高频刺激激活小鼠齿状回的侵袭方法
Summary
该协议说明如何在小鼠中建立可靠的痉挛方法。整个海马齿状回的神经元由痉挛直接和间接的在体内电刺激。神经活动和分子信号由c-fos和 Notch1 免疫荧光染色分别检查;bromodeoxyuridine 标记法对神经发生进行量化。
Abstract
电高频刺激 (痉挛), 使用植入电极靶向不同的大脑区域, 已被证明是一种有效的治疗各种神经和精神疾病。脑深部痉挛, 也称为深脑刺激 (DBS) 在临床试验中变得越来越重要。最近在高频星展 (HF 星展) 手术领域的进展已经开始传播利用这种侵入性技术的可能性到其他情况, 如治疗大萧条症 (MDD), 强迫性障碍 (强迫症), 等等上.
尽管有这些不断扩大的迹象, 但 HF-DBS 的有益影响的潜在机制仍然是谜。为了解决这个问题, 一种方法是使用植入电极, 疏生通过痉挛激活神经元的分散亚群。据报道, 丘脑前核痉挛可用于临床治疗难治性癫痫。潜在的机制可能与增加的神经发生和改变的神经元活动有关。因此, 我们对在痉挛治疗前后小鼠齿状回 (DG) 神经元活性的检测和神经发生发生的生理变化有兴趣。
在这篇手稿中, 我们描述了痉挛的方法, 目的是以急性或慢性方式直接或间接地激活 DG 在小鼠体内的活化。此外, 我们描述了一个详细的协议, 以准备的脑切片的 c-fos 和 Notch1 免疫荧光染色监测神经元活动和信号激活和 bromodeoxyuridine (BrdU) 标记, 以确定高频-星展诱导后神经发生。在 HF-星展治疗后神经元活动和神经发生的激活提供了直接的神经生物学证据和潜在的治疗效果。特别是, 这种方法可以修改和应用于其他感兴趣的大脑区域, 如基底神经节和丘脑地区的特定脑功能紊乱的临床。
Introduction
HF-星展是一种神经外科技术, 在大脑中进行电刺激, 这是自十九世纪七十年代1以来发展起来的。在二十世纪八十年代代末, 痉挛症首次被用作治疗帕金森病和其他运动紊乱的潜在疗法2。在过去的几十年中, HF-星展已越来越广泛地应用于治疗脑部疾病, 目前无法治愈的传统治疗策略。特别是, 由于痉挛电极的准确性提高, 高效率的结果, 和最小的副作用, 在过去几十年中, HF 星展集团治疗的脑部疾病的数量显著增加了3,4, 5。例如, HF-DBS 已被美国食品药品监督管理局 (FDA) 批准, 用于治疗帕金森病 (PD)、阿尔茨海默氏型痴呆、基本震颤和其他类型的运动障碍2,6,7. 在 PD 患者中, 多巴胺能药物在 HF-星展8期间减少到50%。除了成功地治疗运动障碍, HF-星展还显示了它在治疗精神病的临床上的强大效果, 并为认知增强以及2,9,10,11. 值得注意的是, 对治疗其他精神疾病的痉挛的研究处于不同的阶段, 对患者的12提供了很大的承诺。
虽然许多研究表明, 在整个大脑中, 一个焦点痉挛有局部和远程影响13, 影响的神经和分子机制仍然是难以捉摸的2,14。在临床上, 治疗 hf-星展病毒通常用于治疗帕金森病和慢性疼痛,等.许多人提出了一些意见来解释 HF-星展集团治疗所产生的改善, 其中一种可能性痉挛电流调节神经元网络活动, 可能是在植入痉挛电极附近的轴突的重复退极化。或者, HF-DBS 可能会改变输出神经元的放电速率和投射的目标。此外, HF-星展公司可能会导致长期的突触变化, 包括长期增强 (LTP) 和长期抑郁症 (), 这可能有助于改善症状。到目前为止, 还不清楚痉挛是否 influences 了调节细胞过程的关键分子事件, 如成人神经发生在体内。几行研究表明, 啮齿类动物的痉挛可能模仿临床应用的 DBS15,16类似的神经反应。为了了解 HF-DBS 的基本细胞机制, 在本研究中, 我们首先在急性 (一天) 或慢性 (五天) 的小鼠中建立了体内痉挛方法。其次, 我们建立了活化分析方法, 以确定在 HF-星展酶传递后神经元活动和神经发生的改变。
由于神经干细胞在胚胎发育过程中具有丰富的神经元产量, 但在整个成年期仍在继续, 海马 subgranular 区是发生神经再生的主要区域之一。神经发生的过程受许多生理和病理因素的影响。在某些癫痫病例中, 海马神经发生明显减少17,18。此外, 单一的抽搐治疗可以显著增加神经元的生产在齿状回19。这些观察表明, 电生理活性在调节海马神经元神经发生和突触可塑性方面起着至关重要的作用。因此, 为了进一步证明 HF-DBS 对神经元活动和神经发生的影响, 我们首先进行染色的早期基因 (腿部) c-fos 的检测, 这是一个众所周知的短期神经元活动的标志, 导致体验20。还检测到 Notch1 信号, 以监视痉挛传递21、22之后的信号激活。此外, 我们还通过 BrdU 标记分析在不同的行为方式下检测出神经元的产生, 但 BrdU 染色也可以作为 gliogenesis 的标志。
本研究采用两种痉挛方法对海马 DG 的直接和间接活化进行了定位。电极直接植入到 DG 中, 或植入内侧 perforant 路径 (PP) 中, 发送投射激活 dg 神经元。对于 HF-星展的感应, 一个可编程的刺激器被提出为连续刺激通过固定电极到鼠标头上。为了确定痉挛对神经元活化和神经发生的影响, 我们通过免疫荧光染色和海马 DG 区 BrdU 阳性神经元的数目分别检测了 Notch1 的表达, 并在痉挛治疗。特别地, HF-星展集团对 DG 的神经发生的影响比较急性和慢性刺激方式, 或直接和间接刺激方式之间, 分别。
Protocol
Representative Results
Discussion
短波星展卫星技术已被广泛用作治疗自二十世纪九十年代以来许多神经系统疾病的有力工具。目前, 短波星展的标志性工作是治疗帕金森病和原发性震颤, 在临床和科学界引起了广泛的关注和兴趣。在某些神经和精神疾病的治疗应用中, 许多组正在进行的 hf-星展研究有多种类型, 如32,33。其中一些研究目前处于临床试验的不同阶段2,</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
由中国国家自然科学基金资助31522029、31770929和 31371149 (对海涛), 项目 973 (2014CB542203) 从国家重点发展计划为中国的基础研究 (对海涛), 并且授予 Z161100000216154 从北京市科学技术委员会 (对海涛)。作者感谢海涛吴实验室所有成员的鼓励和讨论。作者非常感谢振威刘为他的帮助调试仪器。
Materials
| Brain stereotaxic instrument | Stoelting | 51730D | Stereotactic intracranial implantation for mouse |
| Stimulator | A-M systems | Model 3800 | MultiStim 8-Channel programmable stimulator |
| Dental driller | Saeshin Precision Co., Ltd | STRONG 90 | For drilling and crainiotomy |
| Burr | Meisinger | HM1 005# | For drilling and crainiotomy |
| Digidata 1550 Digitizer | Molecular Devices | AXON 1550 | High-resolution data acquisition |
| Cryotome | Thermo Fisher Scientific | Thermo Cryotome FSE | Cutting frozen sections of specimens |
| Confocal microscope | Olympus | FV-1200 | Japan, with 20x Objective (NA 0.45) |
| Mouse surgery tools | F.S.T. | 14084-08,11254-20,16109-14 | Scissors, forceps, bone cutter, holders etc. |
| Pentobarbital sodium | R&D systems | 4579 | 20-50mg/kg for i.p. injection |
| Penicillin G | Sigma-Aldrich | P3032 | 75,000 U for i.m. injection |
| Carprofen | Sigma-Aldrich | SML1713 | 5-10mg/kg, for s.c. injection |
| 4% Paraformaldehyde (PFA) | Beijing Solarbio Sci-Tech Co. | P1110 | stocking solution for tissue fixation |
| Phosphate buffer (PBS) | Invitrogen | 10010023 | pH7.4, 500ml in stocking |
| Tissue-Tek O.C.T. compound | Sakura | 4583 | Formulation of water-soluble glycols and resins |
| anti-BrdU antibody | Abcam | ab6326 | Dilutions:1/800 |
| anti-c-fos antibody | Abcam | ab209794 | Dilutions:1/500 |
| Goat Anti-Rabbit IgG (Alexa Fluor 568) | Thermo Fisher Scientific | A11036 | Dilutions:1/500 |
| Donkey Anti-Rat IgG (Alexa Fluor 488) | Jackson ImmunoResearch | 712-546-150 | Dilutions:1/500 |
| Antifade mounting medium with DAPI | Vector Laboratories | H-1200 | Counterstaining with DAPI |
| anti-Notch1 antibody (C-20) | Santa Cruz Biotech | sc-6014 | Dilutions:1/50 |
| Donkey Anti-Goat IgG (Alexa Fluor 488) | Abcam | ab150073 | Dilutions:1/1000 |
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