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。     

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     

持续癫痫样放电导致海马神经元钙离子动力学变化的研究

目的：研究低镁介质致痫的培养海马神经元癫痫模型中神经元内游离钙离子（[Ca^2＋]i）的时空分布及其动力学改变,以探讨钙离子在癫痫发病过程中的作用。方法：联合应用共聚焦激光扫描显微镜和膜片钳,运用较高时间分辨率动态观察培养海马神经元癫痫模型[Ca^2＋]i和电生理变化,以及化学门控钙离子通道阻滞剂的影响。结果：致痫后海马神经元胞浆和核内游离钙离子迅速上升到（612±65）nmol／L和（620±69）nmol／L水平,NMDA受体阻断剂MK-801（10μmol／L）和非NMDA受体阻断剂NBQX（10μmol／L）可使[Ca^2＋]i的升高明显减少;升高的[Ca^2＋]i恢复有明显的延迟现象,90min和150min癫痫样放电后[Ca^2＋]i恢复的时间分别为（114．8±5．2）和（135．0±22．7）（P〈0．05）。结论：持续的癫痫样放电可导致海马神经元细胞内钙超载,这个效应可被MK-801阻断,化学门控钙离子通道也参与了细胞外Ca^2＋内流的过程。     

海马中的内源性爆发式放电神经元与颞叶癫痫

颞叶癫痫(temporal lobe epilepsy,TLE)是常见的难治性癫痫,主要累及到海马及海马旁结构等边缘网络。爆发式放电神经元(bursting—firing neurons,BFNs)的活动是促使海马结构产生癫痫电活动及相关病理性改变的重要因素之一。BFNs是一类能够由刺激引起、甚至自发产生成串高频爆发式放电(bursting)的神经元。爆发式放电增加了突触传递的效率,促使突触活动产生短时程和长时程可塑性变化,募集邻近神经元产生同步化放电。BFNs的电活动在癫痫相关性电活动中可能具有起搏点的作用。同时,癫痫电活动也促使内源性BFNs的改变,以及调制非爆发式放电神经元向BFNs的转变,导致海马结构内癫痫电活动的进展和扩散,最终促使癫痫相关性病理性改变和脑的高级功能的损害。     

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。     

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．     

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.     

强直电刺激右背海马诱发双侧海马神经元癫痫相关性单位放电特征

目的：探讨双侧海马细胞癫痫相关性单位放电特征。方法：双玻璃微电极同步记录大鼠44对双侧海马神经元单位放电,每隔5-10min重复强直电刺激右背海马（0.6-0.4mA60Hz2s)一次,共施加10-12个刺激串。结果：强直电刺激可以诱发双侧海马神经元单位放电的原发性和继发性后放,呈现明显的双侧非对称性和动态发展特征,甚至出现双侧交互性改变,与人类颞叶癫痫的病理生理特征相吻合;进行性发展、跨大脑半球扩布和动态变化,强直电刺激对海马细胞单位自发放电具有易化或抑制、调制或解调作用,并取决于这些细胞的基础单位放电,东莨菪碱可以调电刺激引起的海马细胞爆发式单位放电成为紧张性放电,诱导强直电刺激后单位放电频率的抑制效应。结论：强直电刺激右背海马后,双侧海马细胞特征性癫痫相关性单位电活动很可能是颞叶癫痫跨半球脑损伤的病理生理学机制之一。     

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.     

颈动脉内注射腺苷对去缓冲神经大鼠最后区神经元放电的影响

在45只切断双侧缓冲神经的SpragueDawley大鼠,应用细胞外记录方法,观察了颈动脉内注射腺苷对76个最后区(AP)神经元自发放电活动的影响。所得结果如下:(1)在记录到的42个自发放电单位中,颈动脉内注射腺苷(25μg/kg)引起其中29个单位的放电频率由626±075下降至474±076spikes/s(P<001),6个单位放电频率由413±077增加至472±083spikes/s(P<005),另外7个单位放电频率无明显变化,而血压和心率在实验中无变化;(2)在应用非选择性腺苷受体拮抗剂8苯茶碱(8phenyltheophylline,15μg/kg)的10个单位,腺苷对放电的抑制效应可被完全阻断;(3)应用选择性腺苷A1受体拮抗剂8环戊1,3二丙基黄嘌呤(8cyclopentyl1,3dipropylxanthine,50μg/kg)亦可有效地阻断腺苷对12个单位的抑制效应;(4)应用ATP敏感性钾通道阻断剂格列苯脲(500μg/kg)的12个单位,腺苷的上述效应也被消除。以上结果提示,腺苷对AP区神经元自发放电有抑制作用,而此作用与A1受体介导的ATP敏感性钾通道开放有关。     

胍丁胺对大鼠海马 CA1区神经元放电的影响

应用细胞外记录单位放电技术,在大鼠海马脑片上观察了胍丁胺(agmatine,Agm)对CAl区神经元放电的影响。实验结果如下：(1)在47个海马脑片放电单位上灌流Agm(0．1—1．0μmol／L)2min,有38个单位(80．9％)自发放电频率明显降低,且呈剂量依赖性,9个单位(19．1％)无明显的反应;(2)预先用0．2mmol／L的L-谷氨酸(L-glutamate,L-Glu)灌流12个海马脑片放电单位,有9个单位(75％)放电频率明显增加,表现为癫痫样放电,在此基础上灌流Agm(1．0μmol／L)2min,其癫痫样放电被抑制;(3)在7个海马脑片放电单位上给予L型钙通道激动剂Bay K8644(0．1μmoL／L)时,有6个单位(85．7％)放电频率明显增加,另外1个单位(14．3％)无明显变化,再给予Agm(1．0μmol／L)2min,其放电频率被明显抑制;(4)13个CAl放电单位,灌流50μmoL／L一氧化氮合酶(NOS)抑制剂N^G-nitro-L-arginine methyl ester。(L-NAME)5min后其放电频率明显增加,在此基础上再给予Agm(1．0μmol／L)2min,有11个单位(84．6％)的放电频率被抑制,有2个单位(15．4％)的变化不明显。上述结果提示：胍丁胺能抑制海马CAl区神经元自发放电以及由谷氨酸、BayK8644和L-NAME诱发的放电,这一抑制效应可能与胍丁胺阻断CAl区锥体细胞上的NMDA受体,并减少钙离子内流有关。     

最后区注射腺苷对大鼠血压、心率和肾交感神经放电的影响

在 5 3只麻醉Sprague Dawley大鼠观察了最后区内微量注射腺苷 (1ng/ 6 0nl)对平均动脉压 (MAP)、心率(HR)和肾交感神经放电 (RSNA)的影响。实验结果如下 :(1)最后区内微量注射Ado后 ,MAP、HR和RSNA分别由13 76± 0 46kPa、35 6 2 8± 4 2 5bpm和 10 0± 0 %下降至 11 2 3± 0 49kPa (P <0 0 0 1)、336 91± 5 2 3bpm (P <0 0 1)和70 95± 5 19% (P <0 0 0 1) ;(2 )静脉注射非选择性腺苷受体拮抗剂 8 苯茶碱 (8 phenyltheophylline,15 0 μg/kg ,0 2ml)和选择性腺苷A1受体拮抗剂 (8 cyclopentyl 1,3 dipropylxanthine,5 0 0 μg /kg ,0 2ml)后 ,腺苷的上述抑制效应可被完全阻断 ;(3)静脉注射ATP敏感性钾通道阻断剂格列苯脲 (5mg/kg ,0 2ml)后 ,腺苷的上述效应也被消除。以上结果提示 ,最后区微量注射腺苷对血压、心率和肾交感神经放电有抑制作用 ,此作用与A1受体介导的ATP敏感性钾通道开放有关。     

Metabotropic glutamate receptors modulate GABA release from mouse hippocampal slices

The effects of metabotropic glutamate receptor agonists on the basal and potassium (50 mM K⁺)-stimulated release of [³H]GABA from mouse hippocampal slices were investigated using a superfusion system. The group I agonist (1±)-1-aminocyclopentane-trans-1,3-dicarboxylate enhanced the basal GABA release and reduced the K⁺-evoked release by a mechanism antagonized by (RS)-1-aminoindan-1,5-dicarboxylate in both cases. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine failed to have any effect on the basal release, but inhibited the stimulated release. This inhibition was not affected by the antagonist (2S)-2-ethylglutamate. The group III agonists L(+)-amino-4-phosphonobutyrate and O-phospho-L-serine inhibited the basal GABA release, which effects were blocked by the antagonist (RS)-2-cyclopropyl-4-phosphonophenylglycine. Moreover, the suppression of the K⁺-evoked release by L(+)2-amino-4-phosphonobutyrate was apparently receptor-mediated, being blocked by (RS)-2-cyclopropyl-4-phosphonophenylglycine. The results show that activation of metabotropic glutamate receptors of group I is able to potentiate the basal release of GABA, whereas activation of groups I and III receptors reduce K⁺-stimulated release in mouse hippocampal slices.     

5—羟色胺抑制谷氨酸对海马神经元的毒性作用

为探讨5-羟色胺（5-HT）对过量谷氨酸（glutamate,Glu)神经毒性的影响。观察了5-HT存在时,过量Glu对海马细胞存活率、海马脑片CA1区群锋电位（population spike,PS)及神经细胞膜Ga^2 电流的影响。结果发现：5-HT可明显提高过量Glu作用下海马神经细胞的存活率,减缓Glu对海马脑片CA1区PS的降低作用;在细胞膜上,5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用。在细胞膜上5-HT可明显减弱Glu诱导的Ca^2 内向电流,推测,一定浓度的5-HT具有抑制过量Glu神经毒性的作用,其机制可能在于5-HT与细胞膜上特定的受体结合,抑制了Glu诱导的Ca^2 内流。     

Glutamate agonists and [3H]GABA release from rat hippocampal slices: Involvement of metabotropic glutamate receptors in the quisqualate-evoked release

The effects of glutamate agonists and their selective antagonists on the Ca²⁺-dependent and independent releases of [³H]GABA from rat coronal hippocampal slices were studied in a superfusion system. The Ca²⁺-dependent release evoked by glutamate, kainate and N-methyl-D-aspartate (NMDA) gradually declined with time despite the continuous presence of the agonists. Quisqualate (QA) caused a sustained release which exhibited no tendency to decline within the 20-min period of stimulation. This release was enhanced in Ca²⁺-free medium. The release evoked by QA in Ca²⁺-containing medium was significantly inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohept-5,10-imine hydrogen maleate (MK-801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing that QA activates NMDA receptors directly or indirectly through (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The inhibition of MK-801 was slightly diminished and that of CNQX totally abolished in Ca²⁺-free medium. Verapamil inhibited the QA-activated release in both Ca²⁺-containing and Ca²⁺-free media. The effect of QA but not that of AMPA was blocked in Ca²⁺-free medium by L(+)-2-amino-3-phosphonopropionate (L-AP3), a selective antagonist of the metabotropic glutamate receptor. It is suggested that the sustained release of GABA is also mediated partly by activation of metabotropic receptors and mobilization of Ca²⁺ from intracellular stores.     

白藜芦醇抑制大鼠海马 CA1区神经元放电

应用细胞外记录单位放电技术,在大鼠海马脑片上观察了白藜芦醇(resveratrol)对海马CAI区神经元放电的影响。实验结果如下：(1)在52个CAI区神经元放电单位给予白藜芦醇(0．05、0．5、5μmol／L)2min,有46个放电单位(88.5％)放电频率明显降低,且呈剂量依赖性;(2)预先用0．2mmol／L的L-glutamate灌流海码腑片,8个放电单位放电频率明显增加,表现为癫痫样放电,在此基础上灌流白藜芦醇(5μmol／L)2min,其癫痫样放电被抑制;(3)预先用L型钙通道开放剂Bay K8644灌流7个海马5脑片,有6个单位(85.7％)放电增加,在此基础上灌流白藜芦醇(5μmol／L)2min,其放电被抑制;(4)9个放电单位灌流一氧化氮合酶抑制剂L-NAME(N^0-nitro-L-arginine methylester)50μmol／L,有7个单位(77．8％)放电明显增加,在此基础上灌流白藜芦醇(5μmol／L)2min,放电被抑制;(5)10个放电单位灌流人电导钙激活性钾通道阻断剂TEA(tetraethylarnmonium chloride)1mmol/L后,有9个单位(90％)放电增加,在此基础上灌流白藜芦醇(5μmol／L)2min,8个放电单位(88,9％)放电频率明显减低。以上结果提示：白藜芦醇能抑制海马神经元自发放电以及由L-glutamate、L-NAME、Bay K8644和TEA诱发的放电,可能与白藜芦醇抑制L型钙通道,减少钙内流有关;似乎与大电导钙激活性钾通道无关。     

Temperature dependence of synaptic responses in guinea pig hippocampal CA1 neurons in vitro

1. Temperature-dependent properties of synaptic transmission were studied by recording orthodromic responses of the population spike and excitatory postsynaptic potential in CA1 pyramidal neurons of guinea pig hippocampal slices.2. Increasing the temperature of the perfusing medium from 30 to 43°C resulted in a decrease in the amplitude of the population spike (A-PS) and a reduced slope of the excitatory postsynaptic potential (S-EPSP). Bath application of the -aminobutyric acid receptor antagonist, picrotoxin, or a change in the calcium concentration of the perfusate did not affect the A-PS during heating.3. Increasing the strength of the synaptic input to that eliciting a PS with an amplitude 50, 75, or 100% of maximal at 30°C resulted in a significant increase in the A-PS during the middle phase of hyperthermia (35–39°C).4. The long-term potentiation (LTP) induced at either 30 or 37°C showed the same percentage increase in both the amplitude of the population spike and the S-EPSP after delivery of a tetanus (100 Hz, 100 pulses) to CA1 synapses.5. The results of the present study, therefore, indicate that the decrease in CA1 field potential was linearly related to the temperature of the slice preparation, while LTP was induced in these responses during heating from 30 to 37°C.     

Prenatal stress causes dendritic atrophy of pyramidal neurons in hippocampal CA3 region by glutamate in offspring rats

A substantial number of human epidemiological data, as well as experimental studies, suggest that adverse maternal stress during gestation is involved in abnormal behavior, mental, and cognition disorder in offspring. To explore the effect of prenatal stress (PS) on hippocampal neurons, in this study, we observed the dendritic field of pyramidal neurons in hippocampal CA3, examined the concentration of glutamate (Glu), and detected the expression of synaptotagmin‐1 (Syt‐1) and N‐methyl‐D ‐aspartate receptor 1 (NR1) in hippocampus of juvenile female offspring rats. Pregnant rats were divided into two groups: control group (CON) and PS group. Female offspring rats used were 30‐day old. The total length of the apical dendrites of pyramidal neurons in hippocampal CA3 of offspring was significantly shorter in PS than that in CON (p < 0.01). The number of branch points of the apical dendrites of pyramidal neurons in hippocampal CA3 of offspring was significantly less in PS (p < 0.01). PS offspring had a higher concentration of hippocampal Glu compared with CON (p < 0.05). PS offspring displayed increased expression of Syt‐1 and decreased NR1 in hippocampus compared with CON (p < 0.001 and p < 0.01, respectively). The expression of NR1 in different hippocampus subfields of offspring was significantly decreased in PS than that in CON (p < 0.05‐0.01). This study shows that PS increases the Glu in hippocampus and causes apical dendritic atrophy of pyramidal neurons of hippocampal CA3 in offspring rats. The decline of NR1 in hippocampus may be an adaptive response to the increased Glu. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2010