ClC-2对慢性颞叶癫痫大鼠海马CA1区α5亚基-GABA_AR介导紧张性抑制的影响
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摘要
颞叶癫痫是一组以反复癫痫发作、学习记忆功能减退为主要临床表现的疾病。以往的研究表明,它与海马局部神经元退行性变、胶质增生、异常环路重构以及脑内兴奋--抑制作用失衡有关。在成年动物中枢神经系统中,GABA作为主要的抑制性神经递质,通过GABA-R起作用。其中,GABAAR主要通过介导Cl-内流引起细胞超极化而发挥抑制作用。根据GABAAR的分布和激活后的电生理特性,可以将GABAAR粗略地分成两大类：synaptic GABAAR(?>(?)extrasynaptic GABAAR,前者介导GABAergic的时相性抑制(phasic inhibition),后者介导GABAergic的紧张性抑制(tonic inhibition)。近年来,extrasynaptic GABAAR介导的紧张性抑制吸引了越来越多学者的关注。但无论是时相性还是紧张性抑制,均依赖于神经元内低氯状态。
有研究表明,GABAAR激活后,氯离子内流和碳酸氢根离子外流是相藕联的,由于碳酸酐酶的作用可维持细胞内碳酸氢根离子的高浓度,所以,碳酸氢根离子可持续的顺浓度梯度大量外流,同时,氯离子亦大量内流,从而介导突触后抑制作用。如果氯离子在短时间内不能有效排出细胞外,以保持细胞内的低氯环境,反复激活GABAAR会引起细胞内氯离子超载,后者可增加氯离子内流的阻抗,使突触后抑制作用明显减弱。因此,向胞外转运氯离子的能力对于维持神经元正常的GABAergic抑制作用显得尤为重要,氯离子稳态的打破有可能参与了异常放电的形成,而并非仅仅是异常放电的结果。细胞内外氯离子的浓度梯度主要是由-组阳离子-氯离子同向转运体和氯通道共同调节。ClC-2是电压门控性氯通道中的一种,参与调节细胞内外氯离子浓度、维持细胞膜电位等。已有研究表明,在癫痫模型中,作为成熟神经元上主要的氯离子外向转运体KCC2明显下调。考虑到C1C-2本身的特性,即在膜内电位低于膜平衡电位时被激活,产生内向整流,电导大,且该通道的失活无明显时间依赖性。那么,在癫痫病理状态下,C1C-2有何改变?
Rinke用C1C-2基因敲除小鼠证实,在细胞内高氯的状态下,C1C-2有一定的外向转运氯离子的作用,且承担了相当一部分的背景电导以维持膜电位的稳定,但野生型和基因敲除小鼠的mIPSP、sIPSP和PPR均没有显著性差异,说明ClC-2与GABAAR介导的时相性抑制无关。有研究表明,在成年动物海马CA区,GABAergic的紧张性抑制主要是由α5亚基-GABAAR介导。我们要研究的问题是：ClC-2是否与α5亚基-GABAAR介导紧张性抑制有关?即药物阻断α5亚基-GABAAR是否引起C1C-2电流的改变?在癫痫病理状态下,C1C-2电流有何改变?a5亚基-GABAAR介导的紧张性抑制本身有何改变?这种改变对大鼠慢性期SRS发作有何影响?在体阻断a5亚基-GABAAR能否改善大鼠海马的突触可塑性?
第一部分匹罗卡品致痫大鼠的行为学特征及海马神经元损伤的表现
目的：观察匹罗卡品致痫大鼠的行为学特征及海马CA1区神经元损伤的情况。
方法：雄性SD大鼠(180-200g)随机分成对照组、致痫组,为了解大鼠致痫后不同时期的行为学特征、海马神经元的损伤情况以及目的蛋白表达的变化,根据致痫后自然病程的长短,将致痫组分为：急性期组(致痫后24小时)、潜伏期组(致痫后5天)、慢性期组(出现SRS后14天)和晚期组(出现SRS后30天)。致痫组给予匹罗卡品(340mg/kg,i.p.)致痫,致痫前30min给予阿托品(5mg/kg,i.p.)减轻外周胆碱能作用。如果首剂匹罗卡品注射一小时后仍没有点燃,可以追加一次匹罗卡品(170mg/kg,i.p.)。点燃的行为学表现以出现节律性点头、咀嚼、湿狗样抖动、继而直立、跌倒为判断标准。化学点燃后90min给予地西泮(10mg/kg, i.p.)终止发作,之后使用数字摄像机持续视频监测大鼠的行为学表现,并记录潜伏期、首次SRS发作的时间、慢性期SRS的发作频率及发作持续时间等。采用视频采集卡进行后期资料的采集和分析。对照组除了以生理盐水代替匹罗卡品外,其他处理与致痫组完全一致。
应用免疫荧光标记NeuN来观察对照组和致痫后各组大鼠海马CA1区神经元的形态及数量,评估CA1区神经元的存活情况。免疫荧光染色结果的定量分析,采用连续冠状位冰冻切片(20pm),每隔3张脑片取1张,每只大鼠选取5张脑片进行CA1区NeuN阳性细胞的计数,通过计算得出每只大鼠CA1区NeuN阳性细胞的均数及各组大鼠CA1区NeuN阳性细胞的均数±标准误。实验结果均用均数±标准误表示,先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's post hoc tests)检测组间差异。
结果：致痫组大鼠在注射匹罗卡品后24±3min (n=51)被点燃,SE发作(status epilepticus)均达4-5级。注射地西泮并不能立刻终止发作,但可以减轻发作强度、增加存活率。除7只大鼠死亡外,其余致痫组大鼠均存活。在注射地西泮5-7小时后,大鼠SE发作逐渐缓解。随后,致痫组大鼠进入发作后状态,持续约2-3天,期间大鼠仍有自限性的4-5级痫样发作(持续时间：10±3min；平均发作频率：5±2次/天,n=38),需要人工喂食,以流质为主。接下来,致痫大鼠经过一段长约9±1天(n=38)的潜伏期,在这段时期内大鼠无自发性痫样发作,可以自主摄食,但进食量及活动量与对照组相比明显减少,体重不增。首次自主发作(The first spontaneous recurrent seizure, SRS)出现在致痫后第12±1天(n=28)。有4只大鼠因未出现SRS或SRS发作形式无明显丛集性而从实验中删除。其余大鼠均有稳定的SRS发作(平均发作持续时间：4±2min;平均发作频率：4±1次/天,n=28)匹罗卡品致痫模型主要表现为DG门区及CA3区锥体细胞的凋亡,而CA1区的锥体细胞相对被保留。与对照组相比,致痫后各组大鼠海马CA1区NeuN阳性细胞计数无显著统计学差异。
小结：
1、致痫组大鼠的首次自主发作(SRS)出现在致痫后第12±1天(Mean±SEM,n=28)。
2、慢性期和晚期组致痫大鼠均有稳定的SRS丛集性发作,平均发作持续时间4±2min,平均发作频率4±1次/天(Mean±SEM, n=28)
3、与对照组相比,致痫后不同时期组大鼠海马CA1区NeuN阳性细胞计数无显著统计学差异,即致痫后CA1区的锥体细胞相对被保留。
第二部分ClC-2在大鼠海马的定位以及对照组和致痫后各组海马CA1区ClC-2蛋白的定量分析
目的：观察ClC-2在大鼠海马CA1区的定位；比较对照组和致痫后各组大鼠海马CA1区ClC-2的表达量。
方法：应用免疫荧光双染技术对ClC-2进行组织学定位,单染技术比较对照组和致痫后各组ClC-2的表达情况。参考第一部分制做动物模型,分别取对照组、急性期组、潜伏期组、慢性期组和晚期组大鼠进行麻醉(乌拉坦1.5g/Kg,i.p.),经心脏灌注(肝素化生理盐水250ml,后接4%多聚甲醛400ml)。解剖动物,取出大鼠脑组织,4%多聚甲醛后固定,然后转入30%蔗糖溶液中。标本脱水后进行连续冰冻切片,脑片收集于0.01M PBS溶液中,切片厚度均为20μm,脑片用0.01M PBS溶液漂洗后,室温下用封闭液封闭1小时,以结合非特异性结合位点。弃去封闭液后加入一抗4℃过夜。吸去一抗后用0.01M PBS溶液漂洗三次,加入二抗避光室温孵育2小时,0.01M PBS溶液漂洗,避光贴片,置于荧光显微镜下观察并拍照。Western Blot检测对照组和致痫后各组大鼠海马CA1区C1C-2的表达量。取对照组和致痫后各组大鼠,快速断头取脑,将脑组织置于冰上,显微镜下分离出海马CA1区的组织,放入样品缓冲液中,加入蛋白酶抑制剂和磷酸酶抑制剂,冰上匀浆、超声破碎,取上清液分装保存于-80℃。酶联免疫吸附法测定上清液中ClC-2的含量。蛋白免疫印迹实验结果的半定量分析,应用生物凝胶图像分析系统扫描,Genegenius凝胶图像分析系统摄取图片并获得条带灰度值,GeneTool软件进行浓度的定量分析,以(3-actin作为内参。先用单因素方差分析(one-way AN OVA)处理数据,再用Tukey法(Tukey's post hoc tests)比较对照组和致痫后各组的组间差异。
结果：免疫荧光双染结果发现：C1C-2主要定位大鼠海马CA1区锥体细胞的胞体上,在锥体细胞的顶树突近端也有C1C-2相对较低的表达。ClC-2与星型胶质细胞、小胶质细胞之间没有共染。单染结果显示,致痫组大鼠海马CA1区的ClC-2IR增强,尤其是锥体细胞顶树突区的C1C-2IR信号明显增强,而锥体细胞胞体上的C1C-2IR无明显改变,这一现象在进入慢性期和晚期的癫痫大鼠脑片上尤为明显。
Western Blot结果显示,致痫后急性期组大鼠CA1区的C1C-2蛋白表达量与对照组相比无显著差异,慢性期组和晚期组C1C-2蛋白表达量明显高于对照组。
小结：
1、对照组大鼠海马CA1区C1C-2与锥体细胞存在共染,在锥体细胞的胞体高表达,顶树突区近端也有相对较低水平的表达。
2、致痫后,尤其是进入慢性期的大鼠,锥体细胞的顶树突区C1C-2IR明显增强,而锥体细胞的胞体上C1C-2IR无明显改变。
3、与对照组相比,慢性期组和晚期组CA1区ClC-2的表达量明显上调。
第三部分海马CA1区ClC-2功能性上调与α5亚基-GABAAR介导的紧张性抑制有关
目的：研究慢性颞叶癫痫大鼠海马CA1区C1C-2表达上调是否具有功能；该区α5亚基-GABAAR介导的紧张性抑制有何改变；ClC-2与α5亚基-GABAAR介导的紧张性抑制是否有关。
方法：选取对照组和慢性期组大鼠制作海马脑片,全细胞模式记录海马CA1区锥体细胞的C1C-2电流和α5亚基-GABAAR介导的紧张性抑制电流(Itonic),比较对照组和慢性期组C1C-2电流和Itoni。的变化；观察应用特异性α5亚基-GABAARs阻断剂(L-655,708)干预后C1C-2电流和Itoni。的改变。将大鼠快速断头取脑,脑组织浸入4℃切片液中,振动切片机冠状位切片(400μm),将脑片转置32℃的人工脑脊液中孵育1h后记录。切片液、人工脑脊液需事先通混合气(含95%氧气和5%二氧化碳)至少30min。记录槽内持续循环灌注细胞外液(5ml/min)。将脑片置于记录槽中,在红外相差显微镜下,全细胞模式记录CA1区锥体细胞的ClC-2电流(钳制电压：-10mV,测试电压：+40mV--120mV,跃级增量：-20mV),消除液接电位、吉欧封接、破膜、补偿电极电容和膜电容,稳定5min后开始记录,滤波2Hz,采样20Hz。观察对照组和慢性期组大鼠海马CA1区锥体细胞的ClC-2电流的差别。外液中加入L-655,708(100nM)后,记录ClC-2电流的变化。全细胞模式记录Itonic'钳制电压-60mV,在细胞外液加入TTX (200nM)、Kynurenic acid (3mM)和CGP-52432(5μM)分别阻断动作电位、兴奋性谷氨酸受体电流和GABABR,同时补充外源性GAB A (5μM)后,记录GABAAR介导的电流,基线记录平稳后,在细胞外液中加入适当浓度的L-655,708(100nM)选择性阻断介导紧张性抑制的α5亚基-GABAAR,电流平稳后再加入饱和浓度的SR-95531(100gM)完全阻断介导时相性和紧张性抑制的所有GABAAR。选取采样点：加药前100s(t1)、L-655,708加药后200s(t2)和SR-95531加药后100s(t3),采集并计算出加药前后t1、t2、t3电流的平均增量,即ΔIhold t3-tl和ΔIhold t3-t2, ΔIhold t3-t1代表GABAAR介导的总电流,ΔIhold t3-t2代表GABAAR介导的时相性抑制电流的成分。通过计算(|ΔIhold t3-t1-ΔIhold t3-t2|)可以得到α5亚基-GABAAR介导的紧张性性抑制电流Itonic。每组大鼠的ΔIholdt3--t1和t3-t2均以均数±标准误来表示。比较单个神经元加药前后的差异用KS法(Kolmogorov-Smirnov test)；检测组间差异先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's tests)。
手术置入侧脑室给药管(外径0.7mm、内径0.36mm的套管),位置：前囟后0.5mm旁开1.2mm深4mm。用牙托粉固定套管,术后7天匹罗卡品致痫,致痫大鼠进入潜伏期开始侧脑室给予L-655,708,每天两次(9am-9pm),每次侧脑室注射量为5μl,持续给药至SRS出现后14天。实验分为给药组和空白对照组。根据L-655,708浓度的不同,给药组又分为μM、4μM、8μM和16μM组,观察该药对大鼠行为学的影响。记录给药各组首次SRS发作的时间、慢性期SRS发作频率和发作持续时间,与空白组进行比较。
结果：癫痫慢性期组的ClC-2电流较对照组增大了53.2%(对照组：453.1±47.4pA,n=9；癫痫组：694.5±56.2pA,n=12；P<0.05)。外液中加入100nML-655,708后ClC-2电流减小了26.9%(空白组：702.3±63.1pA,n=10；给药组：513.6±50.2pA,n=15；P<0.05)。癫痫组α5亚基-GABAAR介导的紧张性抑制电流(Itonic)明显大于对照组(对照组：132.1±17.9pA,n=12；癫痫组：207.4±13.6pA,n=15；P<0.05)。 L-655,,708可显著抑制癫痫组ITonic(-30.3±7.9%,n=11,P<0.05),但对于对照组Itonic则无明显影响(-21.6±9.2%,n=7,P>0.05)。侧脑室给予致痫大鼠不同浓度的L-655,7088并观察大鼠行为学表现,8gM和16μM组大鼠首次SRS出现的时间早于空白组(给药组9±1天,n=7,空白组：12±1天,n=7；P<0.05),但慢性期SRS发作频率和发作持续时间未见显著组间差异。
小结：
1、癫痫组C1C-2电流较对照组明显增大,说明在病理状态下海马CA1区C1C-2表达上调是有功能的。
2、癫痫组海马CA1区a5亚基-GABAAR介导的紧张性抑制电流(Itonic)显著大于对照组；L-655,708可显著抑制癫痫组Itonic'但对于对照组Itonic则无明显抑制作用。说明对照大鼠脑组织中是以时相性抑制为主,紧张性抑制做为一种背景电流；而在癫痫大鼠脑组织中α5亚基-GABAAR介导的紧张性抑制电流明显增强,可能是对病理状态下时相性抑制减弱的一种代偿现象。
3、离体脑片上应用L-655,708可翻转癫痫组增大的C1C-2电流。说明病理状态下,C1C-2功能性上调与α5亚基-GABAAR介导的Itonic代偿性增强有关。
4、在体动物侧脑室注射L-655,708可使致痫大鼠首次SRS提早出现,但对慢性期SRS发作频率和发作持续时间无明显影响。说明Itonic增强可以延长SE发作后的潜伏期,延缓SRS的出现,即α5亚基-GABAAR介导的紧张性抑制与SRS的形成有关,与慢性期SRS反复发作无关。
第四部分α5亚基-GABAAR介导的紧张性抑制对慢性颞叶癫痫大鼠海马突触可塑性的影响
目的：研究癫痫组(慢性期)大鼠海马CA1区突触可塑性有何改变；应用α5亚基-GABAAR特异性阻断剂对慢性颞叶癫痫大鼠海马LTP/LTD的诱导有何影响。
方法：本部分实验应用在体记录大鼠海马CA1区场电位的技术。以20%乌拉坦(7.5m1/kg i.p.)麻醉动物,将大鼠头部固定于脑立体定位仪上,以H202清除皮下组织和筋膜,充分暴露颅骨中缝和前囟。在颅骨上标记刺激电极(前囟后4.2mm,中线左侧旁开3.8mm,深3200-3500μm)、记录电极(前囟后3.4mm,中线左侧旁开2.5mm,深2200-2500μm)和侧脑室给药管(前囟后0.5mm,中线右侧旁开1.0mm,深4000μm)的位置,用电钻在标记点上钻孔,借助微电极操作仪将电极和给药管缓慢送入目标位置。用牙托粉将不锈钢给药管固定,自然晾干。连续单方波刺激海马Schaffer collateral pathway的同时,在CA1区辐射层记录Schaffer→CA1的兴奋性突触后场电位(field excitatory post-synaptic potentials, fEPSPs)。高频刺激(100Hz,每串50个刺激,每串间隔15s,4串)诱导海马LTP；低频刺激(1Hz,900个刺激,15min)诱导海马LTD。给予条件刺激之前,应用测试刺激至少稳定记录30min做为baseline;侧脑室给药后需继续记录至少30min再诱导LTP/LTD,以观察药物本身对电位是否有影响。给药时先用微量注射器缓慢推注5μ1(L-655,708或vehicle),时间控制在2min左右,再用输液微泵维持至实验结束(推速：0.5μl/h)。该实验以海马CA1区fEPSPs的幅度作为提取参数,每10个连续的测试刺激做一次平均,以加药前或条件刺激前海马fEPSPs幅度的均值做为对照,所得数据由LTP、SPSS10.0软件进行统计分析,比较加药前、后的差异用Wilcoxon signed ranks检验,组间比较用Kruskal-Wallis test检验。Western Blot检测对照组和癫痫组(慢性期)大鼠海马CA1区NR1、NR2A、NR2B、 GluR1、GluR2和GluR3的表达量。在体实验结束后,分别取对照组和癫痫组大鼠脑组织,显微镜下分离,放入样品缓冲液中,加入蛋白酶抑制剂和磷酸酶抑制剂,冰上匀浆、超声破碎,取上清液分装保存于-80℃。酶联免疫吸附法测定上清液中上述目的蛋白的含量。蛋白免疫印迹实验结果的半定量分析,应用生物凝胶图像分析系统扫描,Genegenius凝胶图像分析系统摄取图片并获得条带灰度值,GeneTool软件进行浓度的定量分析,以(3-actin作为内参。先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's post hoc tests)比较对照组和癫痫组的组间差异。
结果：高频刺激(high-frequency stimulation, HFS)诱导癫痫组海马CA1区fEFSPs的幅度显著低于对照组(癫痫组：128.5±9.43%,n=5；对照组：150.9±7.57%,n=5,P<0.05)；低频刺激(low-frequency stimulation, LFS)诱导癫痫组海马CA1区fEFSPs幅度显著高于对照组(癫痫组：117.2±6.22%,n=5；对照组：68.3±7.31%,n=5,P<0.05)。L-655,708对高频刺激诱导海马LTP的fEFSPs幅度无明显影响(给药组：132.4±7.01%,n=5；空白组：130.3±7.36%,n=4,P>0.05)；L-655,708可部分抑制低频刺激诱导海马LTD的fEFSPs幅度(给药组：91.6±6.57%,n=5；空白组：113.7±6.91%,n=4,P<0.05)。应用V(?)estern Blot技术检测对照组和癫痫组大鼠海马CA1区NR1、NR2A、NR2B、 GluR1、GluR2和GluR3的表达量。癫痫组NR1和NR2A(?)的表达量明显高于对照组；而NR2B的表达量显著减低。癫痫组GluR2的表达量明显低于对照组,同时GluR1和GluR3显著增高,癫痫组GluR2/GluR1+GluR3比值显著低于对照组。
小结：
1、高频刺激诱导癫痫组海马CA1区LTP的fEFSPs幅度显著低于对照组；低频刺激诱导癫痫组海马CA1区fEFSPs幅度则明显高于对照组,并翻转形成LTP。说明癫痫组大鼠强化记忆的功能明显减退,同时纠错和删除记忆的功能也有所削弱。
2、L-655,708对高频刺激诱导海马LTP的fEFSPs幅度无明显影响；可部分抑制低频刺激诱导海马LTD的fEFSPs幅度。说明L-655,708可以改善癫痫大鼠的纠错和删除记忆功能,避免LTP过饱和,从而维持相邻突触LTP的诱导阈值。
结论
正常大鼠中,C1C-2主要定位于海马CA1区锥体细胞的胞体上,在锥体细胞顶树突的近端也有相对较低水平的表达。致痫后,尤其是进入慢性期的大鼠,海马CA1区C1C-2的表达量明显上调,且具有功能性,以锥体细胞顶树突区的C1C-2上调显著。
慢性颞叶癫痫大鼠海马CA1区α5亚基-GABAAR介导的紧张性抑制电流(Itonic)显著增大；在离体脑片上应用L-655,708可显著抑制Itonic并翻转增大的C1C-2电流。致痫后,持续侧脑室给予L-655,708可使癫痫大鼠首次SRS提早出现,但对慢性期SRS发作频率和发作持续时间无明显影响；在体应用L-655,708可改善癫痫大鼠海马CA1区LTD的诱导。
Temporal lobe epilepsy, characterized by spontaneous recurrent seizures, learning and memory impairments is associated with neurodegeneration, abnormal reorganization of the circuitry, and loss of functional inhibition in hippocampus. In adult hippocampus, the GABAergic cells mediate the major inhibitory function of the principal neurons, promoting the Cl-entry through the GABAA receptor, whether through phasic (synaptic) or tonic (extrasynaptic) conductance. Aside from classical synaptic component, tonic GABAergic inhibition mediated by extrasynaptic GABAA receptor received increasing attention over the past years. There is growing evidence that tonic inhibition plays an important role in epilepsy, memory and cognition.
Since GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl" concentration at low levels in mature neurons, a shift in Ecl is likely to participate in the generation and not merely a consequence of TLE. As we known, chloride homeostasis is regulated by cation-chloride cotransporters and chloride channels. The transmembrane distribution of chloride determines the direction of the chloride flux gated by GABAA receptor-mediated response in neurons. ClC-2is a member of the supergene family of voltage-gated chloride channels. It is proved to be inwardly rectifying, and plays an important role in setting the intracellular chloride concentration in neurons expressing inhibitory GABAA receptors (GABAARs).
Several observations have shown that repetitive activation of GABAARs leads to a high intracellular chloride load that is generated by a GABAAR-mediated Cl-influx driven by HCO3-efflux via GABAARs, which dose not fade because of rapid replenishment of HCO3-by intracellular carbonic anhydrase (CA) activity. Furthermore, as a main chloride extruder in mature neurons, KCC2has been reported to be downregulated in experimental TLE models. Therefore, the chloride extrusion capacity seems to play a key role in setting the susceptibility of neurons to epileptiform activity. Since the conductance of ClC-2is large and does not display time-dependent inactivation, it is well suited for stabilize ECl. However, whether ClC-2contributes to epilepsy or not is controversial. The studies in human established the link between mutations in ClC-2and epilepsy, while ClC-2KO mice did not lead to higher seizure susceptibility. In addition, Rinke and coworkers used KO mice to demonstrate that ClC-2helps to extrude chloride from neurons under condition of high chloride load and contributes substantially to the background conductance. However, the amplitude and frequency of mIPSC, sIPSC, and PPR (a measure for presynaptic release) show no difference between WT and KO mice. Therefore, the possibility that ClC-2is involved in phasic inhibition by changing the number of postsynaptic GABAARs, inhibitory synapse number, and probability of release could be excluded.
It posed the question whether ClC-2is related to tonic inhibition mediated by extrasynaptic GABAARs. As is known, a5subunit-containing GABAARs have a restricted distribution in dendritic areas of hippocampal CA1and CA3regions, and play a predominant role in tonic inhibition of CA1pyramidal cells. Here we investigated whether the ClC-2expression is altered in CA1pyramidal cells in pilocarpine-treated rats, and whether a5subunit-containing GABAARs could affect ClC-2currents by pharmacological intervention.
Part Ⅰ Behavioural features and the pattern of neuronal loss in the hippocampus of pilocarpine-treated rats
Objective:To observe the behavioural features of pilocarpine-induced rats and neuronal loss in the CA1pyramidal cell layer in pilocarpine-treated rats.
Methods:Male Sprague-Dawley rats weighing180-200g were used. Status epilepticus (SE) was induced by pilocarpine hydrochloride (340mg/kg, i.p.). To lessen peripheral cholinergic effects, atropine methylbromide (5mg/kg, i.p.) was administered30min before pilocarpine. If seizure activity was not initiated within1hour after the initial pilocarpine hydrochloride dose, an additional dose of170mg/kg was given. The onset of SE was defined as the appearance of stage5seizures, followed by continuous and convulsive behaviorally detectable seizure activity. Diazepam (10mg/kg, i.p.) was injected90min after its onset. Rats were hand fed after SE until they were able to drink and eat on their own (2～3d). After SE, rats were killed at different time-point, representative of the different phases of the natural history of the disease:24h immediately followed by the SE (acute phase),5d after SE (latency),14d after the first occurrence of spontaneous recurrent seizure (SRS, chronic epilepsy).30d after the first occurrence of SRS (late chronic epilepsy). Rats were continuously video monitored for the appearance of SRSs, only rats that displayed multiple spontaneous stage3-4seizures in the subsequent month were included into the14d and30d groups. Control rats (con) were treated identically except that saline was substituted for pilocarpine.
Immunofluorescence was adopted to confirm the pattern of neuronal loss in this model. Primary antibody was mouse anti-neuronal nuclei (NeuN, neuron marker,1:500, Millipore). All the sections were treated by a mixture of FITC-conjugated secondary antibodies (1:400, Jackson ImmunoResearch). The quantification of the immunofluorescence staining in CA1area was performed by counting the number of NeuN positive cells per section. In each rat, every fourth section was picked from a series of consecutive hippocampus sections (20μm), and five sections were counted for each rat. An average number of neurons were obtained for each rat across five sections, and then the Mean±SEM across rats was determined. Statistical analysis was performed with SPSS10.0(SPSS Inc, USA). All data was presented as Mean±SEM. Differences in changes of values over times or drugs were tested using one-way ANOVA followed by individual post hoc comparisons (Tukey's post hoc tests).
Results:Convulsive status epilepticus (SE) were observed24±3min (Mean±SEM, n=51) after pilocarpine injection, which was intervened after90min by a low dose of diazepam. It did not stop the SE, but decreased its severity and mortality (7SE rats were dead). SE spontaneously alleviated5～7h after diazepam administration, and then rats entered postictal status, lasting2～3days, with self-limiting and generalized seizures (duration:10±3min; mean frequency:5±2seizures per day, n=38), before undergoing a latent period in which they were apparently normal. The first spontaneous recurrent seizure (SRS) occurred at12±1days after SE (n=32). Rats that did not display any SRS in one month after SE were deleted (n=4), while the others kept experiencing SRSs (duration:4±2min; mean frequency:4±1seizures per day, n=28). According to Racine's classification criteria, partial seizures (stage3-4) were more frequent compared to secondarily generalized seizures (stage5-6), and tended to recur in a cluster way.
Pilocarpine-treated rats displayed marked neuronal loss in the hilus of DG and CA3pyramidal cell layer. However, CA1pyramidal cell layer was well preserved. Compared with control group, the number of NeuN+cells in the CA1pyramidal cell layers in pilocarpine-treated rats did not decreased significantly at different phases.
Summary:
1. The first spontaneous recurrent seizure (SRS) occurred at12±1days after SE.
2. Rats kept experiencing SRSs with a mean frequency of4±1seizures per day and a mean duration of4±2min in the subsequent month.
3. No significant neuronal loss in the CA1pyramidal cell layer in pilocarpine-treated rats.
Part Ⅱ The location of CIC-2IR and the quantification of ClC-2protein level in the CA1pyramidal cells layer in pilocarpine-treated rat
Objective:To investigate the location of ClC-2IR in hippocampus and the change of protein level of ClC-2in CA1region in pilocarpine-induced rats.
Methods:Double immunofluorescence staining was performed to localize ClC-2in hippocampus. Rats from epileptic and control groups were anesthetized with urethane (1.5g/Kg, i.p.) and transcardially perfused with heparinized saline, followed by4%paraformaldehyde (PFA) in0.1M phosphate buffer, PH7.4. The intact brains were removed and post-fixed for3h in the same fixative, then transferred into30%sucrose solution. Transverse brain sections (20μm) were cut using a cryostat. All sections were blocked with3%donkey serum in0.3%Triton X-100for1h at room temperature and incubated with primary antibodies over one night at4℃, then the sections were incubated for2h at room temperature with secondary antibodies. The stained sections were examined with a fluorescence microscope and images were captured with a CCD spot camera.
Western blot was performed to confirm the expression of ClC-2after SE in protein level. Rats were decapitated, brains were immediately removed and frozen on dry ice, then stored at-80℃until processing. The CA1regions were microdissected on dry ice. The tissue samples were homogenated and sonicated in15mmol/L Tris buffer, then centrifuged at13,000g for15min at4℃to isolate the supernatant containing protein samples. The samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was blocked for1h at room temperature in blocking buffer and then incubated with anti-ClC-2antibody (1:200, Alomone Labs) overnight at4℃, followed by incubation in anti-rabbit secondary antibody (1:1000, Cell Signaling Technology). The immune bands were visualized by enhanced chemiluminescence (ECL, Amersham, USA) and film exposure (Kodak, Rochester, NY). The membrane was stripped with stripping buffer for30min at50℃and re-probed with anti-α-actin (1:1000, Santa Cruz), followed by incubation in anti-mouse secondary antibody at a dilution of1:4000(Cell Signaling Technology) as an endogenous control protein to ensure equal loading. Densitometric analysis of bands on the film was conducted with a computer-assisted imaging analysis system, and normalized to β-actin immunoreactivity.
Results:In control rats, ClC-2immunoreactivity (ClC-2IR) was present with high levels of labeling in the cell membrane and perinuclear cytoplasm of pyramidal cells (PCs), and relatively low levels of labeling in the proximal apical dendrites of PCs in CA1regions. Double immunofluorescence staining showed that ClC-2only co-localized with NeuN, neither with GFAP nor with Ibal. In pilocarpine-treated rats, an increased ClC-2IR was present, especially in CA1PCs dendrites (strata radiatum). Meanwhile, ClC-2IR maintained high levels in the pyramidal cell layer.
Immunoblot was performed to quantify this change, ClC-2protein level was significantly higher than that in control group.
Summary:
1. ClC-2IR was present with high levels of labeling in the cell membrane and perinuclear cytoplasm of pyramidal cells (PCs), and relatively low levels of labeling in the proximal apical dendrites of PCs in CA1region.
2. ClC-2upregulated in CA1pyramidal cells (PCs) in the chronic post-SE rats.
3. The obvious enhancement of ClC-2IR during the chronic period is in the apical dendrites of PCs in CA1region.
Part Ⅲ CIC-2alteration is involved in tonic inhibition mediated by a5subunit-containing GABAARs in CA1region
Objective:To investigate whether the upregulation of ClC-2in CA1PCs is functional and whether ClC-2is involved in tonic inhibition mediated by a5subunit-containing GABAARs. Reducing the tonic inhibition would aggravate or improve the generation and propagation of SRS in post-SE rats.
Methods:Whole-cell voltage-clamp recordings in hippocampal slices prepared from epileptic and control rats. Following decapitation, brains were quickly submerged in ice-cold sucrose-ACSF. Transverse slices (400μm) were cut with a Vibratome. Before recording, slices were incubated for30min at32℃in ACSF. Then slices were transferred to the recording chamber and continuously perfused (5ml/min) with recirculating extracellular solution (oxygenated with95%O2-5%CO2). Visualized patch-clamp recordings from the CA1pyramidal neurons were performed by IR-DIC videomicroscopy with an Axopatch200B amplifier, filtered at2kHz and digitized at20kHz. C1C-2currents were evoked at test potentials between+40and-20mV (increment:-20mV; holding potential:-10mV).Itonic were recorded as the change in mean holding currents (ΔIhold) after applying SR-95531(100μM) while voltage clamped at-60mV. The Ihold was sampled and averaged in recordings as follows:100s before (t1) and200s after L-655,708application (t2), and100s after SR-95531application (t3). Mean ΔIhold before and after drugs application were calculated for each neuron. Before and after values for an individual neuron were compared using the Kolmogorov-Smirnov (KS) test. Differences between groups were analyzed with one-way ANOVA followed by individual post hoc comparisons (Tukey's post hoc tests).
A stainless-steel cannula (22gauge,0.7mm outer diameter) was implanted above the right lateral ventricle (0.5mm posterior to the bregma,1.2mm lateral to midline and4mm below the surface of the dura). The cannula was secured by dental cement. A stainless-steel obturator was inserted into the cannula to prevent clogging and infection. Rats were allowed to recover for7days before inducing SE. Intracerebroventricular (i.c.v.) injection was made from the latency till14d after the first occurrence of SRS via an internal cannula (28gauge,0.36mm outer diameter).
Results:C1C-2currents increased by53.2%(control,453.1±47.4pA, n=9; post-SE,694.5±56.2pA, n=12; P<0.05) in polocarpine-treated rats, consistent with the upregulation of C1C-2. And a decrease in L-655,708treatment slices by-26.9%was observed (vehicle,702.3±63.1pA, n=10; L-655,708,513.6±50.2pA, n=15; P<0.05). A significant increase in GABAAR-mediated tonic inhibition (Itonic) in CA1PCs, compared to neurons from control (control:132.1±17.9pA, n=12; post-SE:207.4±13.6pA, n=15; P<0.05). L-655,708(100nM) decreased Itonic in control neurons by-21.6±9.2%(n=7), but produced a significantly greater decrease in post-SE neurons (-30.3±7.9%, n=11).
L-655,708treatment starting from the latency till14d after the first occurrence of SRS in vivo resulted in an earlier manifest of the first occurrence of SRS at9±1days after SE in the8μM and16μM L-655,708groups (n=7each group, P<0.05), but did not show significant effect on the duration and mean frequency of SRSs.
Summary:
1. C1C-2currents increased in CA1PCs in pilocarpine-treated rats.
2. A significant increase in GABAAR-mediated tonic inhibition (Itonic) in CA1PCs, compared to neurons from control. It is probably due to the compensatory increased tonic inhibition for replenishing the decreased synaptic GABAergic innervation at dendrites in TLE model.
3. L-655,708attenuated the increased Itonic in vitro and produces an earlier manifest of the first occurrence of SRS in vivo in pilocarpine-treated rats. We suggest that the chronically raised tonic inhibition mediated by a5subunit-containing GABAARs is associated with the generation of SRS.
4. L-655,708reversed the increase in C1C-2currents in vitro. We suggest that C1C-2modification in CA1PCs is probably to assist the chloride extrusion and maintain the inwardly directed driving force for chloride ions, which is a prerequisite for hyperpolarizing inhibition of extrasynaptic GABAARs.
Part Ⅳ L-655,708attenuates the increased LTD, but produces no significant change in decreased LTP in post-SE rats in vivo.
Objective:To observe what the change of synaptic plasticity of post-SE rats in CA1area, and whether a5subunit-containing GABAARs could affect amplitude of field potentials by pharmacological intervention.
Methods:Experiments were performed on male Sprague-Dawley rats. Urethane (1.5g/kg, ip) was used to induce and maintain anesthesia. Additional doses (0.5g/kg) were given if needed. Surgical level of anesthesia was verified by the stable mean arterial blood pressure during noxious stimulation. The trachea was cannulated, and the animal breathed spontaneously. The left femoral vein and artery were cannulated for intravenous injection and continuous monitoring of blood pressure respectively. Colorectal temperature was kept constant (37-38℃) by means of a feedback-controlled heating blanket. Rats were placed in a stereotaxic frame for all recordings. A small craniotomy (1mm×1.5mm) was performed, and then the dura and arachnoid were carefully removed. A stainless steel guide cannula was implanted in the right lateral ventricle (1mm lateral to midline,0.5mm posterior to bregma and4mm below the surface of dura), an internal cannula was used for intracerebroventricular injections (i.c.v.). The craniotomy hole was sealed with dental cement after implantation procedure. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the dorsal hippocampus in response to stimulation of the ipsilateral Scaffer collateral-commissural pathway. The recording site was located3.4mm posterior to bregma and2.5mm lateral to midline, and the stimulating site was located4.2mm posterior to bregma and3.8mm lateral to midline. The final depth of both electrodes was adjusted to optimize the electrically evoked fEPSPs. The intensity of test stimuli (0.033Hz,0.1ms duration) was adjusted to evoke a fEPSP that was50%of maximum. Conditioning high frequency stimulation (HFS:100Hz,50stimuli per train,4trains at interval of15s) was induced LTP, and LTD was induced by low frequency stimulation (LFS:1Hz,900stimuli15min). Baseline was recorded for>30min prior to injection of drug/vehicle to ensure a steady response. All values were expressed as mean±SEM. The magnitude of LTP/LTD was measured as the percentage of the baseline fEPSP amplitude during30min periods, just prior to HFS/LFS. Before and after values for an individual rat were compared using the Wilcoxon signed ranks test. Differences between groups were analyzed with Kruskal-Wallis test.
Western blot was performed to compare the protein levels of NR1, NR2A, NR2B, GluRl, GluR2and GluR3in CA1area in post-SE rats with that in control ones. Rats were decapitated, brains were immediately removed and frozen on dry ice, then stored at-80℃until processing. The CA1regions were microdissected on dry ice. The tissue samples were homogenated and sonicated in Tris buffer, then centrifuged to isolate the supernatant containing protein samples. The samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was blocked for1h at room temperature in blocking buffer and then incubated with primary antibody overnight at4℃, followed by incubation in secondary antibody. The immune bands were visualized by enhanced chemiluminescence (ECL, Amersham, USA) and film exposure (Kodak, Rochester, NY). The membrane was stripped with stripping buffer for30min at50℃and re-probed with anti-β-actin, followed by incubation in secondary antibody as an endogenous control protein to ensure equal loading. Densitometric analysis of bands on the film was conducted with a computer-assisted imaging analysis system, and normalized to P-actin immunoreactivity.
Results:The amplitude of fEPSPs induced by HFS decreased by14.84%(control:150.9±7.57%, n=5; post-SE:128.5±9.43%, n=5; P<0.05) in post-SE rats, and an increase in the amplitude of fEPSPs induced by LFS was detected (control:68.3±7.31%, n=5; post-SE:117.2±6.22%, n=5; P<0.05) compared to rats from control. L-655,708attenuates the increased LTD (vehicle:113.7±6.91%, n=4; L-655,708:91.6±6.57%, n=5; P<0.05), but produces no significant change in decreased LTP in post-SE rats in vivo (vehicle:130.3±7.36%, n=4; L-655,708:132.4±7.01%,n=5;P>0.05). Immunoblot was performed to quantify the protein levels of NR1, NR2A, NR2B, GluR1, GluR2and GluR3in CA1area. NR2B and GluR2downregulated in CA1pyramidal cells (PCs) in the chronic post-SE rats, and other ones were higher than that in control group.
Summary:
1. The amplitude of fEPSPs induced by HFS decreased significantly in post-SE rats, and an increase in the amplitude of fEPSPs induced by LFS was detected compared to rats from control.
2. L-655,708attenuates the increased LTD, but produces no significant change in decreased LTP in post-SE rats in vivo.
Conclusion
The major findings of this study are that ClC-2upregulated functionally in CA1pyramidal cells (PCs) in the chronic post-SE rats, with a corresponding increase in tonic GABAergic inhibition, and that dampening this tonic inhibition by L-655,708reverses the increase in ClC-2currents in CA1PCs in vitro and in amplitude of fEPSPs induced by LFS in vivo.
有研究表明,GABAAR激活后,氯离子内流和碳酸氢根离子外流是相藕联的,由于碳酸酐酶的作用可维持细胞内碳酸氢根离子的高浓度,所以,碳酸氢根离子可持续的顺浓度梯度大量外流,同时,氯离子亦大量内流,从而介导突触后抑制作用。如果氯离子在短时间内不能有效排出细胞外,以保持细胞内的低氯环境,反复激活GABAAR会引起细胞内氯离子超载,后者可增加氯离子内流的阻抗,使突触后抑制作用明显减弱。因此,向胞外转运氯离子的能力对于维持神经元正常的GABAergic抑制作用显得尤为重要,氯离子稳态的打破有可能参与了异常放电的形成,而并非仅仅是异常放电的结果。细胞内外氯离子的浓度梯度主要是由-组阳离子-氯离子同向转运体和氯通道共同调节。ClC-2是电压门控性氯通道中的一种,参与调节细胞内外氯离子浓度、维持细胞膜电位等。已有研究表明,在癫痫模型中,作为成熟神经元上主要的氯离子外向转运体KCC2明显下调。考虑到C1C-2本身的特性,即在膜内电位低于膜平衡电位时被激活,产生内向整流,电导大,且该通道的失活无明显时间依赖性。那么,在癫痫病理状态下,C1C-2有何改变?
Rinke用C1C-2基因敲除小鼠证实,在细胞内高氯的状态下,C1C-2有一定的外向转运氯离子的作用,且承担了相当一部分的背景电导以维持膜电位的稳定,但野生型和基因敲除小鼠的mIPSP、sIPSP和PPR均没有显著性差异,说明ClC-2与GABAAR介导的时相性抑制无关。有研究表明,在成年动物海马CA区,GABAergic的紧张性抑制主要是由α5亚基-GABAAR介导。我们要研究的问题是：ClC-2是否与α5亚基-GABAAR介导紧张性抑制有关?即药物阻断α5亚基-GABAAR是否引起C1C-2电流的改变?在癫痫病理状态下,C1C-2电流有何改变?a5亚基-GABAAR介导的紧张性抑制本身有何改变?这种改变对大鼠慢性期SRS发作有何影响?在体阻断a5亚基-GABAAR能否改善大鼠海马的突触可塑性?
第一部分匹罗卡品致痫大鼠的行为学特征及海马神经元损伤的表现
目的：观察匹罗卡品致痫大鼠的行为学特征及海马CA1区神经元损伤的情况。
方法：雄性SD大鼠(180-200g)随机分成对照组、致痫组,为了解大鼠致痫后不同时期的行为学特征、海马神经元的损伤情况以及目的蛋白表达的变化,根据致痫后自然病程的长短,将致痫组分为：急性期组(致痫后24小时)、潜伏期组(致痫后5天)、慢性期组(出现SRS后14天)和晚期组(出现SRS后30天)。致痫组给予匹罗卡品(340mg/kg,i.p.)致痫,致痫前30min给予阿托品(5mg/kg,i.p.)减轻外周胆碱能作用。如果首剂匹罗卡品注射一小时后仍没有点燃,可以追加一次匹罗卡品(170mg/kg,i.p.)。点燃的行为学表现以出现节律性点头、咀嚼、湿狗样抖动、继而直立、跌倒为判断标准。化学点燃后90min给予地西泮(10mg/kg, i.p.)终止发作,之后使用数字摄像机持续视频监测大鼠的行为学表现,并记录潜伏期、首次SRS发作的时间、慢性期SRS的发作频率及发作持续时间等。采用视频采集卡进行后期资料的采集和分析。对照组除了以生理盐水代替匹罗卡品外,其他处理与致痫组完全一致。
应用免疫荧光标记NeuN来观察对照组和致痫后各组大鼠海马CA1区神经元的形态及数量,评估CA1区神经元的存活情况。免疫荧光染色结果的定量分析,采用连续冠状位冰冻切片(20pm),每隔3张脑片取1张,每只大鼠选取5张脑片进行CA1区NeuN阳性细胞的计数,通过计算得出每只大鼠CA1区NeuN阳性细胞的均数及各组大鼠CA1区NeuN阳性细胞的均数±标准误。实验结果均用均数±标准误表示,先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's post hoc tests)检测组间差异。
结果：致痫组大鼠在注射匹罗卡品后24±3min (n=51)被点燃,SE发作(status epilepticus)均达4-5级。注射地西泮并不能立刻终止发作,但可以减轻发作强度、增加存活率。除7只大鼠死亡外,其余致痫组大鼠均存活。在注射地西泮5-7小时后,大鼠SE发作逐渐缓解。随后,致痫组大鼠进入发作后状态,持续约2-3天,期间大鼠仍有自限性的4-5级痫样发作(持续时间：10±3min；平均发作频率：5±2次/天,n=38),需要人工喂食,以流质为主。接下来,致痫大鼠经过一段长约9±1天(n=38)的潜伏期,在这段时期内大鼠无自发性痫样发作,可以自主摄食,但进食量及活动量与对照组相比明显减少,体重不增。首次自主发作(The first spontaneous recurrent seizure, SRS)出现在致痫后第12±1天(n=28)。有4只大鼠因未出现SRS或SRS发作形式无明显丛集性而从实验中删除。其余大鼠均有稳定的SRS发作(平均发作持续时间：4±2min;平均发作频率：4±1次/天,n=28)匹罗卡品致痫模型主要表现为DG门区及CA3区锥体细胞的凋亡,而CA1区的锥体细胞相对被保留。与对照组相比,致痫后各组大鼠海马CA1区NeuN阳性细胞计数无显著统计学差异。
小结：
1、致痫组大鼠的首次自主发作(SRS)出现在致痫后第12±1天(Mean±SEM,n=28)。
2、慢性期和晚期组致痫大鼠均有稳定的SRS丛集性发作,平均发作持续时间4±2min,平均发作频率4±1次/天(Mean±SEM, n=28)
3、与对照组相比,致痫后不同时期组大鼠海马CA1区NeuN阳性细胞计数无显著统计学差异,即致痫后CA1区的锥体细胞相对被保留。
第二部分ClC-2在大鼠海马的定位以及对照组和致痫后各组海马CA1区ClC-2蛋白的定量分析
目的：观察ClC-2在大鼠海马CA1区的定位；比较对照组和致痫后各组大鼠海马CA1区ClC-2的表达量。
方法：应用免疫荧光双染技术对ClC-2进行组织学定位,单染技术比较对照组和致痫后各组ClC-2的表达情况。参考第一部分制做动物模型,分别取对照组、急性期组、潜伏期组、慢性期组和晚期组大鼠进行麻醉(乌拉坦1.5g/Kg,i.p.),经心脏灌注(肝素化生理盐水250ml,后接4%多聚甲醛400ml)。解剖动物,取出大鼠脑组织,4%多聚甲醛后固定,然后转入30%蔗糖溶液中。标本脱水后进行连续冰冻切片,脑片收集于0.01M PBS溶液中,切片厚度均为20μm,脑片用0.01M PBS溶液漂洗后,室温下用封闭液封闭1小时,以结合非特异性结合位点。弃去封闭液后加入一抗4℃过夜。吸去一抗后用0.01M PBS溶液漂洗三次,加入二抗避光室温孵育2小时,0.01M PBS溶液漂洗,避光贴片,置于荧光显微镜下观察并拍照。Western Blot检测对照组和致痫后各组大鼠海马CA1区C1C-2的表达量。取对照组和致痫后各组大鼠,快速断头取脑,将脑组织置于冰上,显微镜下分离出海马CA1区的组织,放入样品缓冲液中,加入蛋白酶抑制剂和磷酸酶抑制剂,冰上匀浆、超声破碎,取上清液分装保存于-80℃。酶联免疫吸附法测定上清液中ClC-2的含量。蛋白免疫印迹实验结果的半定量分析,应用生物凝胶图像分析系统扫描,Genegenius凝胶图像分析系统摄取图片并获得条带灰度值,GeneTool软件进行浓度的定量分析,以(3-actin作为内参。先用单因素方差分析(one-way AN OVA)处理数据,再用Tukey法(Tukey's post hoc tests)比较对照组和致痫后各组的组间差异。
结果：免疫荧光双染结果发现：C1C-2主要定位大鼠海马CA1区锥体细胞的胞体上,在锥体细胞的顶树突近端也有C1C-2相对较低的表达。ClC-2与星型胶质细胞、小胶质细胞之间没有共染。单染结果显示,致痫组大鼠海马CA1区的ClC-2IR增强,尤其是锥体细胞顶树突区的C1C-2IR信号明显增强,而锥体细胞胞体上的C1C-2IR无明显改变,这一现象在进入慢性期和晚期的癫痫大鼠脑片上尤为明显。
Western Blot结果显示,致痫后急性期组大鼠CA1区的C1C-2蛋白表达量与对照组相比无显著差异,慢性期组和晚期组C1C-2蛋白表达量明显高于对照组。
小结：
1、对照组大鼠海马CA1区C1C-2与锥体细胞存在共染,在锥体细胞的胞体高表达,顶树突区近端也有相对较低水平的表达。
2、致痫后,尤其是进入慢性期的大鼠,锥体细胞的顶树突区C1C-2IR明显增强,而锥体细胞的胞体上C1C-2IR无明显改变。
3、与对照组相比,慢性期组和晚期组CA1区ClC-2的表达量明显上调。
第三部分海马CA1区ClC-2功能性上调与α5亚基-GABAAR介导的紧张性抑制有关
目的：研究慢性颞叶癫痫大鼠海马CA1区C1C-2表达上调是否具有功能；该区α5亚基-GABAAR介导的紧张性抑制有何改变；ClC-2与α5亚基-GABAAR介导的紧张性抑制是否有关。
方法：选取对照组和慢性期组大鼠制作海马脑片,全细胞模式记录海马CA1区锥体细胞的C1C-2电流和α5亚基-GABAAR介导的紧张性抑制电流(Itonic),比较对照组和慢性期组C1C-2电流和Itoni。的变化；观察应用特异性α5亚基-GABAARs阻断剂(L-655,708)干预后C1C-2电流和Itoni。的改变。将大鼠快速断头取脑,脑组织浸入4℃切片液中,振动切片机冠状位切片(400μm),将脑片转置32℃的人工脑脊液中孵育1h后记录。切片液、人工脑脊液需事先通混合气(含95%氧气和5%二氧化碳)至少30min。记录槽内持续循环灌注细胞外液(5ml/min)。将脑片置于记录槽中,在红外相差显微镜下,全细胞模式记录CA1区锥体细胞的ClC-2电流(钳制电压：-10mV,测试电压：+40mV--120mV,跃级增量：-20mV),消除液接电位、吉欧封接、破膜、补偿电极电容和膜电容,稳定5min后开始记录,滤波2Hz,采样20Hz。观察对照组和慢性期组大鼠海马CA1区锥体细胞的ClC-2电流的差别。外液中加入L-655,708(100nM)后,记录ClC-2电流的变化。全细胞模式记录Itonic'钳制电压-60mV,在细胞外液加入TTX (200nM)、Kynurenic acid (3mM)和CGP-52432(5μM)分别阻断动作电位、兴奋性谷氨酸受体电流和GABABR,同时补充外源性GAB A (5μM)后,记录GABAAR介导的电流,基线记录平稳后,在细胞外液中加入适当浓度的L-655,708(100nM)选择性阻断介导紧张性抑制的α5亚基-GABAAR,电流平稳后再加入饱和浓度的SR-95531(100gM)完全阻断介导时相性和紧张性抑制的所有GABAAR。选取采样点：加药前100s(t1)、L-655,708加药后200s(t2)和SR-95531加药后100s(t3),采集并计算出加药前后t1、t2、t3电流的平均增量,即ΔIhold t3-tl和ΔIhold t3-t2, ΔIhold t3-t1代表GABAAR介导的总电流,ΔIhold t3-t2代表GABAAR介导的时相性抑制电流的成分。通过计算(|ΔIhold t3-t1-ΔIhold t3-t2|)可以得到α5亚基-GABAAR介导的紧张性性抑制电流Itonic。每组大鼠的ΔIholdt3--t1和t3-t2均以均数±标准误来表示。比较单个神经元加药前后的差异用KS法(Kolmogorov-Smirnov test)；检测组间差异先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's tests)。
手术置入侧脑室给药管(外径0.7mm、内径0.36mm的套管),位置：前囟后0.5mm旁开1.2mm深4mm。用牙托粉固定套管,术后7天匹罗卡品致痫,致痫大鼠进入潜伏期开始侧脑室给予L-655,708,每天两次(9am-9pm),每次侧脑室注射量为5μl,持续给药至SRS出现后14天。实验分为给药组和空白对照组。根据L-655,708浓度的不同,给药组又分为μM、4μM、8μM和16μM组,观察该药对大鼠行为学的影响。记录给药各组首次SRS发作的时间、慢性期SRS发作频率和发作持续时间,与空白组进行比较。
结果：癫痫慢性期组的ClC-2电流较对照组增大了53.2%(对照组：453.1±47.4pA,n=9；癫痫组：694.5±56.2pA,n=12；P<0.05)。外液中加入100nML-655,708后ClC-2电流减小了26.9%(空白组：702.3±63.1pA,n=10；给药组：513.6±50.2pA,n=15；P<0.05)。癫痫组α5亚基-GABAAR介导的紧张性抑制电流(Itonic)明显大于对照组(对照组：132.1±17.9pA,n=12；癫痫组：207.4±13.6pA,n=15；P<0.05)。 L-655,,708可显著抑制癫痫组ITonic(-30.3±7.9%,n=11,P<0.05),但对于对照组Itonic则无明显影响(-21.6±9.2%,n=7,P>0.05)。侧脑室给予致痫大鼠不同浓度的L-655,7088并观察大鼠行为学表现,8gM和16μM组大鼠首次SRS出现的时间早于空白组(给药组9±1天,n=7,空白组：12±1天,n=7；P<0.05),但慢性期SRS发作频率和发作持续时间未见显著组间差异。
小结：
1、癫痫组C1C-2电流较对照组明显增大,说明在病理状态下海马CA1区C1C-2表达上调是有功能的。
2、癫痫组海马CA1区a5亚基-GABAAR介导的紧张性抑制电流(Itonic)显著大于对照组；L-655,708可显著抑制癫痫组Itonic'但对于对照组Itonic则无明显抑制作用。说明对照大鼠脑组织中是以时相性抑制为主,紧张性抑制做为一种背景电流；而在癫痫大鼠脑组织中α5亚基-GABAAR介导的紧张性抑制电流明显增强,可能是对病理状态下时相性抑制减弱的一种代偿现象。
3、离体脑片上应用L-655,708可翻转癫痫组增大的C1C-2电流。说明病理状态下,C1C-2功能性上调与α5亚基-GABAAR介导的Itonic代偿性增强有关。
4、在体动物侧脑室注射L-655,708可使致痫大鼠首次SRS提早出现,但对慢性期SRS发作频率和发作持续时间无明显影响。说明Itonic增强可以延长SE发作后的潜伏期,延缓SRS的出现,即α5亚基-GABAAR介导的紧张性抑制与SRS的形成有关,与慢性期SRS反复发作无关。
第四部分α5亚基-GABAAR介导的紧张性抑制对慢性颞叶癫痫大鼠海马突触可塑性的影响
目的：研究癫痫组(慢性期)大鼠海马CA1区突触可塑性有何改变；应用α5亚基-GABAAR特异性阻断剂对慢性颞叶癫痫大鼠海马LTP/LTD的诱导有何影响。
方法：本部分实验应用在体记录大鼠海马CA1区场电位的技术。以20%乌拉坦(7.5m1/kg i.p.)麻醉动物,将大鼠头部固定于脑立体定位仪上,以H202清除皮下组织和筋膜,充分暴露颅骨中缝和前囟。在颅骨上标记刺激电极(前囟后4.2mm,中线左侧旁开3.8mm,深3200-3500μm)、记录电极(前囟后3.4mm,中线左侧旁开2.5mm,深2200-2500μm)和侧脑室给药管(前囟后0.5mm,中线右侧旁开1.0mm,深4000μm)的位置,用电钻在标记点上钻孔,借助微电极操作仪将电极和给药管缓慢送入目标位置。用牙托粉将不锈钢给药管固定,自然晾干。连续单方波刺激海马Schaffer collateral pathway的同时,在CA1区辐射层记录Schaffer→CA1的兴奋性突触后场电位(field excitatory post-synaptic potentials, fEPSPs)。高频刺激(100Hz,每串50个刺激,每串间隔15s,4串)诱导海马LTP；低频刺激(1Hz,900个刺激,15min)诱导海马LTD。给予条件刺激之前,应用测试刺激至少稳定记录30min做为baseline;侧脑室给药后需继续记录至少30min再诱导LTP/LTD,以观察药物本身对电位是否有影响。给药时先用微量注射器缓慢推注5μ1(L-655,708或vehicle),时间控制在2min左右,再用输液微泵维持至实验结束(推速：0.5μl/h)。该实验以海马CA1区fEPSPs的幅度作为提取参数,每10个连续的测试刺激做一次平均,以加药前或条件刺激前海马fEPSPs幅度的均值做为对照,所得数据由LTP、SPSS10.0软件进行统计分析,比较加药前、后的差异用Wilcoxon signed ranks检验,组间比较用Kruskal-Wallis test检验。Western Blot检测对照组和癫痫组(慢性期)大鼠海马CA1区NR1、NR2A、NR2B、 GluR1、GluR2和GluR3的表达量。在体实验结束后,分别取对照组和癫痫组大鼠脑组织,显微镜下分离,放入样品缓冲液中,加入蛋白酶抑制剂和磷酸酶抑制剂,冰上匀浆、超声破碎,取上清液分装保存于-80℃。酶联免疫吸附法测定上清液中上述目的蛋白的含量。蛋白免疫印迹实验结果的半定量分析,应用生物凝胶图像分析系统扫描,Genegenius凝胶图像分析系统摄取图片并获得条带灰度值,GeneTool软件进行浓度的定量分析,以(3-actin作为内参。先用单因素方差分析(one-way ANOVA)处理数据,然后用Tukey法(Tukey's post hoc tests)比较对照组和癫痫组的组间差异。
结果：高频刺激(high-frequency stimulation, HFS)诱导癫痫组海马CA1区fEFSPs的幅度显著低于对照组(癫痫组：128.5±9.43%,n=5；对照组：150.9±7.57%,n=5,P<0.05)；低频刺激(low-frequency stimulation, LFS)诱导癫痫组海马CA1区fEFSPs幅度显著高于对照组(癫痫组：117.2±6.22%,n=5；对照组：68.3±7.31%,n=5,P<0.05)。L-655,708对高频刺激诱导海马LTP的fEFSPs幅度无明显影响(给药组：132.4±7.01%,n=5；空白组：130.3±7.36%,n=4,P>0.05)；L-655,708可部分抑制低频刺激诱导海马LTD的fEFSPs幅度(给药组：91.6±6.57%,n=5；空白组：113.7±6.91%,n=4,P<0.05)。应用V(?)estern Blot技术检测对照组和癫痫组大鼠海马CA1区NR1、NR2A、NR2B、 GluR1、GluR2和GluR3的表达量。癫痫组NR1和NR2A(?)的表达量明显高于对照组；而NR2B的表达量显著减低。癫痫组GluR2的表达量明显低于对照组,同时GluR1和GluR3显著增高,癫痫组GluR2/GluR1+GluR3比值显著低于对照组。
小结：
1、高频刺激诱导癫痫组海马CA1区LTP的fEFSPs幅度显著低于对照组；低频刺激诱导癫痫组海马CA1区fEFSPs幅度则明显高于对照组,并翻转形成LTP。说明癫痫组大鼠强化记忆的功能明显减退,同时纠错和删除记忆的功能也有所削弱。
2、L-655,708对高频刺激诱导海马LTP的fEFSPs幅度无明显影响；可部分抑制低频刺激诱导海马LTD的fEFSPs幅度。说明L-655,708可以改善癫痫大鼠的纠错和删除记忆功能,避免LTP过饱和,从而维持相邻突触LTP的诱导阈值。
结论
正常大鼠中,C1C-2主要定位于海马CA1区锥体细胞的胞体上,在锥体细胞顶树突的近端也有相对较低水平的表达。致痫后,尤其是进入慢性期的大鼠,海马CA1区C1C-2的表达量明显上调,且具有功能性,以锥体细胞顶树突区的C1C-2上调显著。
慢性颞叶癫痫大鼠海马CA1区α5亚基-GABAAR介导的紧张性抑制电流(Itonic)显著增大；在离体脑片上应用L-655,708可显著抑制Itonic并翻转增大的C1C-2电流。致痫后,持续侧脑室给予L-655,708可使癫痫大鼠首次SRS提早出现,但对慢性期SRS发作频率和发作持续时间无明显影响；在体应用L-655,708可改善癫痫大鼠海马CA1区LTD的诱导。
Temporal lobe epilepsy, characterized by spontaneous recurrent seizures, learning and memory impairments is associated with neurodegeneration, abnormal reorganization of the circuitry, and loss of functional inhibition in hippocampus. In adult hippocampus, the GABAergic cells mediate the major inhibitory function of the principal neurons, promoting the Cl-entry through the GABAA receptor, whether through phasic (synaptic) or tonic (extrasynaptic) conductance. Aside from classical synaptic component, tonic GABAergic inhibition mediated by extrasynaptic GABAA receptor received increasing attention over the past years. There is growing evidence that tonic inhibition plays an important role in epilepsy, memory and cognition.
Since GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl" concentration at low levels in mature neurons, a shift in Ecl is likely to participate in the generation and not merely a consequence of TLE. As we known, chloride homeostasis is regulated by cation-chloride cotransporters and chloride channels. The transmembrane distribution of chloride determines the direction of the chloride flux gated by GABAA receptor-mediated response in neurons. ClC-2is a member of the supergene family of voltage-gated chloride channels. It is proved to be inwardly rectifying, and plays an important role in setting the intracellular chloride concentration in neurons expressing inhibitory GABAA receptors (GABAARs).
Several observations have shown that repetitive activation of GABAARs leads to a high intracellular chloride load that is generated by a GABAAR-mediated Cl-influx driven by HCO3-efflux via GABAARs, which dose not fade because of rapid replenishment of HCO3-by intracellular carbonic anhydrase (CA) activity. Furthermore, as a main chloride extruder in mature neurons, KCC2has been reported to be downregulated in experimental TLE models. Therefore, the chloride extrusion capacity seems to play a key role in setting the susceptibility of neurons to epileptiform activity. Since the conductance of ClC-2is large and does not display time-dependent inactivation, it is well suited for stabilize ECl. However, whether ClC-2contributes to epilepsy or not is controversial. The studies in human established the link between mutations in ClC-2and epilepsy, while ClC-2KO mice did not lead to higher seizure susceptibility. In addition, Rinke and coworkers used KO mice to demonstrate that ClC-2helps to extrude chloride from neurons under condition of high chloride load and contributes substantially to the background conductance. However, the amplitude and frequency of mIPSC, sIPSC, and PPR (a measure for presynaptic release) show no difference between WT and KO mice. Therefore, the possibility that ClC-2is involved in phasic inhibition by changing the number of postsynaptic GABAARs, inhibitory synapse number, and probability of release could be excluded.
It posed the question whether ClC-2is related to tonic inhibition mediated by extrasynaptic GABAARs. As is known, a5subunit-containing GABAARs have a restricted distribution in dendritic areas of hippocampal CA1and CA3regions, and play a predominant role in tonic inhibition of CA1pyramidal cells. Here we investigated whether the ClC-2expression is altered in CA1pyramidal cells in pilocarpine-treated rats, and whether a5subunit-containing GABAARs could affect ClC-2currents by pharmacological intervention.
Part Ⅰ Behavioural features and the pattern of neuronal loss in the hippocampus of pilocarpine-treated rats
Objective:To observe the behavioural features of pilocarpine-induced rats and neuronal loss in the CA1pyramidal cell layer in pilocarpine-treated rats.
Methods:Male Sprague-Dawley rats weighing180-200g were used. Status epilepticus (SE) was induced by pilocarpine hydrochloride (340mg/kg, i.p.). To lessen peripheral cholinergic effects, atropine methylbromide (5mg/kg, i.p.) was administered30min before pilocarpine. If seizure activity was not initiated within1hour after the initial pilocarpine hydrochloride dose, an additional dose of170mg/kg was given. The onset of SE was defined as the appearance of stage5seizures, followed by continuous and convulsive behaviorally detectable seizure activity. Diazepam (10mg/kg, i.p.) was injected90min after its onset. Rats were hand fed after SE until they were able to drink and eat on their own (2～3d). After SE, rats were killed at different time-point, representative of the different phases of the natural history of the disease:24h immediately followed by the SE (acute phase),5d after SE (latency),14d after the first occurrence of spontaneous recurrent seizure (SRS, chronic epilepsy).30d after the first occurrence of SRS (late chronic epilepsy). Rats were continuously video monitored for the appearance of SRSs, only rats that displayed multiple spontaneous stage3-4seizures in the subsequent month were included into the14d and30d groups. Control rats (con) were treated identically except that saline was substituted for pilocarpine.
Immunofluorescence was adopted to confirm the pattern of neuronal loss in this model. Primary antibody was mouse anti-neuronal nuclei (NeuN, neuron marker,1:500, Millipore). All the sections were treated by a mixture of FITC-conjugated secondary antibodies (1:400, Jackson ImmunoResearch). The quantification of the immunofluorescence staining in CA1area was performed by counting the number of NeuN positive cells per section. In each rat, every fourth section was picked from a series of consecutive hippocampus sections (20μm), and five sections were counted for each rat. An average number of neurons were obtained for each rat across five sections, and then the Mean±SEM across rats was determined. Statistical analysis was performed with SPSS10.0(SPSS Inc, USA). All data was presented as Mean±SEM. Differences in changes of values over times or drugs were tested using one-way ANOVA followed by individual post hoc comparisons (Tukey's post hoc tests).
Results:Convulsive status epilepticus (SE) were observed24±3min (Mean±SEM, n=51) after pilocarpine injection, which was intervened after90min by a low dose of diazepam. It did not stop the SE, but decreased its severity and mortality (7SE rats were dead). SE spontaneously alleviated5～7h after diazepam administration, and then rats entered postictal status, lasting2～3days, with self-limiting and generalized seizures (duration:10±3min; mean frequency:5±2seizures per day, n=38), before undergoing a latent period in which they were apparently normal. The first spontaneous recurrent seizure (SRS) occurred at12±1days after SE (n=32). Rats that did not display any SRS in one month after SE were deleted (n=4), while the others kept experiencing SRSs (duration:4±2min; mean frequency:4±1seizures per day, n=28). According to Racine's classification criteria, partial seizures (stage3-4) were more frequent compared to secondarily generalized seizures (stage5-6), and tended to recur in a cluster way.
Pilocarpine-treated rats displayed marked neuronal loss in the hilus of DG and CA3pyramidal cell layer. However, CA1pyramidal cell layer was well preserved. Compared with control group, the number of NeuN+cells in the CA1pyramidal cell layers in pilocarpine-treated rats did not decreased significantly at different phases.
Summary:
1. The first spontaneous recurrent seizure (SRS) occurred at12±1days after SE.
2. Rats kept experiencing SRSs with a mean frequency of4±1seizures per day and a mean duration of4±2min in the subsequent month.
3. No significant neuronal loss in the CA1pyramidal cell layer in pilocarpine-treated rats.
Part Ⅱ The location of CIC-2IR and the quantification of ClC-2protein level in the CA1pyramidal cells layer in pilocarpine-treated rat
Objective:To investigate the location of ClC-2IR in hippocampus and the change of protein level of ClC-2in CA1region in pilocarpine-induced rats.
Methods:Double immunofluorescence staining was performed to localize ClC-2in hippocampus. Rats from epileptic and control groups were anesthetized with urethane (1.5g/Kg, i.p.) and transcardially perfused with heparinized saline, followed by4%paraformaldehyde (PFA) in0.1M phosphate buffer, PH7.4. The intact brains were removed and post-fixed for3h in the same fixative, then transferred into30%sucrose solution. Transverse brain sections (20μm) were cut using a cryostat. All sections were blocked with3%donkey serum in0.3%Triton X-100for1h at room temperature and incubated with primary antibodies over one night at4℃, then the sections were incubated for2h at room temperature with secondary antibodies. The stained sections were examined with a fluorescence microscope and images were captured with a CCD spot camera.
Western blot was performed to confirm the expression of ClC-2after SE in protein level. Rats were decapitated, brains were immediately removed and frozen on dry ice, then stored at-80℃until processing. The CA1regions were microdissected on dry ice. The tissue samples were homogenated and sonicated in15mmol/L Tris buffer, then centrifuged at13,000g for15min at4℃to isolate the supernatant containing protein samples. The samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was blocked for1h at room temperature in blocking buffer and then incubated with anti-ClC-2antibody (1:200, Alomone Labs) overnight at4℃, followed by incubation in anti-rabbit secondary antibody (1:1000, Cell Signaling Technology). The immune bands were visualized by enhanced chemiluminescence (ECL, Amersham, USA) and film exposure (Kodak, Rochester, NY). The membrane was stripped with stripping buffer for30min at50℃and re-probed with anti-α-actin (1:1000, Santa Cruz), followed by incubation in anti-mouse secondary antibody at a dilution of1:4000(Cell Signaling Technology) as an endogenous control protein to ensure equal loading. Densitometric analysis of bands on the film was conducted with a computer-assisted imaging analysis system, and normalized to β-actin immunoreactivity.
Results:In control rats, ClC-2immunoreactivity (ClC-2IR) was present with high levels of labeling in the cell membrane and perinuclear cytoplasm of pyramidal cells (PCs), and relatively low levels of labeling in the proximal apical dendrites of PCs in CA1regions. Double immunofluorescence staining showed that ClC-2only co-localized with NeuN, neither with GFAP nor with Ibal. In pilocarpine-treated rats, an increased ClC-2IR was present, especially in CA1PCs dendrites (strata radiatum). Meanwhile, ClC-2IR maintained high levels in the pyramidal cell layer.
Immunoblot was performed to quantify this change, ClC-2protein level was significantly higher than that in control group.
Summary:
1. ClC-2IR was present with high levels of labeling in the cell membrane and perinuclear cytoplasm of pyramidal cells (PCs), and relatively low levels of labeling in the proximal apical dendrites of PCs in CA1region.
2. ClC-2upregulated in CA1pyramidal cells (PCs) in the chronic post-SE rats.
3. The obvious enhancement of ClC-2IR during the chronic period is in the apical dendrites of PCs in CA1region.
Part Ⅲ CIC-2alteration is involved in tonic inhibition mediated by a5subunit-containing GABAARs in CA1region
Objective:To investigate whether the upregulation of ClC-2in CA1PCs is functional and whether ClC-2is involved in tonic inhibition mediated by a5subunit-containing GABAARs. Reducing the tonic inhibition would aggravate or improve the generation and propagation of SRS in post-SE rats.
Methods:Whole-cell voltage-clamp recordings in hippocampal slices prepared from epileptic and control rats. Following decapitation, brains were quickly submerged in ice-cold sucrose-ACSF. Transverse slices (400μm) were cut with a Vibratome. Before recording, slices were incubated for30min at32℃in ACSF. Then slices were transferred to the recording chamber and continuously perfused (5ml/min) with recirculating extracellular solution (oxygenated with95%O2-5%CO2). Visualized patch-clamp recordings from the CA1pyramidal neurons were performed by IR-DIC videomicroscopy with an Axopatch200B amplifier, filtered at2kHz and digitized at20kHz. C1C-2currents were evoked at test potentials between+40and-20mV (increment:-20mV; holding potential:-10mV).Itonic were recorded as the change in mean holding currents (ΔIhold) after applying SR-95531(100μM) while voltage clamped at-60mV. The Ihold was sampled and averaged in recordings as follows:100s before (t1) and200s after L-655,708application (t2), and100s after SR-95531application (t3). Mean ΔIhold before and after drugs application were calculated for each neuron. Before and after values for an individual neuron were compared using the Kolmogorov-Smirnov (KS) test. Differences between groups were analyzed with one-way ANOVA followed by individual post hoc comparisons (Tukey's post hoc tests).
A stainless-steel cannula (22gauge,0.7mm outer diameter) was implanted above the right lateral ventricle (0.5mm posterior to the bregma,1.2mm lateral to midline and4mm below the surface of the dura). The cannula was secured by dental cement. A stainless-steel obturator was inserted into the cannula to prevent clogging and infection. Rats were allowed to recover for7days before inducing SE. Intracerebroventricular (i.c.v.) injection was made from the latency till14d after the first occurrence of SRS via an internal cannula (28gauge,0.36mm outer diameter).
Results:C1C-2currents increased by53.2%(control,453.1±47.4pA, n=9; post-SE,694.5±56.2pA, n=12; P<0.05) in polocarpine-treated rats, consistent with the upregulation of C1C-2. And a decrease in L-655,708treatment slices by-26.9%was observed (vehicle,702.3±63.1pA, n=10; L-655,708,513.6±50.2pA, n=15; P<0.05). A significant increase in GABAAR-mediated tonic inhibition (Itonic) in CA1PCs, compared to neurons from control (control:132.1±17.9pA, n=12; post-SE:207.4±13.6pA, n=15; P<0.05). L-655,708(100nM) decreased Itonic in control neurons by-21.6±9.2%(n=7), but produced a significantly greater decrease in post-SE neurons (-30.3±7.9%, n=11).
L-655,708treatment starting from the latency till14d after the first occurrence of SRS in vivo resulted in an earlier manifest of the first occurrence of SRS at9±1days after SE in the8μM and16μM L-655,708groups (n=7each group, P<0.05), but did not show significant effect on the duration and mean frequency of SRSs.
Summary:
1. C1C-2currents increased in CA1PCs in pilocarpine-treated rats.
2. A significant increase in GABAAR-mediated tonic inhibition (Itonic) in CA1PCs, compared to neurons from control. It is probably due to the compensatory increased tonic inhibition for replenishing the decreased synaptic GABAergic innervation at dendrites in TLE model.
3. L-655,708attenuated the increased Itonic in vitro and produces an earlier manifest of the first occurrence of SRS in vivo in pilocarpine-treated rats. We suggest that the chronically raised tonic inhibition mediated by a5subunit-containing GABAARs is associated with the generation of SRS.
4. L-655,708reversed the increase in C1C-2currents in vitro. We suggest that C1C-2modification in CA1PCs is probably to assist the chloride extrusion and maintain the inwardly directed driving force for chloride ions, which is a prerequisite for hyperpolarizing inhibition of extrasynaptic GABAARs.
Part Ⅳ L-655,708attenuates the increased LTD, but produces no significant change in decreased LTP in post-SE rats in vivo.
Objective:To observe what the change of synaptic plasticity of post-SE rats in CA1area, and whether a5subunit-containing GABAARs could affect amplitude of field potentials by pharmacological intervention.
Methods:Experiments were performed on male Sprague-Dawley rats. Urethane (1.5g/kg, ip) was used to induce and maintain anesthesia. Additional doses (0.5g/kg) were given if needed. Surgical level of anesthesia was verified by the stable mean arterial blood pressure during noxious stimulation. The trachea was cannulated, and the animal breathed spontaneously. The left femoral vein and artery were cannulated for intravenous injection and continuous monitoring of blood pressure respectively. Colorectal temperature was kept constant (37-38℃) by means of a feedback-controlled heating blanket. Rats were placed in a stereotaxic frame for all recordings. A small craniotomy (1mm×1.5mm) was performed, and then the dura and arachnoid were carefully removed. A stainless steel guide cannula was implanted in the right lateral ventricle (1mm lateral to midline,0.5mm posterior to bregma and4mm below the surface of dura), an internal cannula was used for intracerebroventricular injections (i.c.v.). The craniotomy hole was sealed with dental cement after implantation procedure. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the stratum radiatum of the dorsal hippocampus in response to stimulation of the ipsilateral Scaffer collateral-commissural pathway. The recording site was located3.4mm posterior to bregma and2.5mm lateral to midline, and the stimulating site was located4.2mm posterior to bregma and3.8mm lateral to midline. The final depth of both electrodes was adjusted to optimize the electrically evoked fEPSPs. The intensity of test stimuli (0.033Hz,0.1ms duration) was adjusted to evoke a fEPSP that was50%of maximum. Conditioning high frequency stimulation (HFS:100Hz,50stimuli per train,4trains at interval of15s) was induced LTP, and LTD was induced by low frequency stimulation (LFS:1Hz,900stimuli15min). Baseline was recorded for>30min prior to injection of drug/vehicle to ensure a steady response. All values were expressed as mean±SEM. The magnitude of LTP/LTD was measured as the percentage of the baseline fEPSP amplitude during30min periods, just prior to HFS/LFS. Before and after values for an individual rat were compared using the Wilcoxon signed ranks test. Differences between groups were analyzed with Kruskal-Wallis test.
Western blot was performed to compare the protein levels of NR1, NR2A, NR2B, GluRl, GluR2and GluR3in CA1area in post-SE rats with that in control ones. Rats were decapitated, brains were immediately removed and frozen on dry ice, then stored at-80℃until processing. The CA1regions were microdissected on dry ice. The tissue samples were homogenated and sonicated in Tris buffer, then centrifuged to isolate the supernatant containing protein samples. The samples were separated by gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane. The membrane was blocked for1h at room temperature in blocking buffer and then incubated with primary antibody overnight at4℃, followed by incubation in secondary antibody. The immune bands were visualized by enhanced chemiluminescence (ECL, Amersham, USA) and film exposure (Kodak, Rochester, NY). The membrane was stripped with stripping buffer for30min at50℃and re-probed with anti-β-actin, followed by incubation in secondary antibody as an endogenous control protein to ensure equal loading. Densitometric analysis of bands on the film was conducted with a computer-assisted imaging analysis system, and normalized to P-actin immunoreactivity.
Results:The amplitude of fEPSPs induced by HFS decreased by14.84%(control:150.9±7.57%, n=5; post-SE:128.5±9.43%, n=5; P<0.05) in post-SE rats, and an increase in the amplitude of fEPSPs induced by LFS was detected (control:68.3±7.31%, n=5; post-SE:117.2±6.22%, n=5; P<0.05) compared to rats from control. L-655,708attenuates the increased LTD (vehicle:113.7±6.91%, n=4; L-655,708:91.6±6.57%, n=5; P<0.05), but produces no significant change in decreased LTP in post-SE rats in vivo (vehicle:130.3±7.36%, n=4; L-655,708:132.4±7.01%,n=5;P>0.05). Immunoblot was performed to quantify the protein levels of NR1, NR2A, NR2B, GluR1, GluR2and GluR3in CA1area. NR2B and GluR2downregulated in CA1pyramidal cells (PCs) in the chronic post-SE rats, and other ones were higher than that in control group.
Summary:
1. The amplitude of fEPSPs induced by HFS decreased significantly in post-SE rats, and an increase in the amplitude of fEPSPs induced by LFS was detected compared to rats from control.
2. L-655,708attenuates the increased LTD, but produces no significant change in decreased LTP in post-SE rats in vivo.
Conclusion
The major findings of this study are that ClC-2upregulated functionally in CA1pyramidal cells (PCs) in the chronic post-SE rats, with a corresponding increase in tonic GABAergic inhibition, and that dampening this tonic inhibition by L-655,708reverses the increase in ClC-2currents in CA1PCs in vitro and in amplitude of fEPSPs induced by LFS in vivo.
引文
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