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
Summary
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。
Abstract
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.
Introduction
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。
Protocol
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 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。 在产后日 (P) 28 将大鼠幼崽断奶, 以 2-4 的雄性或雌性窝。 用无毒的永久性黑色标记标记雄性大鼠的尾巴。 对雄性大鼠每周3次称重, ?…
Representative Results
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…
Discussion
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…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Spargue-Dawley rat | Charles River Laboratories | ECS supplies | |
| A pulse generator | Ugo Bsile, Comerio, Italy | 57800 | ECS supplies |
| MilliQ water purifying system | EMD Millipore | Z00Q0VWW | Subcellular fractionation supplies |
| Sucrose | Em science | SX 1075-3 | Subcellular fractionation supplies |
| Na4O7P2 | SIGMA-ALDRICH | 221368 | Subcellular fractionation supplies |
| Ethylenediaminetetraacetic acid (EDTA) | SIGMA-ALDRICH | E9884 | Subcellular fractionation supplies |
| HEPES | SIGMA-ALDRICH | H0527 | Subcellular fractionation supplies |
| Okadaic acid | TOCRIS | 1136 | Subcellular fractionation supplies |
| Halt Protease Inhibitor | Thermo Scientific | 78429 | Subcellular fractionation supplies |
| NaVO3 | SIGMA-ALDRICH | 72060 | Subcellular fractionation supplies |
| EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters | Fisher Scientific | SCGPS05RE | Subcellular fractionation supplies |
| Iris Scissors | WPI (World Precision Instruments) | 500216-G | Subcellular fractionation supplies |
| 30 mm tissue culture dish | Fisher Scientific | 08-772B | Subcellular fractionation supplies |
| Glass homogenizer and a Teflon pestle | VWR | 89026-384 | Subcellular fractionation supplies |
| 1.7 mL microcentrifuge tube | DENVILLE SCIENTIFIC INC. | C2170 (1001002) | Subcellular fractionation supplies |
| Sorvall Legend XT/XF Centrifuge | Thermo Fisher | 75004521 | Subcellular fractionation supplies |
| Pierce BCA Protein Assay Reagent A, 500 mL | Thermo Fisher | #23228 | Western blot supplies |
| Pierce BCA Protein Assay Reagent B, 25 mL | Thermo Fisher | #1859078 | Western blot supplies |
| SDS-polyacrylamide gel (SDS-PAGE) | BIO-RAD | #4561086S | Western blot supplies |
| Running Buffer | Made in the lab | Western blot supplies. | |
| Mini-PROTEAN Tetra Vertical Electrophorsis Cell for MiniPrecast Gels, 4-gel | BIO-RAD | #1658004 | Western blot supplies |
| Polyvinyl difluoride (PVDF) membrane | Milipore | IPVH00010 | Western blot supplies |
| Transfer Buffer | Made in the lab | Western blot supplies. | |
| Tris-base | Fisher Scientific | BP152-1 | Western blot supplies |
| Glycine | Fisher Scientific | BP381-5 | Western blot supplies |
| Sodium dodecyl sulfate | SIGMA-ALDRICH | 436143 | Western blot supplies |
| Methanol | Fisher Scientific | A454-4 | Western blot supplies |
| Triton X-100 | Fisher Scientific | BP151-500 | detergent for PSD isolation |
| Mini Trans-Blot Module | BIO-RAD | #1703935 | Western blot supplies |
| Nonfat instant dry milk | Great value | Western blot supplies | |
| Multi-purposee rotator | Thermo Scientific | Model-2314 | Western blot supplies |
| Hyblot CL Autoradiography Film | DENVILLE SCIENTIFIC INC. | E3018 (1001365) | Western blot supplies |
| Enhanced chemifluorescence substrate | Thermo Scientific | 32106 | Western blot supplies |
| a Konica SRX-101A film processor | KONICA MINOLTA | SRX-101A | Western blot supplies |
| Name of Antibody | |||
| PSD-95 | Cell Signaling | #2507 | Antibody dilution = 1:500-1000, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit |
| Synaptophysin | Cell Signaling | #4329 | Antibody dilution = 1:500-1000, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit |
| alpha-Tubulin | Santacruz | SC-5286 | Antibody dilution = 1:500-1000, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Mouse |
| GluN2B | Neuromab | 75-097 | Antibody dilution = 1:500-1000, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Mouse |
| GluA2 | Sigma-aldrich | Sab 4501295 | Antibody dilution = 1:500-1000, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit |
| STEP | Santacruz | SC-23892 | Antibody dilution = 1:200-500, time = 9 – 12 h, Reaction Temperature = 4 °C, Host Species = Mouse |
| Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L) | Jackson ImmunoReserch laboratory | 715-035-150 | Antibody dilution = 1:2000-5000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey |
| Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoReserch laboratory | 711-035-152 | Antibody dilution = 1:2000-5000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey |
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Citar este artigo
Jang, S., Jeong, H. G., Chung, H. J. Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins. J. Vis. Exp. (126), e56016, doi:10.3791/56016 (2017).