外源性锌离子对大鼠海马切片癫痫样放电的起源、传播与频率特性的调节作用

本研究采用多电极记录技术,在离体条件下研究外源性锌离子(Zn2+)对无镁人工脑脊液诱导的Sprague-Dawley大鼠海马切片癫痫样放电的起源、传播与频率特性的调节作用。结果表明:1μmol/L和100μmol/L的Zn2+作用于海马切片,不改变海马切片上癫痫样放电的起始位置,但能够降低癫痫样放电顺行和逆行两个方向的传播速度,并改变癫痫样放电不同频率范围成分所占的比例。以上结果提示,1μmol/L和100μmol/L的Zn2+可以对海马切片上的癫痫样放电起到调节作用,减慢癫痫样放电在网络中的传播速度,同时,可能对神经元放电活动起到去同步化的作用。     

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。     

高频电刺激对大鼠海马脑片癫痫样放电特性影响的研究

本文采用电极阵列检测技术,在大鼠海马脑切片上诱导出稳定的癫痫样放电,分析、研究130 Hz的高频电刺激(high-frequency stimulation,HFS) CA3区时,海马切片在癫痫发作间期放电(inter-ictal discharges,IID)和发作期放电(ictal discharges,ID)的各项参数、癫痫样放电地起始位点、传播方向和传输速率以及各频段的功率谱密度.结果显示:高频电刺激可以有效地降低癫痫发作期的幅值、减少持续时间、增长潜伏时间、抑制癫痫样放电由IID向ID的转变等.提示高频电刺激抑制癫痫的作用机制是通过促进神经元之间的抑制性传输系统,并且抑制海马神经元之间的兴奋性连接,从而达到抑制效果.     

杏仁核点燃模型癫痫样放电传播途径研究

目的 :探讨杏仁核点燃模型癫痫样放电的传播途径。方法 :选择健康Wistar大鼠 3 0只以电刺激杏仁核的方式制作杏仁核点燃癫痫模型 ,于右侧杏仁核、左侧海马及右侧额叶皮质埋植电极记录脑电活动 ,观察电刺激杏仁核时在杏仁核、海马及额叶皮质出现癫痫样放电的潜伏期、最低刺激强度及癫痫样放电的持续时间。结果 :杏仁核出现癫痫样放电时 ,海马及皮质均未记录到癫痫样放电。而当杏仁核、海马及皮质三处出现癫痫样放电时的最低刺激强度依次增大 ,潜伏期依次延长 ,海马处癫痫样放电的持续时间最长。结论 :杏仁核点燃模型癫痫样放电可能由杏仁核经海马传至皮层 ,海马可能为癫痫样放电传播的重要结构     

强直电刺激嗅内皮质-海马环路诱发CA1神经元的癫痫同步性膜电位振荡行为

目的和方法 :4 0 0～ 5 0 0 μm大鼠水平脑切片含有封闭的EC 海马环路。强直电刺激 (60Hz ,2s)海马Schaeffer侧支诱发癫痫放电 ,全细胞记录CA1胞体层单个神经元电活动 ,同步记录相应树突区细胞外场电位 ,探讨单个神经元膜电位振荡特性与细胞外癫痫电活动之间的关系。结果 :①强直电刺激诱发CA1神经元膜电位后放性振荡行为呈宽频特征 (3～ 10 0Hz)。以θ节律多见 ,跟随在刺激引起的膜电位去极化或超极化偏移 (paroxysmaldepolarizingorhyperpolaringshift,PDSorPHS)之后 ,振荡波的上升支和下降支分别由膜电位去极化 超极化或超极化 去极化成分构成 ;②逐渐增强的IPSP构成了膜电位振荡的起搏成分 ,继而反弹形成锋电位和阈下振荡 ,与细胞外癫痫样电活动同步 ,并促成癫痫放电由紧张性向阵挛性形式转变 ;③发现了电偶联电位 (spikelets)以及细胞之间的染料偶联现象。结论 :单个神经元作为振荡器可以启动群体神经元超同步化癫痫样电活动 ;缝隙连接可能参与了膜电位振荡的启动与场电位癫痫样电活动的同步作用。     

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。     

大鼠离体脑片癫痫放电特征及EC—海马环路的作用

目的和方法：采用４００～５００μｍ大鼠水平脑切片强直电刺激海马Ｓｃｈａｅｆｅｒ侧枝（６０Ｈｚ、２ｓ）全细胞、细胞外同步记录ＣＡ１神经元胞体电活动和相应树突区场电位,探讨其在癫痫发生中的作用。结果：①５３片脑片上记录到细胞内、外同步发生的原发性后放,持续２０ｓ以上,放电形式和持续时间常在第６个刺激串后趋于稳定。ＣＡ１神经元的原发性后放常跟在强直电刺激引起的阵发性去极化或超极化偏移之后（ＰＤＳ、ＰＨＳ）。它可以从紧张性放电向爆发性放电转化,振幅逐渐递增并与细胞外癫痫样放电同步,产生癫痫放电极性偏移;②其中８／４０脑片细胞外可记录到继发性后放之后出现的自发性发作样癫痫放电,长达数分钟,与全细胞记录的ＥＰＳＰ同步。切断ＥＣ与海马之间的联系可以易化海马癫痫电活动（３／５）。结论：ＥＣ输入到海马的神经通路可能在封闭的ＥＣ海马环路中起着重要的门控作用     

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.     

豚鼠背侧海马锥体细胞自发放电特征

实验采用细胞外玻璃微电极采集豚鼠海马神经元放电信号,并将信号转化为峰峰间期(interspike interval,ISI)以研究麻醉和清醒状态海马锥体细胞自发放电线性和非线性特点。实验建立了豚鼠海马锥体细胞与中间神经元电生理鉴别标准;麻醉和清醒状态下豚鼠海马CA1和CA3区锥体细胞自发放电频率、时程、复杂度等无显著区别;麻醉组豚鼠海马锥体细胞ISI序列的复杂度小于清醒组,锥体细胞分型和ISI变异度等表现不同。实验表明,麻醉和清醒状态下豚鼠海马锥体细胞自发放电呈不同线性和非线性特征。传统和非线性研究手段的结合,可能较全面地反映海马锥体细胞自发放电特性。     

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…     

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.     

Effects of interleukin-10 on the epileptiform activity development in the hippocampus induced by brief hypoxia, bicuculline and electrical kindling

The comparative effects of antiinflammatory cytokine interleukin-10 on the epileptiform activity development in CA1 hippocampal neurons were studied in different functional models of epileptogenesis that are not accompanied the visible morphological disturbances in the brain cells: --in vitro hypoxic model in the rat hippocampal slices; 2--in vitro disinhibitory model with using GABAA antagonist, bicuculline, in the rat hippocampal slices; 3--partial hippocampal kindling model in freely moving rats. Interleukin-10 (1 ng/ml) depressed the posthypoxic hyperexcitability in CA1 pyramidal neurons of the rat hippocampal slices through a decrease of the effectiveness of hypoxia to depresses the functional neuronal activity in the rat hippocampal slices during hypoxic episode. On the other hand, interleukin-10 (1 ng/ml) did not affect an initiation of epileptiform activity in CA1 pyramidal neurons of the rat hippocampal slices induced by bicuculline. Interleukin-10 (1 ng/5 microl) applied to the dorsal hippocampus in awake rats depressed an initiation of focal seizures ("ictal"-like components of afterdischarges) induced by hippocampal kindling during the first six hours after an application. However, this cytokine did not affect neither the duration of "interictal"-like component of afterdischarges nor motor seizure development. Thus, our findings showed that antiinflammatory cytokine interleukin-10, in addition to its antihypoxic action, exert the neuroprotective effect on the initiation of "ictal"-like, but not "interictal"-like, epileptiform discharges.     

Pattern- and age-dependency of the antiepileptic effects induced by valproic acid in the rat hippocampus.

The effects induced by the antiepileptic drug valproic acid were studied in the CA3 subfield of in vitro hippocampal slices obtained from young (16- to 27-day-old) and adult (over 60-day-old) rats. Spontaneous epileptiform discharges were induced by the addition of the convulsant 4-aminopyridine to the medium. Valproic acid (0.5 mM) selectively blocked the ictal epileptiform discharges in slices obtained from young rats. Interictal epileptiform discharges disappeared during perfusion with higher doses of valproic acid (2 mM). This blockade of interictal epileptiform activity was not observed when valproic acid (0.5-5 mM) was tested in hippocampal slices from adult rats. Thus, in the hippocampus of young rats, 4-aminopyridine-induced ictal activity is more sensitive to valproic acid than are interictal discharges. Moreover, valproic acid is effective in controlling interictal discharges in the young, but not in the adult rat hippocampus.     

Potassium channel blockers tetraethylammonium and 4-aminopyridine fail to prevent microglial activation induced by elevated potassium concentration

The effect of potassium channel blocker tetraethylammonium and 4-aminopyridine was examined on the elevated K+ concentration-induced microglial activation on rat hippocampal slice preparations. Microglial cells were detected by immunohistochemisty with a monoclonal antibody (OX 42) raised against a type 3 complement receptor. During activation the morphology of the microglial cells changes and the staining intensity increases. The degree of microglial activation was determined by measuring the integrated optical density of the cells. Tetraethylammonium and 4-aminopyridine failed to reduce the elevated K+ concentration-induced microglial activation. Both potassium channel blockers, when applied on the hippocampal slices without K+, caused significantly increased microglial activation as compared to the control slices. In order to check whether the functional alteration of the neuronal population induced by 4-aminopyridine caused the activation of the microglial cells, Schaffer collaterals were cut to block spreading of epileptiform hyperactivity of the CA3 pyramidal cells to the CA1 region. No significant differences were found in microglial activation between the CA3 and CA1 regions, indicating that the effect of 4-aminopyridine on microglial cells is independent of the epileptiform activity caused by the drug.     

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAA antagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.     

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。     

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     

Hyperpolarizing potentials in guinea pig hippocampal CA3 neurons

There is a bewildering variety of hyperpolarizing potentials which control activity in hippocampal pyramidal cells. These include an inhibitory postsynaptic potential (IPSP) with early and late components, voltage- and calcium-dependent potassium conductances, a voltage-dependent potassium conductance modulated by muscarinic agents (the M-current), and a complex and poorly understood afterhyperpolarization following epileptiform bursts. In hippocampal CA3 pyramidal cells, mossy fiber stimulation elicits an IPSP which is made up of two readily separable components. Using the in vitro slice preparation, we investigated the underlying ionic basis of these IPSP components and compared them to other hyperpolarizing potentials characteristic of the CA3 neurons. Intracellular recordings were obtained and then tissue was exposed to bathing medium low in chloride concentration or high in potassium concentration; the ion "blockers" EGTA (intracellular); tetraethylammonium (TEA) (intra- and extracellular), and barium and cobalt (extracellular); and the gamma-aminobutyric acid (GABA)/chloride antagonists penicillin, bicuculline and picrotoxin.