We are grateful for the support of the NREF foundation.

注:伦理学声明:本稿件讨论的所有程序都是按照NIH指南动物研究（指南护理和使用实验动物）和哈佛医学院IACUC委员会的批准。 1.预外科制备<ol><li>确保所有的手术器械是由任一高压灭菌或通过用消毒液和/或乙醇作为必要清洁消毒。如果可能的话，使用一次性无菌设备，如手术刀，针头和注射器。 </li><li>覆盖手术单工作台，并确保有一个生物危险废物处置可供。 </li><li>称量大鼠和计算麻醉剂量。用氯胺酮/赛拉嗪混合物（氯胺酮75 mg / kg和赛拉嗪10毫克/千克）麻醉大鼠。 注:介于200-250克是在立体定位框架的动物的正确的固定，以及用于ATN的精确定位的最佳权重。 Isoflur烷也可以被用作麻醉剂。 </li><li>装载和安全两无菌电极上的电极保持器（通过高压灭菌或用环氧乙烷灭菌）立体定向手术帧和用显微镜（倍率10-40X）的帮助下（ 图2），检查电极的正确的提示对齐。固定在两个电极3.0毫米分开。 注:请注意，以避免触及坚硬的表面损坏电极尖端。 </li></ol> 2. DBS手术<ol><li>注射氯胺酮/甲苯​​噻嗪混合腹腔内的动物，确认该动物已达到麻醉的手术平面（通过检查的脚趾捏反射，呼吸频率和深度，呼吸规律）。从将要切开头皮的区域去除毛发和消毒用3交替磨砂每个优碘和任一醇或无菌盐水头皮。固定并且在立体定向FRAM定位动物即</li><li>使用循环热水垫保持动物的体温在最佳水平。适用于眼部润滑剂，以动物的眼睛过度干燥保护。 </li><li>使用下面的立体坐标定位的ATN（前丘脑核）:anterioposterior -1.6毫米，中侧1.5mm，且背腹5.2毫米1。 注:立体坐标基于所述Paxinos和Watson（ 第 6版）的大鼠脑图谱36。 </li><li>使头皮切口矢状露出头骨。使用一对拉钩固定切开头皮暴露颅骨。用无菌盐水冲洗切口。如果切口过湿，用消毒棉签擦干。找到前囟门和带黑色标记标记。从矢状缝和1.6毫米后向科伦引导毛刺孔的位置，使两个标记在约1.5mm mediolaterally两侧人缝合。 </li><li>使用一个手持电钻，使毛刺的孔。确保钻孔的前端是无菌的用乙醇消毒它。钻时，应举行演习以大约45°角的颅骨表面。两毛刺孔之间频繁切换，以避免过度的热在任何钻孔的位置。 </li><li>继续钻进直到硬脑膜露出。使用的针，其前端弯曲类似的"L"的形状，除去任何破骨片，将阻碍电极的插入。小心以避免损坏底层硬脑膜和/或脑组织，同时使用弯曲的针除去骨头碎片。 注意:使用钝针或细钝钳也是一种选择。 </li><li>固定的双重电极组件的立体定位框架的旋转手柄和固定在90°角柄。利用立体定位框架的调整，定位离开电极的前囟门正好上方。 </li><li>使用立体声策略调整对中侧定位，精确地移动所述左电极1.5mm到前囟的左侧，使得现在有两个电极沿冠状缝完全对齐但间隔开在1.5毫米mediolaterally从囟。 </li><li>使用anterioposterior立体定向调整，将电极1.6毫米后到冠状缝。 </li><li>使用背腹的调整，以降低电极以首先检查，如果毛刺孔已在正确的位置，使得电极可插入自如，而不触及毛刺孔的粗糙边缘。如果是这样，插入电极至5.2毫米的颅骨的表面的深度。 </li><li>通过导线将电极连接到刺激器设置为130赫兹，2.5 V和90微秒脉冲宽度1。 </li><li>提供的高频刺激一小时（或时间按照实验装置的所需时间）。在刺激过程中，切记要保证P罗珀麻醉深度经常通过检查的情况下的脚撤回响应于脚趾捏。补充麻醉用约一半用于诱导麻醉的初始剂量。检查麻醉深度时，为了避免您的无菌手套的污染，必要时更换它们。执行基于一个人的实验需要单侧或双侧刺激。包括控制诸如低频刺激（对于如 ，10赫兹）和未刺激的动物（无后续刺激插入电极）。 </li><li>刺激结束后，小心地取出电极和缝合切口缝合3-0或无菌手术主食。 </li><li>施用丁丙诺啡（0.05毫克/千克）经皮下作为镇痛。监测动物，直到它返回到正常活动，然后将其返回到所述壳体设施。 </li><li>后的时间基于所述实验设计的一组时间段（ 例如 ，0，3,6或12小时后的DBS手术），安乐死麻醉过量的动物。确认没有生命体征后，斩首动物。 </li><li>首先去除用剪刀将皮肤剖析了大脑。通过利用沿解剖剪刀矢状缝骨切开。使通过在两个横向侧的骨两个切口（大约一英寸的每个）。使用镊子，解除部分切开骨片从头骨顶部以暴露脑。 </li><li>用细剪刀或镊子，从头骨去除脑和在冰上转移至培养皿用冷PBS。 注:请注意，以避免同时通过骨做切口损伤大脑。 </li></ol> 3.海马隔离注意:请在此部分上的冰的所有后续步骤。 <ol><li>将脑在冰上预冷的丙烯酸类脑基质。用剃刀刀片，从最前编切开大脑冠状在约7-8毫米GE的大脑。使一第二切口冠和后向第一切口，使得约5毫米厚的脑切片可以被除去。 </li><li>脑切片转移至培养皿用冰冷的PBS中。使用刀片，切断两个半球部分，并照顾到注意哪个半分别对应于左，右两侧。这是特别重要的，同时执行单边刺激。 </li><li>用细镊子和剪刀小心地取出海马。 </li><li>闪存冻结的干冰，并存储在-20°C冰箱的海马组织，直到准备随后的RNA提取步骤。 注:对于长期贮存，最好是存储组织在-80℃冷冻机中。 </li></ol> 4. RNA提取和定量PCR <ol><li>确保组织撑干冰冷冻，直到准备同质化。 </li><li>注:执行该步骤在引擎盖。 <ol><li>加入1毫升三试剂来HIPP在1.5ml的离心管ocampal组织和由第一分注多次均化，直到组织中更小的碎片破裂。通过一个注射器通过与地下25针，直到没有不间断组织可见进一步均质化组织。进行冰上同质化的步骤。 </li><li>均质化的组织后，使细胞悬浮液静置在室温下5分钟，以允许细胞裂解。 注:在该点之后的细胞悬浮液可以是快速冷冻并储存在-70℃直到进一步处理。 </li></ol></li><li>中加入0.2ml氯仿，并通过涡旋混合20秒，让溶液静止在室温下15分钟。 </li><li>离心样品在12000×g离心15分钟，在4℃。离心后，小心地取出管，而不会干扰三个独立的层，这将是可见的。 注:底部（红色）相含有蛋白质，中部阴天相含有DNA和顶部清晰的阶段包含řNA。 </li><li>转移的顶部清楚相位（RNA）成一个新的1.5毫升离心管中（通常为500微升含有相RNA的获得）。这种加入0.5毫升异丙醇和涡旋混合。这个加2-3微升糖原（20毫克/毫升）为载体的RNA。使样品休息在桌子上，在室温下放置10分钟。 </li><li>离心样品在12000×g离心1小时，在4℃下。确保该RNA沉淀是在管的底部可见。 </li><li>弃去上清液，并通过加入1ml 75％的乙醇洗涤RNA沉淀（用不含核酸酶的水制成）。倒置样品多次，直到沉淀驱逐出来从管的底部，并漂浮在溶液中。离心在4℃下将溶液在12000×g离心10分钟。 注:在75％乙醇将沉淀可以储存在-20℃直到进一步的步骤。 </li><li>通过使管子打开5分钟，在室温下使RNA沉淀风干。采取预防措施，避免奥雅纳ř干燥，因为这可能会影响其溶解在水中，在随后的步骤中的小球。 </li></ol> 5.从RNA准备删除DNA <ol><li>加入9微升的核酸自由水的RNA颗粒，并确保小球进行轻轻涡旋管之前溶解。向该加1微升10×DNA酶I缓冲液和1μl重组DNA酶（从DNA酶I试剂盒），通过轻弹管混合，短暂离心所述管，并在水浴中孵育在37℃下30分钟。 </li><li>添加2微升DNA酶失活试剂（由DNA酶I盒）到样品并在室温下孵育2分钟，并通过轻弹管经常混合。离心机在12,000rpm离心10分钟，并转移大约10微升上层清液，以一个新的1.5毫升离心管中。 </li><li>使用NanoDrop或分光光度计测量RNA浓度。 </li></ol> 6.从RNA制作的cDNA <ol><li>添加所需量日在含有1微克的RNA和通过加入不含核酸酶的水，使总体积至8微升。这种加1微升10毫米的dNTP混合和1微升从标第一链合成试剂盒随机六聚体。轻轻混合并在任一水浴上或预编程热循环孵育在65℃下5分钟。 </li><li>使含有下列试剂预混物:2微升10×RT缓冲液，4微升25男的MgCl 2，2微升的0.1M DTT和1μlRNA酶OUT的（40U /微升）。 注:卷是给每个反应，用户需要根据自己的实验需要扩大规模。 </li><li>去除含样品从65℃将RNA后，将管在室温下放置一分钟。加9微升预混溶液并在室温下孵育2分钟。加入1微升的Superscript II逆转录酶（50U /微升），然后在室温下孵育10分钟。 </li><li>孵育样品在42℃下为50分的反转录发生。 </li><li>孵育样品在70℃下15分钟以终止反应。 </li><li>将样品在冰上短暂，并添加1μl的RNA酶H（2U /微升）和孵育在37℃下20分钟。 </li><li>短暂离心管在3000 XG降速冷凝液。 注意:这是将用于定量PCR中的cDNA。 cDNA可被存储在-20℃下冷冻直到PCR设置。的cDNA，也可以用无核酸酶的水在进行PCR之前稀释。 </li></ol> 7.定量PCR <ol><li>使含有下列试剂主混合物（体积定为每反应）:6.3微升2倍的SYBR Green混合，0.6微升正向引物（10μM），0.6微升反向引物（10μM）和0.5微升不含核酸酶的水。 注:使用做引物设计的"引物接"在NCBI的核苷酸序列页面选项感兴趣的基因。 </li><li>添加10微升mastermix到96良好PCR板的每个孔中。加入2.5微升cDNA的早期到井含mastermix进行。确保设立一式三份PCR反应每个样本。 </li><li>在完成加入mastermix和cDNA后，盖上使用光学粘合剂片以密封所述孔的PCR板。短暂离心板5分钟，在500 XG在台式离心机沉降所有液体到井的底部。 </li><li>加载PCR板到一个预编程的RT-PCR的机器。使用在表1中提供的PCR参数。 </li><li>数据分析:从定量PCR输出进一步的计算使用了C 吨值。随着测试基因，建立了PCR反应用于内部对照基因，18S rRNA基因（以确保相等的输入）和β-actin（阴性对照）。计算基于ΔΔCT方法37倍的变化。 </li></ol>

The authors have no disclosures.

继Benabid 等具有里程碑意义的工作，用脑深部电刺激治疗帕金森氏病和特发性震颤，在DBS手术技术已经研究了很大的兴趣在过去十年来治疗许多神经系统疾病6,10,43。针对大脑电路的各种神经解剖区域DBS的研究，许多团体正在执行，以解决主要的神经元​​疾病是临床试验的各个阶段。底丘脑核（STN）或苍白球（GPI）的内段的刺激被FDA批准并用于治疗运动障碍在帕金森氏病...

背后深部脑刺激的发展作为一个神经外科技术的历史可以追溯到19世纪70年代时的电刺激大脑电路的可能性进行了探讨2。采用慢性高频刺激作为治疗神经疾病始于20世纪60年代3。后来在上世纪90年代慢性植入电极DBS 4-6的来临，这是由DBS治疗神经疾病的人数不断增加。深部脑刺激，第一次在美国用作用于原发性震颤6的处理。今天的手术被广泛用于治疗神经元疾病是目前无法治疗由药物干预。 DBS是目前用于治疗帕金森氏病和肌张力障碍7-9的运动障碍。阿耳茨海默氏型痴呆，亨廷顿氏病，癫痫症，疼痛和神经精神疾病，例如抑郁症，强迫症，图雷特7综合症和成瘾是一些适合治疗由DBS 10-12的条件。而DBS手术是FDA批准的用于治疗帕金森氏症，肌张力障碍和原发性震颤，利用DBS用于治疗上面提到的其他条件都在实验室将提供很大希望给患者13,14的各个阶段和临床研究。 在临床上，DBS手术是在两个阶段中进行。使用放射性定位的组合的第一阶段包括通过外科手术定位DBS电极在所述靶向解剖结构的位置，CT，MRI以及微电极读数提高精度。第二阶段涉及植入在病人的胸部上的脉冲发生器和从头皮到脉冲发生器安装延伸引线。基于所述神经系统疾病，用于脉冲发生器编程几个方案已被标准化，并将被用来提供所需的电压。最终卷塔格达到以逐步的方式，以便接收与 ​​最小电压15的最好的临床响应。然而，在我们的研究中，不像慢性DBS植入物应用于临床，为简单起见，我们已采取在我们的动物模型研究的一次性高频刺激（1小时）。 我们组的研究的一部分集中在研究使用DBS手术治疗难治性癫痫。采用高频电刺激立体定向手术方法已探索了许多人作为一种有效的选择治疗构成约30％的癫痫10,16,17的各个发病的医学难治性癫痫。小脑刺激靶向皮质表面以及深层小脑核已经使用在过去作为目标，以治疗癫痫10,18,19。此外，海马的刺激也已试过，但用不同的结果20,21。一些其它的研究DBS目标癫痫包括大脑皮质，丘脑，丘脑下核和迷走神经8。然而，从在过去的几年中的几项研究，结果如下，前丘脑核（ATN）已经成为最常见的DBS目标为癫痫的治疗10,22。建立在有关神经解剖学电路和从动物模型的研究结果，一些研究已经在治疗癫痫23-26集中的ATN的深部脑刺激的治疗效果。使ATN是边缘电路的一部分，并位于影响发作频率的大脑区域。所学专业哈马尼等人 ，已经测试ATN-DBS的疗效在毛果芸香碱诱发癫痫模型，发现双侧ATN刺激潜伏期延长的毛果芸香碱诱发的癫痫发作和癫痫持续状态24。此外，ATN的高频刺激发现，以减少在戊四氮（PTZ）模型EP的发作频率ilepsy 25,27-29。 Lee 等人 ，已经报道经ATN慢性脑深部刺激的平均降低发作频率由约75％在治疗顽固性部分癫痫30。 在治疗难治性癫痫最近的临床研究表明DBS手术后有希望的结果针对前丘脑核（ATN）22。多中心的随机临床试验110例患者接受了ATN的双边DBS治疗难治性癫痫（SANTE试行）表示癫痫发作频率下降了约40％31。从本研究的结果还暗示上在2-3个月后的手术观察延迟最优抗癫痫作用。户田等人的进一步研究。，证实与这些发现，他们表现出神经发生发生在稍后的时间交 ​​的DBS（3-5天）在动物模型1。此外，恩西纳斯。等，已经报道海马neurogene矽统后的ATN 32的高频刺激成年小鼠海马齿状回。以前的研究33-35报道下滑在某些情况下，癫痫海马神经发生，如慢性颞叶癫痫和学习赤字的关联，记忆障碍和自发的反复运动性发作。另外，还有在神经干细胞祖因素如FGF2和IGF-1中的动物模型33长期痫海马的降低。考虑到这一点，介入策略，如星展，显示神经发生在齿状回的增强是令人兴奋的途径进行研究。这些发现促使我们进一步深入探索到底层机制的神经发生后DBS治疗癫痫。我们有针对性的ATN单方面双方（未报告数据），以及双边（有代表性的结果），看到高架神经营养因子（BDNF）的表达在大鼠海马齿状回。我们的C光凭目前的假设是，BDNF表达启动基因表达的级联高潮在神经发生该转换为DBS手术的抗癫痫作用。在本文中，我们提出我们的方法DBS手术针对ATN大鼠其次是基因表达分析作为一个有吸引力的方法来研究DBS的好处背后的机制。

<table><tbody><tr><td>Deep Brain Stimulation Surgery</td><td></td><td></td><td></td></tr><tr><td>Stereotactic frame</td><td>Kopf Instruments</td><td>Model 900</td><td></td></tr><tr><td>Drill&amp;nbsp;</td><td>Dremmel</td><td>7700, 7.2 V</td><td></td></tr><tr><td>Scalpel</td><td>BD</td><td>372610</td><td></td></tr><tr><td>Ketamine</td><td>Patterson Veterinary</td><td>07-803-6637</td><td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td></tr><tr><td>Xylazine</td><td>Patterson Veterinary</td><td>07-808-1947</td><td></td></tr><tr><td>Buprenorphine</td><td>Patterson Veterinary</td><td>07-850-2280</td><td>Schedule III Controlled Substance, procurement, use and storage according to institutional rules</td></tr><tr><td>Surgical staples</td><td>ConMed Corporation</td><td>8035</td><td></td></tr><tr><td>Sutures (3-0)&amp;nbsp;</td><td>Harvard Apparatus</td><td>72-3333</td><td></td></tr><tr><td>Syringe (1 ml, 29 1/2 G)</td><td>BD</td><td>329464</td><td>Sterile, use for Anesthesia administration intraperitoneally</td></tr><tr><td>Syringe (3 ml, 25 G)</td><td>BD</td><td>309570</td><td>Sterile, use for Analgesia administration subcutaneously</td></tr><tr><td>Needles</td><td>BD</td><td>305761</td><td>Sterile, use for clearing broken bone pieces from the burr holes</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>S25309B</td><td>Use for general sterilization&amp;nbsp;</td></tr><tr><td>Eye Lubricant</td><td>Fisher Scientific</td><td>19-898-350</td><td></td></tr><tr><td>Stimulator</td><td>Medtronic</td><td>Model 3628</td><td></td></tr><tr><td>DBS electrodes</td><td>Rhodes Medical Instruments, CA</td><td>SNEX100x-100 mm</td><td>Electrodes are platinum, concentric and bipolar</td></tr><tr><td>Betadine (Povidone-Iodine)&amp;nbsp;</td><td>PDI</td><td>S23125</td><td>Single use swabsticks, use for sterilizing the scalp before making incision&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Brain Dissection and Hippocampal tissue isolation</td><td></td><td></td><td></td></tr><tr><td>Acrylic Rodent Brain Matrix</td><td>Electron Microscopy Sciences</td><td>175-300</td><td>www.emsdiasum.com</td></tr><tr><td>Razor Blade</td><td>V W R</td><td>55411-050</td><td></td></tr><tr><td>Guillotine Scissors</td><td>Clauss</td><td>18039</td><td>For decapitation, make sure these scissors are maintained in clean and working condition</td></tr><tr><td>Scissors</td><td>Codman Classic</td><td>34-4098</td><td>Use for removing the brain from the skull</td></tr><tr><td>Forceps</td><td>Electron Microscopy Sciences</td><td>72957-06</td><td>Use for removing the brain from the skull and for handling during dissection</td></tr><tr><td>Phosphate Buffered Saline&amp;nbsp;</td><td>Boston Bioproducts</td><td>BM-220</td><td></td></tr><tr><td>&amp;nbsp;RNA Extraction and cDNA Preparation</td><td></td><td></td><td></td></tr><tr><td>Tri Reagent</td><td>Sigma</td><td>T9424</td><td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td></tr><tr><td>Syringe (3 ml, 25 G)</td><td>BD</td><td>309570</td><td>Use for tissue homogenization</td></tr><tr><td>Chloroform</td><td>Fisher Scientific</td><td>BP1145-1</td><td>Always use in a fume hood and wear protective goggles while handling; avoid contact with skin</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A416-1</td><td></td></tr><tr><td>Glycogen</td><td>Thermo Scientific</td><td>R0561</td><td></td></tr><tr><td>Dnase I Kit</td><td>Ambion</td><td>AM1906</td><td></td></tr><tr><td>Superscript First Strand Synthesis Kit</td><td>Invitrogen</td><td>11904-018</td><td></td></tr><tr><td>Tabletop Microcentrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>&amp;nbsp;Quantitative PCR</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>SYBR Green PCR Kit</td><td>Qiagen</td><td>204143</td><td></td></tr><tr><td>Custom Oligos</td><td>Invitrogen</td><td>10668051</td><td></td></tr><tr><td>PCR Plates (96 wells)</td><td>Denville Scientific</td><td>C18080-10</td><td></td></tr><tr><td>Optical Adhesive Sheets</td><td>Thermo Scientific</td><td>AB1170</td><td></td></tr><tr><td>Nuclease free Water</td><td>Thermo Scientific</td><td>SH30538-02</td><td></td></tr><tr><td>Real Time PCR Machine</td><td>Applied Biosystems</td><td>7500</td><td></td></tr></tbody></table>

analysis of gene expression changes in the rat hippocampus after deep brain stimulation of the anterior thalamic nucleus

深部脑刺激（DBS）的手术，靶向脑的各个区域，例如基底神经节，丘脑和丘脑底区域，是一种有效的治疗该未能对药物响应的几个运动障碍。在DBS外科领域的最新进展已经开始扩展这个手术技术的其他条件等不同的病态肥胖，抑郁症和强迫症的应用。尽管有这些迹象不断扩大，鲜为人知的是，潜在的生理机制，以促进DBS手术的有利影响。一种方法这个问题是在接受电刺激的神经元进行基因表达分析。以前的研究已经表明，神经发生在大鼠海马齿状回被引发在DBS丘脑1的前核的定位。 DBS手术针对ATN被广泛用于治疗难治性癫痫。因此，这是多大的INTERES吨为我们探索诱发电刺激ATN的转录变化。在这个手稿中，我们描述了立体定位引导下DBS手术针对ATN成年雄性Wistar大鼠我们的方法。我们还讨论了组织解剖，RNA分离，cDNA的制备和定量RT-PCR的随后步骤，用于测量基因表达的改变。这种方法可用于修改用于刺激基底节和脑的其它区域通常临床目标。这里所描述的基因表达研究假设的发现分子球员可以指挥机制DBS一个候选目标基因的方法。

图1A和1B示出BDNF和GABRD相对于对照基因β肌动蛋白的相对表达。 BDNF，神经营养蛋白经常与在许多神经元疾病38-41神经保护作用相关联。因此，有趣的分析响应于它产生于癫痫患者的治疗益处的ATN刺激BDNF的表达谱。在图1A中示出了其中的BDNF横跨指示时间点的基因表达图谱发表的DBS刺激，BDNF上调立即（0小时）的DBS手术后第3小时观察到随着峰值表达式（3?...

Watch this Scientific Journal Video about Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus at JoVE.com

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

analysis-gene-expression-changes-rat-hippocampus-after-deep-brain

Research

JoVE Journal

Neuroscience

10.9K 次观看.  Brigham & Women's Hospital, Harvard Medical School. The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

视频: Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Intra-Operative Behavioral Tasks in Awake Humans Undergoing Deep Brain Stimulation Surgery

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through implanted electrodes. Deep brain stimulation was first implemented as a therapeutic modality by Benabid et al. in the late 1980s, when he used this technique to stimulate the ventral intermediate nucleus of the thalamus for the treatment of tremor 1. Currently, the procedure is used to treat patients who fail to respond adequately to medical management for diseases such as Parkinson's, dystonia, and essential tremor. The efficacy of this procedure for the treatment of Parkinson's disease has been demonstrated in well-powered, randomized controlled trials 2. Presently, the U.S. Food and Drug Administration has approved DBS as a treatment for patients with medically refractory essential tremor, Parkinson's disease, and dystonia. Additionally, DBS is currently being evaluated for the treatment of other psychiatric and neurological disorders, such as obsessive compulsive disorder, major depressive disorder, and epilepsy. 
DBS has not only been shown to help people by improving their quality of life, it also provides researchers with the unique opportunity to study and understand the human brain. Microelectrode recordings are routinely performed during DBS surgery in order to enhance the precision of anatomical targeting. Firing patterns of individual neurons can therefore be recorded while the subject performs a behavioral task. Early studies using these data focused on descriptive aspects, including firing and burst rates, and frequency modulation 3. More recent studies have focused on cognitive aspects of behavior in relation to neuronal activity 4,5. This article will provide a description of the intra-operative methods used to perform behavioral tasks and record neuronal data with awake patients during DBS cases. Our exposition of the process of acquiring electrophysiological data will illuminate the current scope and limitations of intra-operative human experiments.

Deep brain stimulation surgery offers a unique opportunity to examine information encoding in the awake human brain. This article will describe intra-operative methods used to perform cognitive and behavioral tasks while simultaneously acquiring physiological data such as EMG, single-unit neuronal activity and/or local field potentials.

在接受深部脑刺激手术术中唤醒人类行为任务

Deep brain stimulation (DBS) is a surgical procedure that directs chronic, high frequency electrical stimulation to specific targets in the brain through ...

科研

医学

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

一种新的方法，以评估在帕金森病大鼠脑深部电刺激效应的运动结果:楼梯和缸测试

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

行为学

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

重复经颅磁刺激对大鼠大脑的单边半球

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

神经科学

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

高频刺激激活小鼠齿状回的侵袭方法

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Transcranial Direct Current Stimulation (tDCS) in Mice

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for several neuropsychiatric diseases. The biological effects of tDCS are not fully understood, which is in part explained due to the difficulty in obtaining human brain tissue. This protocol describes a tDCS mouse model that uses a chronically implanted electrode allowing the study of the long-lasting biological effects of tDCS. In this experimental model, tDCS changes the cortical gene expression and offers a prominent contribution to the understanding of the rationale for its therapeutic use.

Transcranial Direct Current Stimulation tDCS in Mice

Transcranial direct current stimulation (tDCS) is a therapeutic technique proposed to treat psychiatric diseases. An animal model is essential for understanding the specific biological alterations evoked by tDCS. This protocol describes a tDCS mouse model that uses a chronically implanted electrode.

经颅直流电刺激 (tDCS) 在小鼠中的作用

Transcranial direct current stimulation (tDCS) is a non-invasive neuromodulation technique proposed as an alternative or complementary treatment for ...