁺) on voltage-gated sodium channel currents (I _Na) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La³⁺ reversibly enhanced I _Na in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC₅₀) of La³⁺ on I _Na was 9.93 μM. In addition, 10 μM La³⁺ shifted the steady state activation curve of I _Na towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La³⁺ could increase the amplitudes of I _Na and change the activation and inactivation courses of I _Na even in very low concentration.     

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

The equilibrium potential for GABA-A receptor mediated currents (E_GABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K⁺/Cl^- co-transporter (KCC2) but a relatively high expression of Na⁺-K⁺-Cl^- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in E_GABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on E_GABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus E_GABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in E_GABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in E_GABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in E_GABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.     

Inhibiting the Activity of CA1 Hippocampal Neurons Prevents the Recall of Contextual Fear Memory in Inducible ArchT Transgenic Mice

The optogenetic manipulation of light-activated ion-channels/pumps (i.e., opsins) can reversibly activate or suppress neuronal activity with precise temporal control. Therefore, optogenetic techniques hold great potential to establish causal relationships between specific neuronal circuits and their function in freely moving animals. Due to the critical role of the hippocampal CA1 region in memory function, we explored the possibility of targeting an inhibitory opsin, ArchT, to CA1 pyramidal neurons in mice. We established a transgenic mouse line in which tetracycline trans-activator induces ArchT expression. By crossing this line with a CaMKIIα-tTA transgenic line, the delivery of light via an implanted optrode inhibits the activity of excitatory CA1 neurons. We found that light delivery to the hippocampus inhibited the recall of a contextual fear memory. Our results demonstrate that this optogenetic mouse line can be used to investigate the neuronal circuits underlying behavior.     

Mdivi-1 Protects Epileptic Hippocampal Neurons from Apoptosis via Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress in Vitro

Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of the mitochondrial fission protein dynamin-related protein 1, has been proposed to have a neuroprotective effect on hippocampal neurons in animal models of epilepsy. However, the effect of mdivi-1 on epileptic neuronal death in vitro remains unknown. Therefore, we investigated the effect of mdivi-1 and the underlying mechanisms in the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) in vitro. We found that mitochondrial fission was increased in the HNC model of AE and inhibition of mitochondrial fission by mdivi-1 significantly decreased neuronal apoptosis induced by AE. In addition, mdivi-1 pretreatment significantly attenuated oxidative stress induced by AE characterized by decrease of reactive oxygen species (ROS) production and malondialdehyde level and by increase of superoxide dismutase activity. Moreover, mdivi-1 pretreatment significantly decreased endoplasmic reticulum (ER) stress markers glucose-regulated protein 78, C/EBP homologous protein expression and caspase-3 activation. Altogether, our findings suggest that mdivi-1 protected against AE-induced hippocampal neuronal apoptosis in vitro via decreasing ROS-mediated oxidative stress and ER stress.     

The Reduction of EPSC Amplitude in CA1 Pyramidal Neurons by the Peroxynitrite Donor SIN-1 Requires Ca2+ Influx Via Postsynaptic Non-L-Type Voltage Gated Calcium Channels

The peroxynitrite free radical (ONOO^?) modulation of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs) was investigated in rat CA1 pyramidal neurons using the whole-cell patch clamp technique. SIN-1(3-morpholino-sydnonimine), which can lead the simultaneous generation of superoxide anion and nitric oxide, and then form the highly reactive species ONOO^?, induced dose-dependent inhibition in amplitudes of both mEPSCs and sEPSCs. The SIN-1 action on mEPSC amplitude was completely blocked by U0126, a selective MEK inhibitor, suggesting that MEK contributed to the action of ONOO^? on mEPSCs. The effect of SIN-1 was completely occluded either in the presence of the calcium chelator EGTA or the non-selective calcium channel antagonist Cd²⁺. Furthermore, the application of nifedipine (20 μM), the L-type calcium channel blocker, had no effect on the ONOO^?-induced decrease in mEPSC amplitude, excluding a role for L-type voltage-gated Ca²⁺ channels in this process. SIN-1 inhibited the frequency of sEPSCs but had no effect on mEPSC frequency, which suggested a presynaptic action potential-dependent the action of ONOO^? at CA1 pyramidal neuron synapses. The best-known glutamatergic input to CA1 pyramidal neurons is via Schaffer collaterals from CA3 area. However, no changes were observed in slices treated with SIN-1 on the spontaneous firing rates of CA3 pyramidal neurons. These findings suggested that SIN-1 inhibited glutamatergic synaptic transmission of CA1 pyramidal neurons by a postsynaptic non-L-type voltage gated calcium channel-dependent mechanism.