海马脑片癫痫样放电时空特性的多电极记录研究

为了探索离体条件下癫痫样放电的时空特性,本研究采用多电极记录系统记录高钾人工脑脊液(artificial cerebrospinal fluid,ACSF)诱导的幼年大鼠海马脑片的自发放电活动。在成功诱导出癫痫样的簇样放电后,加入苯巴比妥钠以观察其对脑片各区域放电的压抑作用。结果显示:(1)高钾ACSF持续灌流脑片15min左右,多电极阵列上可记录到海马CA3(a~c)和CA1区反复出现同步节律的癫痫样簇样放电,与脑电信号中的发作间期痫性放电相似;定量分析结果提示,CA区各个亚区锥体细胞的活动特性无明显差异(P0.05),而齿状回(dentate gyrus,DG)颗粒细胞层没有出现簇样放电,仅有少量动作电位发放,发放频率远低于CA区(P0.05);(2)在持续高钾灌流下,稳定的簇样放电一旦建立即可至少持续40min;(3)簇样放电发放稳定后给予60μmol/L苯巴比妥钠,发现同步化放电的区域逐渐缩小,CA1和CA3c区的放电活动首先被压抑,而CA3a和b区的部分锥体细胞在加药10min后仍有较强的簇样放电。以上结果提示,多电极阵列能够有效地用于研究离体条件下癫痫样放电的时空特性,并可探索抗癫痫药对脑片不同区域癫痫样活动的作用。     

马桑内酯在Wistar大鼠海马脑片上引起的痫样放电及SC_(1001)钠盐的对

马桑内酯(CoriariaLactone,CL)是从抗精神病中草药—马桑寄生中提取的有效成分,它具有致痫作用。目前,运用CL在整体动物身上建立急、慢性癫痫模型的实验已获成功。为了进一步从细胞水平探讨CL的致痫作用及可能的电生理机制,并观察抗癫新药SC1001钠盐对CL所致痫样放电活动的抑制作用,本实验采用离体海马脑片技术,在59例Wistar大鼠的海马脑片标本上进行了以下研究:以海马CA1区锥体细胞诱发场电位为指标,观察了不同刺激强度时顺向、逆向刺激与诱发场电位之间的关系;观察了持续灌流人工脑脊液(ACSF)对诱发场电位的影响和双脉冲易化,频率…     

钩藤对致痫大鼠海马脑片诱发场电位的影响

目的研究钩藤对癫痫模型海马脑片诱发场电位的影响。方法以毛果芸香碱致痫大鼠为实验对象,采用脑片旁滴注给药,用细胞外玻璃微电极记录方法,观察钩藤对癫痫模型离体海马脑片CA1区锥体细胞诱发群锋电位（populationspike,PS）的影响。结果给予钩藤后使致痫大鼠海马脑片PS幅度平均降低27.64％,平均8.71min恢复(n=14,P<0.01)。结论钩藤能降低致痫大鼠海马脑片CA1区顺向诱发PS幅度,提示钩藤对中枢神经系统的突触传递过程有明显的抑制效应,具有抗癫痫作用。     

羟基马桑毒素所致的大鼠海马锥体细胞痫样放电活动

本研究采用离体海马脑片电生理研究技术,细胞外记录海马锥体细胞群体锋电位(population spike,PS),观察羟基马桑毒素(tutin)对大鼠海马脑片CA1区锥体细胞电活动的影响,探讨tutin是否具有致痛作用及其致痫机制。结果如下:(1)用40、30和20μg/ml浓度的tutin灌流海马脑片,可显著增高由顺向刺激Schaffer侧支所诱发的PS的幅度,灌流tutin 30min时,PS第一个波的幅度分别为对照的(388.7±20.1)%、(317.2±19.1)%和(180.9±11.6)%(各组n=5,P<0.05)。(2)伴随PS波幅的增高,可出现成串痫样放电波,波数4～11个不等。(3)灌流tutin后的部分脑片(n=9/34),在未刺激Schaffer侧支时也出现自发的成串、高幅痫样放电。(4)灌流CNQX阻断非NMDA受体后,再灌流tutin,PS幅度和放电波数均无显著性变化,即CNQX可完全抑制tutin所致的痫样放电;灌流AP-5阻断NMDA受体后,tutin仍可使PS幅度增高但放电波数无显著性增加,即AP-5可部分抑制tutin所致的痫样放电。上述结果表明,tutin可使海马脑片锥体细胞兴奋活动增强,具有致痫作用;兴奋性谷氨酸受体尤其是非NMDA受体可能介导tutin的致痫作用。     

氟桂利嗪对青霉素致痫大鼠皮层及海马癫痫样放电的影响

目的:观察氟桂利嗪对青霉素致痫大鼠皮层和海马癫痫样放电的影响.方法:选取Wistar大鼠60只制作青霉素致痫模型,在大鼠海马、颞叶、额叶皮层埋置电极,记录氟桂利嗪(20 mg/kg)灌胃后大鼠癫痫样放电的变化.结果:实验组动物皮层及海马癫痫样放电潜伏期明显延长,持续时间缩短,单位时间内癫痫样放电数明显减少,与对照组相比有显著差异.结论:氟桂利嗪具有明显抗癫痫作用,可显著抑制青霉素致痫鼠皮层及海马区癫痫样放电.     

介绍一种电药理实验的4孔脑片浴槽

目的：介绍一种同时进行４路流体灌流的多孔脑片浴槽。方法：用大鼠朋脑皮层脑片细胞外电极记录，观察自发性癫痫样放电。结果：通过染料试验证明４路液体互不通连。用无Ｍｇ＾２＋和含３０μｍｏｌ／Ｌ４－氨基吡喧的人工脑脊液灌流１ｈ后，各孔脑片均可出现自发性放电，并可维持稳定达３ｈ以上。结论：此装置可以同时观察４种抗癫痫药物对脑片自发放电的抑制作用。     

柴胡对癫痫模型电活动的调制

目的 :研究柴胡对癫痫发作的影响。方法 :以家兔和大鼠为实验对象 ,用毛果芸香碱致痫 ,采用脑电图和细胞外玻璃微电极记录技术 ,观察柴胡对癫痫模型大脑皮层放电及海马脑片场电位的影响。结果 :腹腔注射柴胡后可使癫痫发作次数及发作持续时间显著减少 ,发作间隔时间显著延长 ,(P <0 .0 5 ) ,脑片旁滴注柴胡后使致痫大鼠海马脑片诱发场电位幅度平均降低 2 0 .4 1% ,恢复时间平均为 6 .86min ,(P <0 .0 1)。结论 :柴胡注射液能明显抑制癫痫模型电活动 ,提示柴胡具有抗痫作用     

咖啡因对大鼠杏仁核点燃模型的作用研究

目的:观察兴奋药咖啡因对点燃癫痫模型的作用.方法:建立大鼠杏仁核点燃模型,观察不同剂量的咖啡因及与抗癫痫药物联用对杏仁核点燃大鼠的后放电时程(ADD)和Racine's分级的影响.结果:咖啡因提高点燃大鼠的ADD,但联用抗苯妥英钠和苯巴比妥可降低ADD(P＜0.01)及Racine's分级(P＜0.01).结论:小剂量的咖啡因与抗癫痫药物能产生协同作用.     

一氧化氮的释放对海马脑片CAl区痫样放电的影响

用自制的一氧化氮（NO）敏感电极──Nation-壳聚糖合镍修饰铂电极（Nation-CTS（Ni）-Pt）连续测定了青霉素致痫海马脑片CAl区锥体层神经元NO的释放,并同时观察了NO合酶抑制剂7-nitro-indaole（7-NI）及Nω-nitro-L-arginine（L-NNA）对诱发痫波及NO释放量的影响。研究观察到：（1）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,与此同时,NO释放量亦增加并存在剂量反应关系;（2）NO合酶抑制剂7-NI及L-NNA抑制海马脑片CAl区NO的产生的同时,可部分翻转青霉素的致痫作用;（3）NO敏感电极检测线性范围为4．5×10－4～1．0×10－8mol／L,可用于生物组织如脑片的NO释放量测定。结果提示,NO可能有致痫作用;L-NNA及7-NI对癫痫有抑制作用;NO敏感电极的应用为生物体NO的连续实时测定提供了有效的实验工具。     

一氧化氮对谷氨酸诱发的大鼠海马脑片CA1区神经元活动的抑制

用细胞外记录单位放电技术,在大鼠海马脑片上观察了Ｌ－精氨酸（Ｌ－ａｒｇ）、Ｎ－硝基Ｌ－精氨酸（Ｌ－ＮＮＡ）及ＳＩＮ－１对谷氨酸（ｇｌｕｔａｍａｔｅ,Ｇｌｕ）诱导的ＣＡ１区神经元放电的影响。旨在了解Ｌ－精氨酸：ＮＯ通路在谷氨酸诱发的海马放电中的作用及其可能的机制。结果如下：（１）用ＧｌＵ（０．５ｍｍｏｌ／Ｌ）灌流海马脑片１ｍｉｎ,１２个放电单位放电频率明显增加,表现为癫痫样放电;（２）海马脑片２ｍｉ     

Time-related antiepileptic effects of the synthetic glucorticoid dexamethasone in rat hippocampal slices

The in vitro antiepileptic activity of the synthetic glucocorticoid dexamethasone (DEX) was tested in rat hippocampal slices on the CA1 epileptiform activity induced by sodium penicillin (PEN). Slice perfusion with 1 mM PEN produced within 60 min the development of a CA1 epileptiform bursting made up of an increase of the primary CA1 population spike followed by the appearance of secondary epileptiform population spikes. Slice perfusion with 100 μM DEX together with PEN (1 mM) partially prevented but did not block the expression of the CA1 epileptiform bursting as evidenced by a significant (P < 0.05) reduction of the duration of the bursting due to the epileptogenic agent. Slice perfusion with 50 μM DEX together with PEN (1 mM) failed to prevent or block the expression of the CA1 penicillin-induced epileptiform bursting. A 60 min slice pretreatment with 50–100 μM DEX followed by a slice perfusion with 50–100 μM DEX together with PEN (1 mM) prevented the expression of the CA1 epileptiform bursting. Cycloheximide (1 μM), a protein synthesis inhibitor, perfused together with DEX reverted the inhibitory effects of dexamethasone on the expression of the penicillin-induced CA1 epileptiform bursting. The results indicate that the synthetic glucocorticoid DEX presents concentration- and time-related in vitro. antiepileptic effects. In addition, the data suggest that this inhibitory effect occurs via a protein synthesis-dependent mechanism.     

生长抑素对实验性癫痫大鼠海马CA1区的作用

在大鼠海马ＣＡ１区微量注射０．０３－０．３ｎｍｏｌ生长抑素（ｓｏｍａｔｏｓｔａｔｉｎ,ＳＳ）后,皮层脑电出现单个或成串的棘尖波,平均脑电总功率著升高,并且在一定范围内（０．００６－０．１５ｎｍｏｌ）具有剂量依赖性。在海马ＣＡ１微量注射０．０３－０．３ｎｍｏｌＳＳ可诱发大鼠表现出痫样行为,并可加重红藻氨酸诱导的大鼠痫样活动。在９５个大鼠海马脑片上细胞外记录ＳＳ对ＣＡ１区青霉素诱导的１１４个痫样放电单     

Neuron to astrocyte communication via cannabinoid receptors is necessary for sustained epileptiform activity in rat hippocampus

Astrocytes are integral functional components of synapses, regulating transmission and plasticity. They have also been implicated in the pathogenesis of epilepsy, although their precise roles have not been comprehensively characterized. Astrocytes integrate activity from neighboring synapses by responding to neuronally released neurotransmitters such as glutamate and ATP. Strong activation of astrocytes mediated by these neurotransmitters can promote seizure-like activity by initiating a positive feedback loop that induces excessive neuronal discharge. Recent work has demonstrated that astrocytes express cannabinoid 1 (CB1) receptors, which are sensitive to endocannabinoids released by nearby pyramidal cells. In this study, we tested whether this mechanism also contributes to epileptiform activity. In a model of 4-aminopyridine induced epileptic-like activity in hippocampal slice cultures, we show that pharmacological blockade of astrocyte CB1 receptors did not modify the initiation, but significantly reduced the maintenance of epileptiform discharge. When communication in astrocytic networks was disrupted by chelating astrocytic calcium, this CB1 receptor-mediated modulation of epileptiform activity was no longer observed. Thus, endocannabinoid signaling from neurons to astrocytes represents an additional significant factor in the maintenance of epileptiform activity in the hippocampus.     

Rosiglitazone Suppresses In Vitro Seizures in Hippocampal Slice by Inhibiting Presynaptic Glutamate Release in a Model of Temporal Lobe Epilepsy

Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg²⁺ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg²⁺ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.     

Electrophysiological approach of the antiepileptic effect of dexamethasome on hippocampal slice culture usihg a multirecording system: The physiocard®

The in vitro antiepileptic activity of the synthetic glucocorticoid dexamethasone (DEX) was tested in rat hippocampal organotypic cultures on the field potential epileptiform activity induced by picrotoxin (PTX). Spontaneous as well as evoked electrophysiological activities have been studied through the extracellular multirecording Physiocard® system. PTX typically elicited seizure-like discharges (epileptiform bursts) in the hippocampus neurons. Those epileptiform bursts can be divided in two groups, one rhythmic which lasted 43 ± 24s (mean ± sd) at a frequency of 4.6 ± 1.9Hz and the other arhythmic composed of population spikes, which occurred during 14.3 ± 6.9min. In the presence of DEX at different concentrations, results obtained were: 1) DEX 1μM decreased the occurrence of the two different groups of spontaneous epileptiform bursts, most of the time to zero. 2) DEX 50μM prevented totally the occurrence of epileptiform bursts. 3) DEX 50μM contrarily to DEX 1μM avoided the decrease of evoked field potentials' amplitude induced by PTX 3μM on all simultaneous recorded points. Those results suggest that synthetic glucocorticoid DEX presents an acute antiepileptic effect in a dose dependent manner on the hippocampus tissue.     

三种抗癫痫药对癫痫患者脑电图的背景影响比较研究

目的：研究对比三种抗癫痫药（苯妥因钠、丙戊酸钠、卡马西平）对癫痫患者脑电图的背景影响。方法：选取我院于2009年3月至2011年2月收治的60例癫痫患者,随机分为苯妥因钠（PHT）、卡马西平（CBZ）和丙戊酸钠（SVP）组各20例,动态观察各组患者于治疗期间痫样波放电的频度和EEG背景的变化。结果：EEG痫样波放电的抑制率以SVP最为明显,而CBZ在EEG背景活动影响方面均比其他两组显著。结论：三种药物对癫痫波放电的抑制顺序是SVP〉PHT〉CBZ,SVP组明显优于其他两组。     

Effect of lamotrigine on a novel model of epilepsy

The effect of lamotrigine (LTG) on evoked and spontaneous seizure-like activity induced by veratridine, was investigated. Rat brain slices were examined using conventional electrophysiological intracellular techniques. Alteration of sodium channel function by veratridine (0.3 microM) induced spontaneous seizure-like activity in the hippocampal CA1 pyramidal neurons. Therapeutic concentrations of LTG (5-10 microM) inhibited both evoked and spontaneous bursting induced by veratridine. This inhibition was voltage-dependent indicating possible interaction between the drug and the inactivated state of sodium channels. There was an increase in the firing threshold of the bursting but no change in the resting membrane potential (RMP) and membrane input resistance. Results from this work suggest that the veratridine model of epilepsy is very sensitive to drugs which act on sodium channels. These data make the veratridine model a suitable tool for screening potential sodium channel-dependent antiepileptic drugs.     

应用多电极记录技术研究海马切片癫痫样放电的起始点和传播方向

弄清癫痫样放电的起始位置和传播方向对研究癫痫机制及其临床治疗有重要意义．为了解决这一问题,应用微电极阵列对低镁人工脑脊液诱导的Sprague-Dawley (SD)大鼠海马切片的癫痫样放电进行记录．分别用癫痫样放电的两种成分：场电位和多单元信号来确定癫痫样放电的起始位置和传播方向．首先计算并比较了海马切片锥体细胞层位置电极记录的癫痫样放电场电位的起始时间,由起始时间的先后关系确定癫痫样放电在锥体细胞层的起始位置和传播方向．然后用整个切片上记录的癫痫样放电的多单元信号动作电位序列进行互相关分析,进一步确定了癫痫样放电在整个海马切片内的起始位置和传播方向．结果显示,CA3区的癫痫样放电具有比CA1区更高的幅度和更长的持续时间,表明CA3区有更高的兴奋性．对于记录到的同步癫痫样放电,CA3b区场电位和多单元信号均比CA3c和CA1区出现更早,起始位置和其随后位置之间的传播延　时与二者之间的距离成正相关．因此,在低镁模型的大鼠海马切片中,癫痫样放电起始于CA3b区并分别向CA3c和CA1区传播．     

Repeated inhibitory effects of NPY on hippocampal CA3 seizures and wet dog shakes

Intracerebroventricular injection of NPY inhibits epileptiform seizures and seizure-related "wet dog shakes" (WDS) following electrical stimulation of the dentate gyrus or subiculum. This study examined the effects of NPY on seizures and WDS elicited in hippocampal CA3. Like in the other hippocampal regions, NPY significantly inhibited both seizures and accompanying WDS consistent with in vitro data. The identification of an additional antiepileptic hippocampal target for NPY could prove therapeutically relevant considering that the hippocampal formation is a frequent seizure focus in human epilepsy. The effects of NPY were found to persist on seven repeated NPY injection days. Thus tolerance to the anti-seizure effects of NPY does not appear to develop rapidly. Tolerance being a problem with several current antiepileptic drugs, this further strengthens the concept of NPY receptors as a potential future antiepileptic target.     

Negative occurrence between hippocampal rhythmic slow activity and epileptiform spikes: influence of the striatum

The effects of caudate and septal stimulation on hippocampal activity were studied. Sodium penicillin was intravenously injected in order to induce a steady rate of interictal epileptic activity. Penicillin injection caused significant reduction of the rate of occurrence and duration of hippocampal rhythmic slow activity (RSA). Spontaneous RSA as well as RSA-eliciting caudate and septal stimulation induced a marked inhibition on frequency of epileptiform complexes. Lesions of the medial septal nucleus made it impossible to record RSA or to observe any sort of influence on hippocampal epileptiform activity by caudate stimulation. This result suggests that the septum is important for RSA genesis in the striato-hippocampal pathway or in the reciprocal septo-hippocampal connections.