Name: electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins
Uploaded: 2017-08-15T14:00:00.000+00:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

作者感谢 Dr. 埃里克 c. 博尔顿允许我们使用他的离心机为分馏和 Dr. 格雷厄姆 h. Diering 在 Dr. 理查德 l. 胡加尼尔的实验室在约翰的霍普金斯大学为我们提供了 small-scale 协议的 PSD 分馏。

所有试验程序, 包括动物实验, 都已由伊利诺伊大学香槟分校的机构动物保育和使用委员会批准。
1. 维持鼠窝
<ol><li>养殖大大鼠 (参见材料表), 并在标准条件下保持它们的12小时暗循环和 ad 随意获得食物和水。</li>
	<li>在产后日 (P) 28 将大鼠幼崽断奶, 以 2-4 的雄性或雌性窝。</li>
	<li>用无毒的永久性黑色标记标记雄性大鼠的尾巴。</li>
	<li>对雄性大鼠每周3次称重, 并记录其 bodyweights。</li>
</ol>2. 一台 ECS 机的研制
<ol><li>在上午7:30, 在动物准备室消毒长凳, 并在长凳上放置一个 ECS 机 (脉冲发生器)。</li>
	<li>将一只雄性老鼠在一个有盖子的干净的空笼子里重 200-250 克。重复这一点, 所有的雄性大鼠都要接受 ECS 诱导。让老鼠习惯30分钟。</li>
	<li>当老鼠被 habituating 在他们的笼子里时, 设置一个脉冲发生器为 ECS 感应到频率100脉冲/秒, 脉冲宽度为0.5 毫秒, 休克持续时间为0.5 秒, 电流55毫安 (图 1a)。</li>
	<li>通过按下 "复位" 按钮来准备脉冲发生器, 并确保 "就绪" 按钮已亮起。确保耳夹没有连接到脉冲发生器, 然后按下 "休克" 按钮几秒钟。 注意: 在这一点上, 脉冲发生器已准备好 ECS 感应。</li>
	<li>将耳夹插入脉冲发生器。</li>
</ol>3. 诱导急性 ECS
注意: 请参阅 图 1B, 顶部面板。
<ol><li>用无菌盐水浸湿耳夹, 确保其饱和。</li>
	<li>用盐水浸泡过的纱布把老鼠的耳朵弄湿。一旦他们是湿的, 去除纱布。</li>
	<li>每只耳朵附上一个夹子, 位置在主要软骨带之外。</li>
	<li>确认在 ECS 机器上建立一个真正的回路;否则, 计算机上将出现错误消息或读取 "1"。</li>
	<li>穿厚的非金属手套。当小鼠用戴着手套的手轻轻握住时, 按下 "休克" 按钮几秒钟, 慢慢松开对老鼠的抓地力, 观察癫痫发作。对于假的 "无癫痫" (NS) 控制, 处理大鼠相同, 但不提供电流。</li>
	<li>在挛开始时断开 ear 剪辑, 并根据修订后的拉辛的比例22记录检出行为, 其中包括 "口腔和面部运动" (阶段 1)、"头点头" (阶段 2)、"前肢挛" (阶段 3)、"与前肢挛一起饲养"(阶段 4) 和 "养育和下落与前肢挛" (阶段 5)。扣押应持续大约十年代;使用定时器记录扣押时间。</li>
	<li>在癫痫终止后, 将鼠返回其家笼。监测鼠的另5分钟, 以确保恢复的老鼠从癫痫。把它单独存放在笼子里, 然后把笼子还给恢复室。</li>
	<li>在下一只老鼠身上重复 ECS 感应</li>
	<li>在当天的剩余时间和至少一天的早晨和第二天的下午, 对老鼠进行监测, 直到它们被安乐死, 进行实验。 注意: ECS 诱导方法可能导致临床症状作为附带副作用, 需要注意。例如, ECS 诱导协议可能会诱发持续时间长于十五年代的癫痫发作, 给大鼠造成不必要的痛苦。在这种情况下, 终止扣押使用安定 (10 毫克/千克, ip) 或戊巴比妥 (25-30 毫克/千克, ip)。如果老鼠在癫痫停止后出现呼吸窘迫或严重的行为异常, 安乐他们被二氧化碳吸入后被斩首。</li>
</ol>4. 慢性 ECS 的诱导
注意: 请参见底部面板图 1B。
<ol><li>每天在同一时间诱导一个 ECS, 如步骤 1-3, 上面所述, 连续七天。</li>
	<li>在老鼠回到自己的笼子后, 每天两次监视它们。</li>
</ol>5. 大鼠海马的均匀化和分馏
注意: 请参见图 2。
<ol><li>准备一个新的均匀化缓冲液 (溶液 a), 含有320毫米蔗糖, 5 毫米焦磷酸钠, 1 毫米 EDTA, 10 毫米 HEPES pH 7.4, 200 nM 冈田酸, 和蛋白酶抑制剂。过滤器-使用带有0.22 µm 孔径的过滤器对缓冲器进行灭菌, 并将其放在冰上。</li>
	<li>在一个特定的时间点后, 急性 ecs 或慢性 ecs (图 1), 安乐鼠通过 CO2吸入 5-10 分钟, 其次是斩首使用断头台。</li>
	<li>卸下大脑并解剖在冰上放置的金属板上的海马, 如前所述23,24。</li>
	<li>将两个海马从一只老鼠放到一个30毫米的组织培养皿中, 用剪刀把它们切成小块。</li>
	<li>使用1毫升吸管将剁碎的海马到手动玻璃匀浆器, 并加入1毫升的 ice-cold 均质缓冲液 (溶液 a, 步骤 5.1)。将圆杵插入玻璃匀浆机。而玻璃匀浆机是在冰上, 轻轻地和稳步地在杵 10-15 次的1分钟, 直到小块的海马组织消失。</li>
	<li>使用1毫升吸管将匀浆转移到1.7 毫升离心管, 并将匀浆在 800 x g 处离心10分钟, 在4° c 下, 将 postnuclear 上清 (S1 分数) 从含有不溶性组织和细胞核的颗粒中分离出来 (P1 分数)。转移50µL 和950µL 的 S1 分数到两个独立的, 新的1.7 毫升离心管使用1毫升吸管和储存这些管冰。在冰上保存 P1 分数颗粒。</li>
	<li>离心 S1 分数 (950 µL) 10 分钟, 在 13800 x g 和4° c 分离上清 (S2 分数), 富含胞浆可溶性蛋白, 和颗粒 (P2 分数), 富含膜结合蛋白, 包括触蛋白。使用1毫升吸管将 S2 分数转移到新的1.7 毫升离心, 并将其储存在冰上。</li>
	<li>用1毫升的吸管在498µL 的 ice-cold 纯化水中重颗粒 (P2 分数)。添加2µL 1 米 HEPES (ph 7.4) 使用20µL 吸管达到最后浓度 4 mM HEPES (ph 7.4)。在4° c 孵育, 搅拌30分钟, 在冰上储存悬浮 P2 分数。</li>
	<li>使用免疫分析法测定 S1、S2 和 P2 组分的蛋白质浓度。增加50毫米 HEPES (pH 7.4) 到每个分数, 以达到1毫克/毫升浓度和存储在-80 ° c, 直到使用, 或处理的 P2 分数, 以隔离 PSD。</li>
</ol>6. 从粗膜蛋白 (P2) 馏分中分离 PSD
<ol><li>离心 P2 分数 (500 µL) 为20分钟在 2.5万 x g 和4° c 分离裂解上清 (LS2 分数) 和裂解颗粒 (LP1 分数)。使用1毫升吸管将 LS2 分数转移到新的1.7 毫升离心管上, 并将其储存在冰上。</li>
	<li>重 LP1 颗粒在250µL 50 毫米 HEPES (pH 7.4) 混合与250µL 1% 洗涤剂在 1x PBS 缓冲区使用1毫升吸管。孵育在4° c 与温和的鼓动15分钟。</li>
	<li>离心悬浮 LP1 颗粒3小时在 2.5万 x g 和4° c 分离上清 (非 psd 分数) 从颗粒 (psd 分数)。使用200µL 吸管将上清液移至1.7 毫升离心管, 并重 PSD 颗粒在100µL 50 mM HEPES (pH 7.4) 中。</li>
	<li>使用免疫分析法测定 LS2、非 psd 和 psd 组分的蛋白质浓度。增加50毫米 HEPES (pH 7.4), 以达到1毫克/毫升浓度和存储在-80 ° c, 直到使用。 注意: 在步骤 5-6 (即解决方案 A、HEPES 缓冲区和 PBS 缓冲区) 中使用的所有解决方案都应使用无离子和有机污染物和微粒的纯净水进行处理。</li>
</ol>7. 西部印迹
<ol><li>解冻每个蛋白质组分在冰。将每个分数 (S2、P2 和 PSD 在1毫克/毫升) 中的12µL 转移到新的1.7 毫升离心管中, 使用 20-µL 吸管。</li>
	<li>在水浴中加入3µL 5x SDS 样品缓冲液, 在75° c 下孵育30分钟。将样品冷却至室温 (RT)。</li>
	<li>在4-20% 梯度 15-井梳 sds-聚丙烯酰胺凝胶电泳 (sds 页) 中, 用20µL 吸管将蛋白质样本的10µL。在 sds 页装置中运行凝胶, 在运行缓冲液中80-100 伏 (25 毫米三, 190 毫米甘氨酸, 0.1% sds; pH 8.3)。 注: 每种凝胶应在急性或慢性 ecs 后不同时间点包含 NS 大鼠和 ecs 鼠的蛋白质样本。</li>
	<li>转移的蛋白质从 SDS 页凝胶到聚乙烯氟 (PVDF) 膜在转移装置在 25-30 V (60 mA) 为 9-12 h 在调动缓冲 (25 毫米三, 190 毫米甘氨酸和20% 甲醇; pH 8.3)。</li>
	<li>将 PVDF 膜从转移装置中取出, 并在 RT 的多用途旋转器上用三缓冲盐水 (TBS) 清洗5分钟。</li>
	<li>在5% 牛奶和 0.1% Tween-20 在 tbs 的1小时内, 将膜在洗涤缓冲液 (1% 牛奶和 0.1% Tween-20) 在一个多用途的旋转器上过夜, 在4° c (参见材料表稀释) 中孵育。</li>
	<li>清洗缓冲膜4次, 每10分钟清洗一次, 然后用辣根过氧化物酶 (HRP)-在 RT 的多用途转子的洗涤缓冲液中孵育 1 h 的共轭二次抗体。</li>
	<li>洗膜4次, 每10分钟洗涤缓冲区, 然后与 TBS 5 分钟。</li>
	<li>用增强的 chemifluorescence 基质孵育膜1分钟, 并将其暴露在 X 射线胶片上。用胶片处理器开发曝光的胶片。</li>
</ol>8. 西方印迹的量化
<ol><li>将西部印迹扫描为 TIFF 文件, 并将此文件保存到计算机。</li>
	<li>在 ImageJ 程序中打开西部印迹文件作为灰度图像, 在 "文件"、"打开图像"、"右箭头"、"灰度" 下。</li>
	<li>从 "ImageJ" 工具栏中选择 "矩形选择" 工具, 并绘制一个矩形, 它覆盖了一个有兴趣的蛋白质的单个西方印迹带。</li>
	<li>在 "分析," 命中 "测量" 获得的面积和平均密度的一个选定的波段。</li>
	<li>将矩形移动到背景区域, 而不更改其大小和形状。重复步骤8.4 到区域和背景的平均密度。</li>
	<li>减去一个背景带的平均密度值, 从一个西方的印迹带, 给予背景减去带密度的蛋白质的兴趣。</li>
	<li>重复步骤 8.2-8.6 的所有西方印迹波段的兴趣。</li>
	<li>按蛋白的一个管家基因产品的背景减去带密度除以感兴趣蛋白质的背景减去带密度;这一步产生了感兴趣的蛋白质的正常化值。</li>
</ol>

作者声明他们没有竞争的金融利益。

在这里, 我们描述一个 ECS 诱导方法的大鼠, 诱发全球刺激神经元活动在他们的海马。ECS 是一种动物模型的电治疗, 这是临床用于治疗药物难治性抑郁症的人1,2,3。尽管使用电疗法治疗严重抑郁症, 但确切的基础机制仍不清楚。由于 ecs 诱发啮齿动物的抑郁样行为并刺激海马神经发生4,32, ecs 已被广泛用于研究是否新的成年出生的神经元在海马齿状回有助于抑郁行为5,32。
电流通过 stereotaxically 植入电极、角膜电极或耳夹电极33,34, 在啮齿类动物中诱发轻度到严重的广义强直挛发作。患者脑电图记录显示, 双侧电刺激引起比单边刺激更突出和更长的广泛性癫痫发作35,36。在我们的 ECS 诱导方法, 强直性挛惊厥是诱导的电流传递通过无创耳夹电极连接到两个耳朵, 模拟癫痫发作在人类诱发的双边刺激36。虽然我们还没有检查在使用镇静或麻醉的大鼠中的 ECS 的功效, 但对大鼠给予麻醉药物可以减少电刺激引起的癫痫的程度是可行的, 这可能是因为在大脑网络中, 麻醉状态与增强抑制和挫伤的兴奋性音调有关。此外, 在 ECS 诱导方法的关键步骤是实验拨电流强度, 以引出阶段 4-5 的广义补药挛癫痫的年龄, 体重, 和物种的啮齿动物。 我们的 ECS 诱导方法已优化诱导阶段 4-5 发作在几秒钟内唤醒雄性大大鼠体重 200-250 克。如果在麻醉状态下的大鼠被用于 ECS 诱导, 则应修改传递电流的强度、持续时间和频率, 以便成功地从4阶段到5级的癫痫发作。
ECS 还提供了刺激神经元多动的优势, 并导致急性短暂性发作, 死亡率非常低33, 与 chemoconvulsants, 包括匹罗卡品和 kainite, 导致癫痫持续状态和造成自发性复发性发作和严重组织学改变的慢性表现37,38。虽然 ecs 已被常规用于筛选抗药物33,34, 急性 ecs 和慢性 ecs 不会导致慢性癫痫的产生, 因此不能用于动物模型研究癫痫。相反, ECS 已被广泛使用, 以研究广泛的大脑活动的高度改变表达和/或后修饰的突触蛋白在体内, 这有助于持续的变化突触强度和结构 (图 3和图 4)10,40。在这项研究中, 我们只用雄性大鼠来排除雌性大鼠发情周期阶段对 ECS 后 PSD 蛋白量的意想不到的影响。然而, 据观察, 没有性别差异的支架蛋白, 包括 PSD-95 和 SAP102 在 PSD 区域的额叶皮质和海马39, 暗示荷尔蒙变化控制发情周期可能不会影响基础PSD 蛋白的数量。
采用多种方法检查 ECS 诱发神经发生、突和突触可塑性变化的程度5、6、7、8、9、11,40. 兴奋性突触后电流 (") 的电生理记录被广泛用于检测谷氨酸兴奋神经键的突触强度的变化21。例如, 微型 "记录显示了在急性 ECS41后的小鼠皮层锥体神经元的兴奋性突触强度的稳态缩小。然而, 识别的突触蛋白, 有助于 ECS 诱导的突触可塑性是有挑战性的, 因为电生理记录必须配对的基因敲除或击倒特定的候选突触蛋白。在 PSD 中丰富的突触后膜上的谷氨酸受体水平的变化, 以及对兴奋性突触传递的强度和功效进行了调节17,18,25。虽然免疫组化可用于检测 ECS 诱导的突触蛋白表达的变化, 但这种技术可以一次只检查 1-2 候选突触蛋白, 并且需要 well-validated 抗体来明确识别它们不引起非特异性染色。
脑组织与西方印迹结合的亚细胞分型, 在识别由 ECS 改变的突触蛋白方面具有优于电生理学和免疫组织化学的优势。脑组织亚细胞分型是一种快速而粗糙的生化方法, 可以分离出从粗膜 (P2 分数) 中的可溶性胞浆蛋白 (S2 分数), 包括 ER 和高尔基体膜、细胞膜结合器、等离子膜和突触终端膜, 重新形成突42,43,44。PSD 分数可以进一步从 P2 部分分离, 以丰富突触蛋白42,43,44。psd 分数的无偏蛋白质组分析可以确定所有 psd 蛋白的水平被 ECS 改变。西方印迹可以快速地进行, 以检测 PSD 蛋白的表达变化, 这可以很容易地识别从非特异的波段。
先前的脑部分离方法需要大量的啮齿动物脑组织42,43,44, 使得这种技术在检测个体的可溶性或膜蛋白时具有挑战性。啮齿动物大脑或单个大脑的特定脑区有越来越多的需求, 定量比较蛋白质组从一个脑区到另一个和从控制动物的转基因动物或动物, 已经经过了具体的治疗。因此, 我们对传统的脑分馏方法进行了修正和优化, 从两个单一的海马中分离出粗可溶性和膜组分。我们的 small-scale 粗分馏协议成功地分离了从胞浆 S2 可溶性组分的粗 P2 膜组分, 表示由于缺乏细胞质蛋白α-蛋白在 P2 分数, 而膜结合的 PSD95 检测P2 但不 S2 分数 (图 3A和图 4a)。使用这里描述的方法, 我们定量地表明, 急性 ECS 显著增加步骤61表达式, 并降低其基底、GluN2B 和胞外信号调节激酶1/2 在原油中的酪氨酸磷酸化。急性 ECS10后48小时的大鼠海马膜 P2 分数。
意想不到地, S2 分数包含了突;步骤61;而且, 在很小的程度上, GluA2 (图 3a和图 4a)。步骤61是与内质网 (ER) 和 PSD45相关的糖化积分膜蛋白26 。离心分离粗膜颗粒 (P2 分数) 可能不足以使所有突触泡中含有突, 以及包含 GluA2 和步骤61的体和溶酶体。然而, GluN2B、GluA2 和 PSD95 的明显富集以及 P2 馏分中缺乏α-蛋白, 表明我们的分馏协议可以丰富与突触泡无关的膜结合蛋白和跨膜蛋白,体在粗膜 P2 馏分中。
虽然粗膜 P2 分数含有突, 但在 P2 馏分中检测突触蛋白的活动依赖性改变的一个限制是, 不能确定其变化的确切位置。如果在 psd 中丰富的突触蛋白被确定, 那么进一步的生物化学分馏可以用来隔离 psd。以往的 PSD 分离方法需要大量的啮齿动物脑组织(即, 10-20 啮齿动物的大脑) 和蔗糖梯度42,43,44。由于这种传统方法不足以分离出一只老鼠的两个海马的 psd 分数, 我们已经采用了一种简单的方法, 直接分离 psd 分数, 而没有蔗糖梯度20,21.这种方法能产生大约 30-50 µg 的 PSD 蛋白, 足以用于几种生化化验, 包括印迹。我们的方法丰富了 PSD95, GluN2B, 和 GluA2, 这是已知的集中在 psd 分数, 并检测在慢性 ECS 诱导后 psd 分数 GluN2B 表达的变化 (图 3和图 4)。
在这里, 我们的定量西方印迹分析显示, 在急性 ECS (图 3C) 后的3ĥ和 24 h 的 P2 和 PSD 分数中, GluA2 表达没有显著变化。GluN2B 表达式在 P2 分数中是不变的, 但在急性 ECS (图 3B) 后, PSD 分数在3ĥ和24小时内呈上升趋势, 提示可能的突触与外的差异调节GluN2B-containing NMDA 受体。我们还观察到, GluN2B 表达在慢性 ecs 后的 P2 分数呈下降趋势, 并且在24和 96 h 以下的慢性 ecs (图 4B) 上的 PSD 分数显著降低。虽然高度投机, 这样的 GluN2B 从 psd 的重复诱导后, 可由多个机制, 包括直接内化从 psd 膜或横向扩散到外的地点。考虑到我们先前的报告, 神经元活动的延长明显增加了步骤61表达, 并减少了培养海马神经元 P2 分数的 GluN2B 的 Tyr 磷酸化作用46, 我们的研究结果表明慢性 ECS 后 P2 和 PSD 分数的 GluN2B 表达减少可能是由于增强的步骤61表达式和随后从外膜中去除 GluN2B。
总之, 我们已经证明, 我们的 small-scale PSD 分馏方法, 结合 ECS 感应协议, 使我们能够区分在体内谷氨酸受体的活动依赖性调节在突触后膜与全细胞膜, 包括外膜。该协议可以很容易地应用于任何突触蛋白的兴奋性突触, 并可以修改的其他啮齿动物海马或其他大脑地区的调整体积, 每个解决方案的基础上的组织重量。因此, 我们的 small-scale PSD 分馏方法是多才多艺的, 并可用于未来的应用, 以检查的体内改变后突触蛋白在每个动物的遗传, 药理, 或机械治疗。

电疗法已被用于治疗抑郁症患者, 包括严重的耐药抑郁症, 双相抑郁症, 帕金森氏病和精神分裂症1,2。在这种治疗中, 癫痫发作是由电刺激传递给麻醉病人的头部通过 epicranial 电极1,2,3。重复性管理的 ECS 已临床有益的耐药抑郁症的疾病1,2,3。然而, 确切的机制的基础上长期疗效的抗抑郁药物的人仍然难以捉摸。ECS 是一种电治疗的动物模型, 广泛用于研究其治疗机制。在啮齿类动物中, 急性 ecs 和慢性 ecs 治疗促进了海马的成人神经发生, 并重组了神经元网络4,5, 这很可能有助于提高认知灵活性。此外, 全球范围内的大脑活动的提升改变了大量的成绩单, 如大脑衍生神经因子6, 和多种蛋白质, 包括代谢谷氨酸受体 17和 n-甲基-d-天门冬氨酸(NMDA) 型谷氨酸受体亚基7。这些变化涉及调解长期修改突触数, 结构和强度在海马7,8,9。
在 ECS 模型中, 通过 stereotaxically 植入电极、角膜电极或耳电极将电刺激传递给啮齿动物, 以唤起全身强直-挛发作10,11。立体定向植入电极涉及脑部手术, 需要很大的时间来提高实验者的手术技能, 以减少伤害。较少侵入性角膜电极可引起角膜磨损和干燥, 需要麻醉。耳夹电极的使用绕过这些限制, 因为它们可以用于啮齿目动物没有手术或麻醉, 并造成最小的伤害。事实上, 我们发现, 目前交付给清醒大鼠通过耳夹电极可靠地诱导阶段4-5 挛发作和改变突触蛋白在其海马10。
为了研究在啮齿动物的特定脑区内的突触蛋白诱导丰度, 选择最适合于它们的检测和定量的实验方法是非常重要的。亚细胞分馏的大脑允许的可溶性胞浆蛋白的粗分离;膜蛋白;细胞界限蛋白;甚至蛋白质的特殊亚细胞结构, 如 PSD12,13,14。PSD 是一个稠密和组织良好的亚细胞域的神经元, 其中突触蛋白高度集中在和附近的突触膜12,13,15。psd 的分离是有用的研究突触蛋白丰富的 psd, 因为动态变化的丰度和功能的突触后的谷氨酸受体, 支架蛋白, 信号转导蛋白在 psd12,15,16,17与突触可塑性和在几个神经紊乱中观察到的 synaptopathy 相关,17,18。以前的亚细胞分馏法用于纯化 PSD 涉及的蔗糖梯度的差异离心分离的洗涤剂不溶性分数从大脑的粗膜分数14,19. 这一传统方法的主要挑战是它需要大量的啮齿动物的大脑14,19。10-20 啮齿目动物的制备隔离 PSD 的每一次治疗需要大量的成本和时间的投资, 并没有实际可行的, 如果有很多的治疗。
为了克服这一挑战, 我们已经采用了一种简单的方法, 直接分离 psd 分数, 没有蔗糖梯度离心20,21, 并将其修改为适用于单鼠海马的 psd 隔离大脑.我们的 small-scale psd 分馏方法的产量约 30-50 µg 的 psd 蛋白从2海马, 足以用于几个生物化学的化验, 包括沉淀和西部印迹。西方印迹表明, 我们的方法成功地分离 PSD 通过揭示丰富的突触后密度蛋白 95 (PSD-95) 和排除前标记突和可溶性细胞质蛋白α蛋白。我们的 ecs 诱导和 small-scale psd 分馏方法很容易适应其他啮齿动物的脑区, 并提供了一个相对简单和可靠的方法来评价 ecs 对 psd 蛋白表达的影响。

<table><tbody><tr><td>Spargue-Dawley rat</td><td>Charles River Laboratories</td><td></td><td>ECS supplies</td></tr><tr><td>A pulse generator</td><td>Ugo Bsile, Comerio, Italy</td><td>57800</td><td>ECS supplies</td></tr><tr><td>MilliQ water purifying system</td><td>EMD Millipore</td><td>Z00Q0VWW</td><td>Subcellular fractionation supplies</td></tr><tr><td>Sucrose</td><td>Em science</td><td>SX 1075-3</td><td>Subcellular fractionation supplies</td></tr><tr><td>Na&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>221368</td><td>Subcellular fractionation supplies</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>SIGMA-ALDRICH</td><td>E9884</td><td>Subcellular fractionation supplies</td></tr><tr><td>HEPES</td><td>SIGMA-ALDRICH</td><td>H0527</td><td>Subcellular fractionation supplies</td></tr><tr><td>Okadaic acid</td><td>TOCRIS</td><td>1136</td><td>Subcellular fractionation supplies</td></tr><tr><td>Halt Protease Inhibitor</td><td>Thermo Scientific</td><td>78429</td><td>Subcellular fractionation supplies</td></tr><tr><td>NaVO&lt;sub&gt;3&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>72060</td><td>Subcellular fractionation supplies</td></tr><tr><td>EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters</td><td>Fisher Scientific</td><td>SCGPS05RE</td><td>Subcellular fractionation supplies</td></tr><tr><td>Iris Scissors</td><td>WPI (World Precision Instruments)</td><td>500216-G</td><td>Subcellular fractionation supplies</td></tr><tr><td>30 mm tissue culture dish</td><td>Fisher Scientific</td><td>08-772B</td><td>Subcellular fractionation supplies</td></tr><tr><td>Glass homogenizer and a Teflon pestle</td><td>VWR</td><td>89026-384</td><td>Subcellular fractionation supplies</td></tr><tr><td>1.7 mL microcentrifuge tube</td><td>DENVILLE SCIENTIFIC INC.&amp;nbsp;</td><td>C2170 (1001002)</td><td>Subcellular fractionation supplies</td></tr><tr><td>Sorvall Legend XT/XF Centrifuge&amp;nbsp;</td><td>Thermo Fisher</td><td>75004521</td><td>Subcellular fractionation supplies</td></tr><tr><td>Pierce BCA Protein Assay Reagent A, 500 mL</td><td>Thermo Fisher</td><td>#23228</td><td>Western blot supplies</td></tr><tr><td>Pierce BCA Protein Assay Reagent B, 25 mL</td><td>Thermo Fisher</td><td>#1859078</td><td>Western blot supplies</td></tr><tr><td>SDS-polyacrylamide gel (SDS-PAGE)</td><td>BIO-RAD</td><td>#4561086S</td><td>Western blot supplies</td></tr><tr><td>Running Buffer</td><td>Made in the lab</td><td></td><td>Western blot supplies.&amp;nbsp;</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophorsis Cell for MiniPrecast Gels, 4-gel</td><td>BIO-RAD</td><td>#1658004</td><td>Western blot supplies</td></tr><tr><td>Polyvinyl difluoride (PVDF) membrane&amp;nbsp;</td><td>Milipore</td><td>IPVH00010</td><td>Western blot supplies</td></tr><tr><td>Transfer Buffer</td><td>Made in the lab</td><td></td><td>Western blot supplies.&amp;nbsp;</td></tr><tr><td>Tris-base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Western blot supplies</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td>Western blot supplies</td></tr><tr><td>Sodium dodecyl sulfate</td><td>SIGMA-ALDRICH</td><td>436143</td><td>Western blot supplies</td></tr><tr><td>Methanol&amp;nbsp;</td><td>Fisher Scientific</td><td>A454-4</td><td>Western blot supplies</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td>detergent for PSD isolation</td></tr><tr><td>Mini Trans-Blot Module&amp;nbsp;</td><td>BIO-RAD</td><td>#1703935</td><td>Western blot supplies</td></tr><tr><td>Nonfat instant dry milk</td><td>Great value</td><td></td><td>Western blot supplies</td></tr><tr><td>Multi-purposee rotator&amp;nbsp;</td><td>Thermo Scientific</td><td>Model-2314</td><td>Western blot supplies</td></tr><tr><td>Hyblot CL Autoradiography Film</td><td>DENVILLE SCIENTIFIC INC.&amp;nbsp;</td><td>E3018 (1001365)</td><td>Western blot supplies</td></tr><tr><td>Enhanced chemifluorescence substrate&amp;nbsp;</td><td>Thermo Scientific</td><td>32106</td><td>Western blot supplies</td></tr><tr><td>a Konica SRX-101A film processor</td><td>KONICA MINOLTA</td><td>SRX-101A</td><td>Western blot supplies</td></tr><tr><td>&lt;strong&gt;Name of Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PSD-95</td><td>Cell Signaling</td><td>#2507</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>Synaptophysin</td><td>Cell Signaling</td><td>#4329</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>alpha-Tubulin</td><td>Santacruz</td><td>SC-5286</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>GluN2B</td><td>Neuromab</td><td>75-097</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>GluA2</td><td>Sigma-aldrich</td><td>Sab 4501295</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>STEP</td><td>Santacruz</td><td>SC-23892</td><td>Antibody dilution = 1:200 - 500, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoReserch laboratory</td><td>715-035-150</td><td>Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey</td></tr><tr><td>Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoReserch laboratory</td><td>711-035-152</td><td>Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey</td></tr></tbody></table>

electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins

电癫痫 (ECS) 是一种实验动物模型的电治疗, 最有效的治疗严重抑郁症。在低死亡率和神经元死亡的挛癫痫的诱导下, 是一种广泛应用的筛选抗药物的模型。在这里, 我们描述一个 ECS 感应方法, 其中一个简短的55毫安电流是交付 0.5 s 到雄性大鼠 200-250 克重量通过耳夹电极。这种双边刺激产生的阶段 4-5 挛癫痫发作持续了大约十年代。在急性或慢性 ECS 停止后, 大多数老鼠恢复了与假 "无癫痫" 大鼠行为的区别。由于 ECS 在全球范围内提升了大脑活动, 它也被用来检测突触蛋白的活动依赖性的变化及其对突触强度的影响, 使用多种方法。特别是, 突触后密度 (PSD) 与西方印迹结合的亚细胞分馏允许在这个专门的突触结构中定量地测定突触蛋白的丰度。与以前的分馏方法相比, 需要大量的啮齿动物大脑, 我们这里描述了一个 small-scale 分离方法, 以隔离 PSD 从海马的单一大鼠, 没有蔗糖梯度离心。利用这种方法, 我们发现分离的 PSD 分数含有突触后的膜蛋白, 包括 PSD95、GluN2B 和 GluA2。前标记突和可溶性细胞质蛋白α-蛋白被排除在 psd 的分数, 表明成功的 psd 隔离。此外, 慢性 ECS 降低 GluN2B 表达在 psd, 表明我们的 small-scale psd 分馏方法可用于检测的变化, 海马 psd 蛋白从一大鼠后, 遗传, 药理, 或机械治疗.

使用详细的程序在这里提出, 一个电击 (55 毫安, 100 脉冲/s 0.5 s) 通过耳夹电极诱导非期4-5 挛发作大鼠 (图 1A-b) 传递。总8的大鼠接受急性 ECS 诱导和显示阶段4-5 强直性挛发作。癫痫发作持续了大约十年代, 所有的老鼠在 1-2 分钟内恢复了癫痫停止。假 "没有癫痫" 大鼠没有受到电击, 因此没有显示癫痫发作。共使用了4只假鼠。对于慢性 ECS 诱导, 大鼠每天接受一次电击, 连续7天, 并在每次电击时显示4-5 挛发作 (图 1A-b)。共有8的大鼠接受了慢性 ECS 诱导, 总共4只假鼠被用作 "无癫痫" 大鼠。虽然大多数接受慢性 ECS 的老鼠与假的 "无癫痫" 大鼠行为难以区分, 但有少数发育的身体震颤, 并减少了 7th电击的探索活动。
为了检查 ecs 诱发的全球活动海拔是否改变了海马中的蛋白质表达, 大鼠在急性 ecs 后的3小时和24小时内被牺牲, 在慢性 ecs 治疗的最后 ecs 后24小时和96小时 (图 1B)。每只老鼠的两个海马被迅速解剖, 并服从我们优化的 small-scale 分馏协议 (图 2)。初始匀浆两个海马不溶性组织和细胞核 (S1 分数) 是 re-centrifuged 在 13800 x g 10 分钟, 以分离的上清含有可溶性细胞质蛋白 (S2 分数) 从粗膜颗粒含有突 (P2 分数) (图 2)。我们的西部印迹检测到 S2 分数的细胞质蛋白α-蛋白, 但不是 P2 的分数, 海马从大鼠急性和慢性 ECS 治疗 (图 3和图 4), 表明成功的分馏从粗膜颗粒中胞浆可溶性蛋白。
PSD95 是膜相关的鸟激酶 (MAGUK) 家族的成员和 PSD12、18、25的核心组件。PSD95 和 NMDA 受体亚基 GluN2B 只在 P2 分数中检测到, 而不是 S2 分数 (图 3和图 4)。在 P2 (图 3和图 4) 中的 S2 分数中检测到了另一种谷氨酸α-氨基-3-羟基-5-甲基-4-isoxazolepropionic 酸 (AMPA) 受体亚基 GluA2 的更强表达。表明, 突触后膜蛋白在粗膜 P2 分数颗粒中富集。有趣的是, 突触囊泡蛋白突和整体膜蛋白纹富酪氨酸磷酸酶 61 (步骤61)26在 S2 和 P2 分数 (图 3和图 4) 中同样被检测到。考虑到小尺寸的突触泡-平均直径为 39 nm27和体, 离心分离的粗膜颗粒 (P2 分数) 可能没有足够的颗粒所有突触泡和体。这些结果共同表明, 我们的粗分馏协议, 可以丰富膜结合蛋白和跨膜蛋白, 不与突触泡, 可能与体在粗膜 P2 分数。
为了检查 ECS 诱发的全球活动度是否改变了海马中突触后蛋白质的表达, 粗膜 P2 分数裂解在 ice-cold 水中, 悬浮在 HEPES 缓冲液中, 离心在 2.5万 x g 处为20分钟, 以隔离裂解颗粒 (LP1) (图 2)。LP1 颗粒是悬浮在 HEPES 缓冲含有1% 海卫 X-100 洗涤剂和离心在 2.5万 X g 以上 3 h 获得 PSD 颗粒 (图 2)。随后的 PSD 分数的西部印迹检测 PSD95, GluN2B, 和 GluA2 (图 3和图 4), 这是已知的后突的蛋白质17,25,28。然而, PSD 分数缺乏α-蛋白, 重要的是, 前标记突 (图 3和图 4)。步骤61, 其中 dephosphorylates GluN2B 和 GluA2 并充当其负稳压器29、30、31, 在 PSD 分数中未找到 (图 3和图 4),与最近的报告演示了步骤61的突触排除, PSD95 在 PSD26中进行了丰富的介导。总之, 这些结果表明, 我们的 small-scale 分馏方法成功地分离了 PSD 蛋白从粗膜 P2 分数。
西方印迹的定量显示, GluN2B 表达式在 P2 分数中是不变的, 但在急性 ECS (n = 4) (图 3B) 后, PSD 分数在3ĥ和 24 h 上呈上升趋势。在急性 ECS (图 3C) 后, 在3ĥ和 24 h 的 P2 和 PSD 分数中, GluA2 表达不变。在慢性 ECS 后24小时和96小时 GluN2B 表达在 P2 分数中呈下降趋势, PSD 分数显著降低 (24 小时: p < 0.05, n = 4; 48 h: p < 0.01, n = 4) (图 4B)。相比之下, 慢性 ECS 不影响 GluA2 表达在 P2 和 PSD 分数 (n = 4) (图 4C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56016/56016fig1.jpg"> 图 1: 用于诱导急性 ecs 和慢性 ecs 的模式.(a) 一只老鼠通过耳夹电极连接到脉冲发生器, 电击 (55 毫安, 100 脉冲/s 0.5 秒) 被应用于诱发4-5 级挛发作。(B) 急性 ECS 是由一个电击诱发的。连续7天每天有一次电击引起慢性 ECS。所示的时间点代表的持续时间后, 急性 ecs 或最后 ecs 的慢性 ecs 诱导前解剖的海马。假 "不癫痫" (NS) 控制大鼠处理相同, 但没有收到电击。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56016/56016fig2.jpg"> 图 2: 从单个大鼠的海马中分离出 S2、P2 和 PSD 分数的亚细胞分馏工作流.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56016/56016fig3.jpg"> 图 3:从接受 NS 或急性 ECS 的大鼠的海马 S2、P2 和 PSD 分数中检测突触蛋白.(A) 有代表性的西方印迹显示了 NMDA 受体亚基 GluN2B、AMPA 受体 GluA2 的蛋白表达, 以及 S2、P2 和从海马接受急性 ECS 的假 "无癫痫" (NS) 大鼠和大鼠的 PSD 分数中的步骤61 。胞质可溶性蛋白α-蛋白在 S2 馏分中富集。突是一种前囊泡蛋白, 在粗膜 P2 馏分中富集, 但在 PSD 分数中不丰富。PSD-95 是丰富的 P2 和 PSD 分数。(B-C)量化的 GluN2B (B) 和 GluA2 (C) 在 P2 和 PSD 分数在3和24小时后急性 ECS (n = 4 鼠每时间点)。P2 分数中的 GluN2B 和 GluA2 表达被归一化为 S2 馏分中的α-蛋白。psd 馏分中的 GluN2B 和 GluA2 表达被归一化为 psd 分数的 PSD-95。数据显示为百分比± SEM.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56016/56016fig4.jpg"> 图 4:从接受 NS 或慢性 ECS 的大鼠的海马 S2、P2 和 PSD 分数中检测突触蛋白.有代表性的蛋白的印迹;突;步骤61;和突触后的蛋白质, 包括 PSD95, GluN2B, 和 GluA2, 在 S2, P2, 和 PSD 分数从海马的假 "无癫痫" (NS) 大鼠和接受慢性 ECS。胞质可溶性蛋白α-蛋白在 S2 馏分中富集。突是一种前囊泡蛋白, 在粗膜 P2 馏分中富集, 但在 PSD 分数中不丰富。PSD-95 是丰富的 P2 和 PSD 分数。(B-C)定量的 GluN2B (B) 和 GluA2 (C) 在 P2 和 PSD 分数在24和96小时后, 慢性 ECS (n = 4 鼠每时间点)。P2 分数中的 GluN2B 和 GluA2 表达被归一化到 S2 馏分中的α-蛋白。psd 馏分中的 GluN2B 和 GluA2 表达被归一化为 psd 分数的 PSD-95。数据显示为百分比± SEM;* p < 0.05, ** < 0.01 与对照组比较 (方差分析和 Tukey 的事后测试)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>分数</td>
			<td>蛋白质含量</td>
			<td>蛋白质标记</td>
		</tr>
<tr>
<td>P1</td>
			<td>核</td>
			<td>Histon H1</td>
		</tr>
<tr>
<td>S1</td>
			<td>胞/膜</td>
			<td>蛋白 (细胞骨架) 和 GAPDH (胞)</td>
		</tr>
<tr>
<td>P2</td>
			<td>粗突</td>
			<td>AMPA 和 NMDA 受体亚基</td>
		</tr>
<tr>
<td>S2</td>
			<td>胞/轻膜</td>
			<td>GAPDH (胞) 和 LAMP1 (溶)</td>
		</tr>
<tr>
<td>触膜</td>
			<td>体/线粒体</td>
			<td>AMPA 和 nmda 受体亚基, 突 (前标记)</td>
		</tr>
<tr>
<td>psd</td>
			<td>psd</td>
			<td>AMPA 和 NMDA 受体亚基, PSD95, PICK1, CaMKII</td>
		</tr>
</tbody></table>表 1: 蛋白质标记物和抗体的列表, 以区分亚细胞组分。

Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

电癫痫 (ECS) 是一种电治疗严重抑郁症的实验动物模型。ECS 全球刺激海马活动, 导致突和突触可塑性。在这里, 我们描述的方法的 ECS 诱导大鼠和亚细胞分型的海马, 以检查癫痫诱导的变化, 突触蛋白。

大鼠电癫痫发作与海马的分离检测突触后密度蛋白的变化

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

electroconvulsive-seizures-rats-fractionation-their-hippocampi-to

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.9K 次观看.  University of Illinois at Urbana-Champaign. 电癫痫 (ECS) 是一种电治疗严重抑郁症的实验动物模型。ECS 全球刺激海马活动, 导致突和突触可塑性。在这里, 我们描述的方法的 ECS 诱导大鼠和亚细胞分型的海马, 以检查癫痫诱导的变化, 突触蛋白。

视频: Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

科研

神经科学

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

从海马CA1区片核富含分的分离，研究突触核信使蛋白质的活性依赖进口核

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

采用高分辨率脑电图记录脑源成像局灶性癫痫的临床前模型大鼠

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

医学

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

非约束脑电遥测:硬膜外和脑深部脑电立体定位放置电极

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/58455/microdialysis-excitatory-amino-acids-during-eeg-recordings-freely'>Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)</a>This video demonstrates the assessment of extracellular excitatory neurotransmitter levels in rats with chronic epilepsy induction. After implanting a microdialysis probe into the brain and confirming the absence of seizures using electroencephalography (EEG), extracellular fluid is collected to evaluate elevated neurotransmitter levels in epileptic rats. A high-potassium solution is then infused to sustain neuronal excitability and trigger an increased release of neurotransmitters, with samples collected to assess the potassium-induced peak in excitatory neurotransmitters.

Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

癫痫诱发大鼠癫痫发作的脑电图记录

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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