Name: an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation
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Description: Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

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。

动物实验程序遵循北京基础医学研究所 (北京, 中国) 的机构指南和中国政府对实验室动物的护理和使用规定。小鼠 (成年男性, 26 ~ 30 克) 被安置并且保持在恒定的温度23°c, 与水和食物ad 随意, 在12小时 light/12-h 黑暗的周期 (灯在上午7:00)。所有实验程序都是在光周期中进行的。
1. 外科准备
注意: 自定义电极是使用从 Halpern 的报告23中修改的方法自制的。
<ol><li>使用2铜微丝 (外径为200µm) 作为平行双极电极。将电极缠绕在一起, 形成圆柱电极进行刺激。 注: 在这里, 在 PP 或 DG 内的电极长度根据细胞核的解剖深度而变化。</li>
	<li>使用环氧乙烷消毒柜对电极进行杀菌。处理和储存自制的2通道微丝电极, 不受任何污染。在任何手术植入前都应严格消毒电极。</li>
	<li>高压釜手术器械。</li>
	<li>手术前, 用戊巴比妥 (20-50 毫克/千克) 腹腔注射小鼠, 以达到麻醉的手术平面。</li>
	<li>用无菌青霉素 G 普鲁卡因悬浮液 (7.5万 U, 即时链) 和镇痛剂 (卡洛芬, 0.05-1 毫克/千克, SC) 注射小鼠, 以减少感染和疼痛的风险, 分别为24。</li>
	<li>在动物麻醉后, 用眼睛润滑剂两眼, 以防止眼部干燥。确保诱导镇静和麻醉深度的动物的反射反应脚趾捏, 并进行手术时, 没有反应发生。 注意: 兽医软膏是防止眼部干燥的另一种选择。</li>
	<li>用电剪或剃刀刀片刮掉老鼠头顶部的大部分皮毛, 用三交替磨70% 酒精和 betadine 溶液消毒剃须部位, 并在 betadine 皮肤干燥后进行手术。</li>
</ol>2. 高频刺激手术25,26
注意: 在这个步骤中, 电极被单边植入到背 DG 或内侧 PP 区域, 这是海马体的一部分, 在情景学习和记忆中扮演重要角色。
<ol><li>将麻醉的鼠标放到立体定向的框架上。要保持鼠标的头部不动, 正确地对齐耳棒并快速安装它们。轻轻拧紧耳棒, 使其支持中心头, 并稍微保护鼠标。</li>
	<li>使用恒温垫, 以保持动物的体温在 37 0.5 摄氏度。</li>
	<li>使用镊子抓住老鼠耳朵前的皮肤, 用消毒剪刀剪掉, 以去除老鼠头骨顶部大约1厘米2的皮肤区域。 注: 少量出血可沿切口皮肤的边缘观察。</li>
	<li>用棉签去除头骨上的肌肉和其他附着物, 露出头骨表面。用棉签和解剖凿子完全清除颅骨表面的所有骨膜、肌腱和结缔组织, 以识别矢状和 lambda 缝合线。</li>
	<li>识别 bregma 和 lambda 点。冠状缝合和矢状缝合的交叉点在颅骨的上半部分是 bregma 的位置。lambdoid 缝线与矢状缝合的交点是可见的。使用 bregma 作为原点, 设置其他核位置。</li>
	<li>在头骨的 bregma 和 lambda 位置的识别之后, 在水平水平上调整这两个点。确保这两点的高度在背腹方向上几乎相同 (任何误差应 < 0.05 毫米)。另外, 请确保头骨在内侧侧向方向上的水平与26相同。</li>
	<li>对于单边手术, 将自制的2通道铜微丝电极安装在立体定向手术架上的旋转电极支架上。</li>
	<li>首先将电极垂直于 bregma 点上方, 并将其设置为原始位置。使用数字显示立体定向手术框架, 以表明前后 (AP), 内侧侧 (ML) 和 dorso 腹 (DV) 位置。 注意: 为了防止电极弯曲, 在手术过程中不要触摸电极尖端到头骨上。</li>
	<li>将电极轻轻移动到正确的位置 (图 1A, ap-2.1/毫升1.0 为 DG 和 AP-3.8/毫升 @ 3.0 为 PP, 分别)27以上的头骨没有任何接触。当所需的站点被确定为电极插入, 使用 dorsoventral 立体定向调整到更高的电极持有者, 并标记的位置与黑色标记。 注: 在参考立体定向坐标的确切位置可能会因小鼠的应变、体重和性别而异。同一性别的成年小鼠应该被用来最小化位置的任何变化。如果可能, 应使用预操作解剖扫描来识别单个主题基础27上的位置。</li>
	<li>使用手持机器人辅助钻头 (毛刺大小 = 0.5 毫米), 使毛刺孔在标记的位置精确。在钻孔前用70% 乙醇消毒毛刺尖端。沿 Z 轴 0.8-1.0 毫米移动小钻头穿过头骨, 而不改变其 X Y 位置。在手术过程中, 用无菌棉花止血。 注: 为了避免钻井过程中过度钻孔和过热, 确保钻井速度为每分钟 1.5万-1.8万转 (RPM), 并且每 8-十年代经常从毛刺孔中提取钻头。</li>
	<li>继续钻直到硬脑膜暴露出来。使用弯曲的针去除任何在钻探过程中产生的骨屑, 并在硬脑膜上做一个针孔, 而不会损害底层的软组织。 注意: 为了避免损伤脑组织, 弯曲钝针也可以用一个好的钝钳代替。</li>
	<li>为了确认毛刺孔已正确地在所需位置, 通过调整立体定向手术框架降低电极高度, 并确保电极可以顺利地通过毛刺孔插入, 而不触及任何障碍。如果是这样, 将电极缓慢地插入到1.8 毫米的深度为 dg (或3.0 毫米为 PP) 从头骨的表面位于中间 PP 到 dg (pp-dg)。 注: 在电极植入过程中, 用无菌棉擦拭毛刺孔中的任何脑脊液或血流出。</li>
	<li>将微丝电极插入颅骨孔后, 用小刮刀将其置于适当的牙科水泥处。随着水泥的加厚, 模具周围的插入电极和螺丝形成一个漂亮的平滑帽。Debride 和消毒伤口边缘。</li>
	<li>从电极支架上松开电极。将鼠标从立体定向的框架中移开, 并将其返回到温暖的笼子。 注意: 手术后, 等待动物回到笼子里, 直到完全从麻醉中恢复到足够的意识。</li>
	<li>让鼠标自由移动, 让它在笼子里恢复2天。然后, 将在鼠标大脑中植入的电极连接到可编程的刺激器通过引线。设置图 2中所示的软件接口。在400赫兹 (图 1B, 100 毫秒之间的列车之间, 二十年代之间的系列)28, 将痉挛参数设置为6系列6列的6个脉冲。将刺激强度设置为200μA。</li>
	<li>在所设定的时间段内提供痉挛。每天上午8:00 和8:00 提供刺激2x</li>
	<li>对于对照组, 在小鼠体内植入电极, 不进行刺激。</li>
	<li>对于c-fos染色, 刺激实验鼠敏锐 (每天 2x)。对于 BrdU 标记, 将实验小鼠分为急性刺激组 (每天应用 2x) 和一个慢性刺激组 (应用10x 在5天, 2x 每天)。在刺激过程中, 确保老鼠是清醒的。记住要确保引线没有扭曲。 注意: 在手术的最后一天, 急性刺激组的小鼠同时受到刺激。</li>
	<li>当刺激完成时, 小心地断开电极销与电极架和录音系统的连接器。</li>
</ol>3. 免疫荧光染色和 Bromodeoxyuridine 标记29
<ol><li>对于c-fos和 Notch1 染色, 在上一次 HF-星展集团刺激后约3小时, 麻醉小鼠注射戊巴比妥 (20-50 毫克/千克, ip), 以达到麻醉的手术平面。</li>
	<li>对于 BrdU 标记, 约12小时后, 最后 HF-星展集团刺激, 给予6注射 BrdU 的控制和受刺激的小鼠在2小时间隔 (50 毫克/千克0.9% 生理盐水, ip)。36小时后的最后一次注射, 麻醉小鼠注射戊巴比妥 (20-50 毫克/千克, ip), 以实现一个外科飞机。</li>
	<li>使用手术刀通过肋骨笼, 使切口暴露心脏, 然后通过一个22口径钝灌注针到左心室。在右心房做一个小切口。Transcardially 灌注它与50毫升冷磷酸盐缓冲盐水 (PBS) 跟随50毫升冷4% 多聚甲醛 (煤灰) 在 1x PBS (pH 7.4)。 注意: 当鼠标血液中的体内被替换并且流体运行清晰时, 将 PBS 切换到煤灰。良好的灌注可以通过肝脏的清除来判断。一旦观察到固定震颤30, 应停止灌注。</li>
	<li>用眼剪刀斩首老鼠, 在皮肤上切开, 露出头骨。用镊子, 微切头骨, 仔细解剖整个老鼠的大脑。后ﬁx 大脑在4% 多聚甲醛额外2天, 并将其转移到30% 蔗糖溶液在4摄氏度过夜。</li>
	<li>使用恒温器, 将大脑切成20µm 冠状部分。用刷子将部分转移到玻璃滑梯上, 并将其存储在-20 摄氏度。 注意: 有电极 misplacements 或过度机械损伤的动物将被排除在随后的分析中。</li>
	<li>将滑块转移到正常 PBS (0.1 米, pH 值 7.4), 不含固定剂。用 PBS 清洗切片 3-4x, 每5分钟取出多余的固定剂。对于 BrdU 标记组, 在37摄氏度的 2-N HCl 中孵育切片, 并在0.1 米硼酸盐缓冲液中冲洗 (pH 8.4; 10 分钟)。然后在 PBS 中清洗2x 片5分钟。 注意: 然后执行染色。</li>
	<li>在室温 (RT) 中, 以1% 正常山羊血清、3% 牛血清白蛋白 < BSA >、0.5% 的海卫 X-100 和0.02% 叠氮化钠在 1 X PBS 中的培养来治疗切片。</li>
	<li>用抗 c-fos 多克隆抗体 (1:500; 兔), anti-Notch1 抗体 (1:50; 山羊), 或抗 BrdU 抗体 (1:800; 鼠) 在4摄氏度的阻塞溶液中孵化.</li>
	<li>在 PBS 中冲洗3x 片, 然后用 alexa 568 共轭抗兔 (1:500)、alexa 氟488共轭抗山羊 (1:1、000)、或 alexa 氟488共轭抗鼠 (1:500) 二级抗体在 RT 中进行孵化。</li>
	<li>清洗样品3x 在 PBS (每次10分钟)。使用含有 DAPI 的防褪色试剂安装培养基覆盖免疫标记的大脑部分。让切片风干, 并保存在4摄氏度的光保护。</li>
	<li>用高分辨率多通道 (顺序) 扫描共聚焦显微镜从大脑的刺激和静态的侧面获取海马区的图像。在控件和实验组之间一致设置成像参数。</li>
	<li>使用高放大目标 (20X 空气目标, NA 0.45) 执行马赛克成像, 利用机动化阶段获取相邻视场的连续 3D (XYZ) 图像, 并利用适当的软件将它们缝合在一起, 使大型复合材料图像。 注: 平铺功能包括用于改进无缝图像的真实拼接和平滑选项。复合图像可以快速、方便地用拼接功能进行制备, 从而在很大范围内形成图像。</li>
	<li>对于c-fos和 Notch1 染色, 对缝合图像的免疫荧光密度21进行正核定量分析。随机选择3个区域在延髓, 背部和腹侧海马 DG 分区域, 并以双盲方式测量免疫荧光密度使用图像 J软件 (http://rsb.info.nih.gov/ij/)31。</li>
	<li>对于 BrdU 标记, 通过双盲方式分析在钻点周围通过背海马 (-1.7 毫米到-2.7 毫米相对于 bregma) 的切片。</li>
	<li>仅计数 DG 中的 BrdU 阳性神经元。在计数中包括位于颗粒细胞2细胞直径内的细胞。 注意: 在这里, 计数是使用40X 的空气目标进行的。随机地计算每只动物的 3-5 个部分, 计数是平均的, 并表示为每个 DG19的平均值。</li>
</ol>

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作者没有什么要申报的。

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2,34。虽然 HF-星展集团应该带来显著的好处, 刺激副作用可能发生, 即使在大脑的一个完美的位置, 如情绪和思维的负面变化,等等, 这是可逆的, 可以避免修改刺激设置。
除了在过去数十年的临床试验中积累的 hf 星展技术的重大进展外, 对活体动物的 hf-星展集团的研究也提供了了解生理变化和神经生物学的机会。电刺激引起的机制。在这篇手稿中, 我们描述了一个改良的 HF-星展集团方法在小鼠进行刺激海马 DG 分区域直接或间接痉挛。虽然描述的协议是在啮齿类动物中进行的, 但在其他模型生物 (包括猪35) 中也成功地进行了痉挛反应研究。对于这种技术, 立体定向手术也可以用于潜在的核靶向刺激, 以测试的效果, 痉挛使用某些动物模型的神经和精神疾病。从这些研究得出的结论应该容易地转化为诊所, 特别是为了改善现有目标的痉挛。请注意, 在本方法研究中, 与 HF-星展集团在临床上多次暴露的系列相比, 我们选择了一次性和短期的痉挛暴露, 刺激和诱发海马 DG 的神经元活动, 参数和范式的一部分以前报告的痉挛在海马切片21,28中诱发了 LTP。
HF-DBS 的一般限制在其他地方被广泛地审查了24,25。这里的实验的挑战之一是需要一个优秀的外科技术和持续护理的外科解剖轨迹。为了正确准确地将电极植入靶核进行刺激, 关键过程之一是由熟练的技师成功地进行手术, 以避免不必要的血管损伤和电极的错位。此外, 还需要对脑标本进行进一步的解剖和组织学分析, 以确定痉挛后电极的正确靶向。这也是这一方法的优点之一, 以清楚地显示纠正靶向电极后, 在一个完整的动物大脑操作。本协议中提供的许多细节可以很容易地适应长期刺激通过在动物体内植入的刺激器。应该指出的是, 它也可以替代刺激参数的潜在治疗脑目标与不同的痉挛分娩模式。然而, 本文报告了一个单一单位急性痉挛提供的可编程刺激单元, 以了解细胞变化后, 电刺激。
虽然 HF-DBS 作为治疗神经系统和精神疾病的替代选择, 在临床上的几年, 其有效性的机制仍然缺乏了解。考虑到 HF-DBS 的使用和未来的改进, 需要更务实的方法。作为一种发展的工具, 痉挛技术需要进一步的技术创新和机械发现。为了研究 hf-星展集团的细胞和分子机制, 基于高通量测序技术的全球转录分析将为了解 hf-dbs 诱导后的分子事件和信号变化提供重要信息。36. 除了无偏见的筛查策略外, 根据已证明对神经元退极化反应的现有分子候选者的检查, 它可能还会提供重要的线索来识别关键的效应因素。负责痉挛。c-fos是针对固有的神经元活动上调的, 因此, 看它的表达式至少可以为我们提供最近37发射的神经元的景观。但是, c-fos不仅应用于仅反映活动, 因为它还会标记正在进行 LTP37的神经元。以前的著作, 包括我们的, 已经证明 Notch1 在成熟的海马神经元突触可塑性中起着关键的作用, 对成人神经发生在体内21,22至关重要。在这种情况下, 关键候选者的详细定量和时空定位应被仔细描述, 并通过对c-fos表达式的免疫组化分析来说明前和后痉挛诱导。这样的研究将提供直接的证据来证明不同脑区基因表达模式的变化, 以应对痉挛的21, 这也有利于揭示细胞的变化, 如神经元活化,神经发生, 或变性。
还应指出的是, 尽管在临床上有一个 HF-星展卫星应用的优点, 由于刺激效果的结果与大脑中的刺激参数和目标位置紧密相关, 细胞和分子痉挛的影响机制可能是非常复杂的。在本研究中, 我们进一步进行了 BrdU 标记, 以研究痉挛对成人神经发生的急性和慢性影响。尽管 BrdU 染色可以作为神经发生和 gliogenesis 的标志, 但根据以前的报告, 在高频星展细胞治疗后 SGZ 观察到的 BrdU 阳性神经元的大部分增加是增生成人神经祖细胞21,38,39. 无论痉挛治疗的机制是否复杂, 我们在本手稿中描述的方法和结果都直接证明了重复高频电刺激激活海马 DG可以显著诱发神经元活动和神经发生的激活在体内。
总之, 我们证明, hf-星展集团可以改善小鼠的神经活动和神经发生, 与没有接受 HF 星展的控制小鼠的神经元活动和神经发生相比较。这可能提高对痉挛性体内的认知效应和刺激模式的科学理解。急性 HF-星展集团直接或间接治疗后神经元活化的鉴定表明, 痉挛治疗对局部和远程连接的突触传递有显著影响. 在 HF-星展治疗后神经元活化的快速诱导, 提供了一个强大的工具来操纵受损的突触传播或同步在许多神经和精神疾病, 如 MDD。另一方面, 在慢性和直接的, 但不是急性或间接的 HF-星展治疗后发生神经发生的诱导表明, 不同的电刺激方式之间的显著疗效和功能变异性。这些发现为神经调节和未来痉挛的潜在治疗效果提供了直接证据。综合而言, HF-星展集团将为治疗神经和精神疾病提供巨大优势, 方法是将临床上取得的技术进步和实验室的机械进展结合起来。

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。

<table><tbody><tr><td>Brain stereotaxic instrument</td><td>Stoelting</td><td>51730D</td><td>Stereotactic intracranial implantation for mouse</td></tr><tr><td>Stimulator</td><td>A-M systems</td><td>Model 3800</td><td>MultiStim 8-Channel programmable stimulator</td></tr><tr><td>Dental driller</td><td>Saeshin Precision Co., Ltd</td><td>STRONG 90</td><td>For drilling and crainiotomy&amp;nbsp;</td></tr><tr><td>Burr</td><td>Meisinger</td><td>HM1 005#</td><td>For drilling and crainiotomy&amp;nbsp;</td></tr><tr><td>Digidata 1550 Digitizer</td><td>Molecular Devices</td><td>AXON 1550</td><td>High-resolution data acquisition</td></tr><tr><td>Cryotome</td><td>Thermo Fisher Scientific</td><td>Thermo Cryotome FSE</td><td>Cutting frozen sections of specimens</td></tr><tr><td>Confocal microscope</td><td>Olympus</td><td>FV-1200</td><td>Japan, with 20x Objective (NA 0.45)</td></tr><tr><td>Mouse surgery tools</td><td>F.S.T.</td><td>14084-08,11254-20,16109-14</td><td>Scissors, forceps, bone cutter, holders etc.</td></tr><tr><td>Pentobarbital sodium</td><td>R&amp;amp;D systems</td><td>4579</td><td>20-50mg/kg for i.p. injection</td></tr><tr><td>Penicillin G&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3032</td><td>75,000 U for i.m. injection</td></tr><tr><td>Carprofen</td><td>Sigma-Aldrich</td><td>SML1713</td><td>5-10mg/kg, for s.c. injection</td></tr><tr><td>4% Paraformaldehyde (PFA)</td><td>Beijing Solarbio Sci-Tech Co.&amp;nbsp;</td><td>P1110</td><td>stocking solution for tissue fixation</td></tr><tr><td>Phosphate buffer (PBS)</td><td>Invitrogen</td><td>10010023</td><td>pH7.4, 500ml in stocking</td></tr><tr><td>Tissue-Tek O.C.T. compound</td><td>Sakura</td><td>4583</td><td>Formulation of water-soluble glycols and resins</td></tr><tr><td>anti-BrdU antibody</td><td>Abcam</td><td>ab6326</td><td>Dilutions:1/800</td></tr><tr><td>anti-c-fos antibody</td><td>Abcam</td><td>ab209794</td><td>Dilutions:1/500</td></tr><tr><td>Goat Anti-Rabbit IgG (Alexa Fluor 568)</td><td>Thermo Fisher Scientific</td><td>A11036</td><td>Dilutions:1/500</td></tr><tr><td>Donkey Anti-Rat IgG (Alexa Fluor 488)</td><td>Jackson ImmunoResearch</td><td>712-546-150</td><td>Dilutions:1/500</td></tr><tr><td>Antifade mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>Counterstaining with DAPI</td></tr><tr><td>anti-Notch1 antibody (C-20)</td><td>Santa Cruz Biotech</td><td>sc-6014</td><td>Dilutions:1/50</td></tr><tr><td>Donkey Anti-Goat IgG (Alexa Fluor 488)</td><td>Abcam</td><td>ab150073</td><td>Dilutions:1/1000</td></tr></tbody></table>

an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation

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。

继 HF-星展集团对海马 DG 分区域直接或 PP 分区域的刺激后, 通过立体定向调整将 DG 间接的通过插入电极激活, 这些啮齿动物被戊巴比妥麻醉并取样3小时。在最后的 HF-星展集团刺激后的c-fos和 Notch1 染色。对于 BrdU 染色, 36 小时后的最后 BrdU 注射后1天或5天的 HF-星展集团的刺激, 啮齿类动物被麻醉的戊巴比妥用于准备大脑部分。图 3显示了在 DG 的同侧一侧, c-fos的表达式明显增加。此外, 在 DG 的对侧一侧的c-fos表达式也上调与无 HF-星展集团刺激控制相比, 它们几乎没有显示c-fos信号的表达。基于 Notch1-specific 免疫荧光染色的脑切片, 如补充图 1所示, 我们进一步证明, Notch1 信号也可以激活后, 成年海马 DG 的痉挛刺激诱导在 PP 中神经元的退极化 (也指作为补充文件 1提供的原始数据)。
除了在 DG 分区域进行直接的 HF-星展集团的刺激外,图 4A还显示了电极尖端的位置和 hf-星展网刺激点, 这是安装在内侧 PP 中的.图 4B显示 HF-dbs在 PP 中的刺激不仅显著提高了 dg 同侧一侧的c-fos表达式的水平, 而且增加了在 dg 侧侧的 c-fos 的表达, 而非 HF-星展集团的刺激 控制。
基于对脑切片的 BrdU 标记分析, 如图 5A-5C所示, 我们进一步证明, 在直接对 DG 进行慢性 (5 天) HF-DBS 刺激后, 成人海马 DG 也可以激活神经发生。慢性 hf-星展集团在 DG 的刺激不仅显著上调的神经发生在同侧, 但也增加了与非 HF-星展集团刺激控制的对侧神经发生。然而, 急性 (1 天) 刺激未能诱发神经发生的上调在同侧或在侧侧的 DG。如图 5D中所示, 无论是在急性刺激组中还是在慢性刺激组中, PP 中的间接 HF-DBS 都不能改变海马 DG 的神经发生水平。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57857/57857fig1.jpg"> 图 1.电极尖端和痉挛参数的脑靶的示意图表示.(A) 表示内侧 perforant 路径到齿状回的下叶片的神经元投射。入的电极 ( 紫色 ) 的位置被表明直接地刺激 DG 或 PP 分区域。(B) 痉挛参数被表示为6系列6列6脉冲在400赫兹, 与100毫秒之间的列车和二十年代之间的系列。钙 = Ammonis;有齿状的回;PP = perforant 路径。<a href="/files/ftp_upload/57857/57857fig1large.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/57857/57857fig2.jpg"> 图 2.multistimulator 的接口和参数。该刺激器连接到数字模拟转换程序, 并由专有软件激活。刺激类型的痉挛是单相。<a href="/files/ftp_upload/57857/57857fig2large.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/57857/57857fig3.jpg"> 图 3.神经元活化在 DG 反应的 HF-星展集团直接治疗.(A) 电极尖端 (白色未填充盒) 直接位于 DG 区域的上方 (AP-2.1/毫升±1.0/DV + 1.8), 有或没有痉挛。此面板显示了脑切片冠状部分的染色, 其中c-fos抗体在控制和痉挛组。DAPI 蓝染色用于指示海马核的位置。刻度条 = 100 µm (B) 此面板显示了对免疫荧光信号密度的定量分析。在 dg 的同侧一侧, 神经元以高表达式c-fos显着激活, 而 dg 的对侧侧的c-fos表达式也相对增加, 与c-fos表达式控制小鼠没有痉挛治疗。意味着 SEM, 单向方差分析, F224 = 216, P < 0.0001, Bonferroni后端测试, ***P < 0.001, n无性病= 9 (3 切片从3小鼠), n痉挛抵消 = 9 (3 切片/鼠标从3小鼠), n痉挛 IPS = 9 (3 切片/鼠标从3小鼠)。<a href="/files/ftp_upload/57857/57857fig3large.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/57857/57857fig4.jpg"> 图 4.神经元在 DG 中激活, 以响应 PP 分区域的痉挛.(A) 电极尖端 (白色未填充盒) 位于中间 PP 位置的 AP-3.8, ML ±3.0, 和 DV + 3.0, 有或没有痉挛。(B) 此面板显示脑切片的冠状部分的染色, 其中c-fos抗体在控制和痉挛组。DAPI 蓝染色用于指示海马核的位置。刻度条 = 100 µm (C) 此面板显示了对免疫荧光密度的定量分析。在 PP 中的痉挛治疗可以显著地激活 DG 中的同侧神经元, 并具有较高的c-fos表达。与对照组小鼠的 c-fos表达相比, 对侧 DG 的 c-fos 表达式也相对增加, 而没有痉挛治疗。意味着 SEM, 单向方差分析, F224 = 189.3, P < 0.0001, Bonferroni后端测试, ***P < 0.001, n无性病= 9 (3 切片从3小鼠), n痉挛抵消 = 9 (3 切片/鼠标从3小鼠), n痉挛 IPS = 9 (3 切片/鼠标从3小鼠)。<a href="/files/ftp_upload/57857/57857fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57857/57857fig5.jpg"> 图 5.在 dg 和 PP 的痉挛反应中, dg 中神经发生的明显活化.(A) 此面板显示了在 DG 中进行痉挛治疗和生成 BrdU 标记增殖神经元的实验范式。这些动物被分为急性和慢性组。在急性组, 小鼠在5天受到痉挛治疗2x。在慢性组, 小鼠受痉挛治疗5天 (从1天到每天 5, 2x 每天)。然后, 两组动物在6天注射了 BrdU (50 毫克/千克, 6x 2 小时间隔)。在8天, 动物被麻醉和灌流, 以准备恒温器切片。(B) 此面板显示了 BrdU (绿色) 后 HF-星展集团在 DG 中的免疫荧光染色。(C) 对神经元产量的定量分析表明, 急性 (1 天) 的刺激在 dg 中不能增强神经发生, 而慢性 (10x 在5天内) 刺激的 dg 显著上调神经发生。与对照组相比, 同侧半球的慢性组 BrdU 标记细胞数量显著增加。刻度条 = 100 µm. 双向方差分析, 刺激类型效应 f2, 14 = 97.09, P < 0.0001, 半球效应 f1, 14 = 1.137, P = 0.3044, Bonferroni后临时测试, ***P < 0.001, n控件= 3 (3 切片从3小鼠), n1 天= 4 (4 切片从3小鼠), n5 天= 3 (3 切片从3小鼠)。(D) 此面板显示了 BrdU (在绿色) 后 HF-星展集团在 PP 分区域的免疫荧光染色。(E) 对神经元生产的定量分析表明, 无论是急性 (一天两次) 还是慢性 (5 天中的 10x) 刺激, 都不能显著增强神经发生。BrdU 标记细胞数量相对稳定, 或无痉挛治疗。刻度条 = 100 µm. 方法是: 扫描电镜, 双向方差分析, 刺激类型效应 F2, 16 = 0.9025, P = 0.4252, 半球效应 f1, 16 = 1.402, p = 0.2537, Bonferroni后临时测试, P > 0.05, n控件= 3 (3 切片从3小鼠), n1 天= 3 (3 切片从3小鼠), n5 天= 5 (5 切片从3小鼠)。<a href="/files/ftp_upload/57857/57857fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary Figure 1" src="/files/ftp_upload/57857/57857supfig1.jpg"> 辅助图1。Notch1 在 DG 中的表达对 HF-星展治疗的反应.(A) 此面板显示了 DG 中 HF-星展集团后 Notch1 (绿色) 3 小时的免疫荧光染色。此数字是从冯的报告21中引用和修改的。(B) 对荧光的定量分析表明, 在 DG 中的痉挛可以增强 Notch1 的表达。刻度条 = 100 µm. 指电子显微镜, 学生t-测试, t = 12, ***P < 0.001, n无性病= 9 (3 切片/鼠标从3小鼠), n痉挛= 9 (3 切片/鼠标从3小鼠)。<a href="/files/ftp_upload/57857/57857supfig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplementary File 1" src="/files/ftp_upload/57857/57857supfig2.jpg"> 辅助文件1。原始数据.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57857/Supplementary_raw_data_revised.xlsx" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation at JoVE.com

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

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。

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 ...

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Universidad San Sebastian

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JoVE Journal

Neuroscience

7.7K 次观看.  Beijing Institute of Basic Medical Sciences. 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。

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

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential cellular mechanism underlying learning and information storage, have long been used to elucidate the physiology of various neuronal circuits in the hippocampus, amygdala, and other limbic and cortical structures. With this in mind, transgenic mouse models of neurological diseases represent useful platforms to conduct long-term&#160;potentiation (LTP) studies to develop a greater understanding of the role of genes in normal and abnormal synaptic communication in neuronal networks involved in learning, emotion and information processing. This article describes methodologies for reliably inducing LTP in the freely behaving mouse. These methodologies can be used in studies of transgenic and knockout freely&#160;behaving mouse models of neurodegenerative diseases.

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of neuropathology.

穿通通路 - 齿状回突触在自由活动的小鼠长时程增强

Studies of long-term potentiation of synaptic efficacy, an activity-dependent synaptic phenomenon having properties that make it attractive as a potential ...

科研

行为学

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

神经科学

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

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 ...

Non-invasive Strategies for Chronic Manipulation of DREADD-controlled Neuronal Activity

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by designer drugs (DREADDs) have become the most popular chemogenetic approach used in modern neuroscience. Most studies deliver the ligand clozapine-N-oxide (CNO) using a single intraperitoneal injection, which is suitable for the acute activation/inhibition of the targeted neuronal population. There are, however, only a few examples of strategies for chronic modulation of DREADD-controlled neurons, the majority of which rely on the use of delivery systems that require surgical intervention. Here, we expand on two non-invasive strategies for delivering the ligand CNO to chronically manipulate neural population in mice. CNO was administered either by using repetitive (daily) eye-drops, or chronically through the animal&#39;s drinking water. These non-invasive paradigms result in robust activation of the designer receptors that persisted throughout the CNO treatments. The methods described here offer alternatives for the chronic DREADD-mediated control of neuronal activity and may be useful for experiments designed to evaluate behavior in freely moving animals, focusing on less-invasive CNO delivery methods.

Here we describe two non-invasive methods to chronically control neuronal activity using chemogenetics in mice. Eye-drops were used to deliver clozapine-N-oxide (CNO) daily. We also describe two methods for prolonged administration of CNO in drinking water. These strategies for chronic neuronal control require minimal intervention reducing animals&#8217; stress.

DREADD控制神经活动慢性操作的非侵入性策略

Chemogenetic strategies have emerged as reliable tools for remote control of neuronal activity. Among these, designer receptors exclusively activated by ...

用低成本的 EEG 和毫米级线圈刺激小鼠大脑

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

作者聚焦: 低成本脑电图记录系统结合毫米级线圈经颅刺激小鼠体内大脑  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp (2018)
This video demonstrates a protocol to modulate theta oscillations in a genetically engineered mouse model with light-sensitive ion channels in its GABAergic medial septum neurons. By delivering light pulses to the hippocampus, the protocol aims to synchronize neuronal activity and generate theta oscillations, which are recorded as local field potentials.

小鼠模型中 Theta 振荡的选择性作

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

高频刺激激活小鼠齿状回的侵袭方法

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此视频中的章节

穿通通路 - 齿状回突触在自由活动的小鼠长时程增强

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

听觉神经的光遗传学刺激

Optogenetic 在行为小鼠海马θ振荡中的作用

经颅直流电刺激 (tDCS) 在小鼠中的作用

DREADD控制神经活动慢性操作的非侵入性策略

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

小鼠模型中 Theta 振荡的选择性作