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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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,</...

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 border="0" cellpadding="0" cellspacing="0" style="width:975px;" width="975">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">Brain stereotaxic instrument</td>
			<td style="width:215px;">Stoelting</td>
			<td style="width:199px;">51730D</td>
			<td style="width:307px;">Stereotactic intracranial implantation for mouse</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Stimulator</td>
			<td style="width:215px;">A-M systems</td>
			<td style="width:199px;">Model 3800</td>
			<td>MultiStim 8-Channel programmable stimulator</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Dental driller</td>
			<td style="width:215px;">Saeshin Precision Co., Ltd</td>
			<td style="width:199px;">STRONG 90</td>
			<td>For drilling and crainiotomy&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Burr</td>
			<td style="width:215px;">Meisinger</td>
			<td style="width:199px;">HM1 005#</td>
			<td>For drilling and crainiotomy&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:255px;">Digidata 1550 Digitizer</td>
			<td style="width:215px;">Molecular Devices</td>
			<td style="width:199px;">AXON 1550</td>
			<td>High-resolution data acquisition</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Cryotome</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">Thermo Cryotome FSE</td>
			<td>Cutting frozen sections of specimens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Confocal microscope</td>
			<td style="width:215px;">Olympus</td>
			<td style="width:199px;">FV-1200</td>
			<td>Japan, with 20x Objective (NA 0.45)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:255px;">Mouse surgery tools</td>
			<td style="width:215px;">F.S.T.</td>
			<td style="width:199px;">14084-08,11254-20,16109-14</td>
			<td style="width:307px;">Scissors, forceps, bone cutter, holders etc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Pentobarbital sodium</td>
			<td style="width:215px;">R&#38;D systems</td>
			<td style="width:199px;">4579</td>
			<td>20-50mg/kg for i.p. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin G&#160;</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">P3032</td>
			<td>75,000 U for i.m. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Carprofen</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:199px;">SML1713</td>
			<td>5-10mg/kg, for s.c. injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">4% Paraformaldehyde (PFA)</td>
			<td style="width:215px;">Beijing Solarbio Sci-Tech Co.&#160;</td>
			<td style="width:199px;">P1110</td>
			<td>stocking solution for tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate buffer (PBS)</td>
			<td style="width:215px;">Invitrogen</td>
			<td style="width:199px;">10010023</td>
			<td>pH7.4, 500ml in stocking</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue-Tek O.C.T. compound</td>
			<td style="width:215px;">Sakura</td>
			<td style="width:199px;">4583</td>
			<td>Formulation of water-soluble glycols and resins</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-BrdU antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab6326</td>
			<td>Dilutions:1/800</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-c-fos antibody</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">ab209794</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:255px;">Goat Anti-Rabbit IgG (Alexa Fluor 568)</td>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:199px;">A11036</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Donkey Anti-Rat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Jackson ImmunoResearch</td>
			<td style="width:199px;">712-546-150</td>
			<td>Dilutions:1/500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Antifade mounting medium with DAPI</td>
			<td style="width:215px;">Vector Laboratories</td>
			<td style="width:199px;">H-1200</td>
			<td>Counterstaining with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">anti-Notch1 antibody (C-20)</td>
			<td style="width:215px;">Santa Cruz Biotech</td>
			<td style="width:199px;">sc-6014</td>
			<td>Dilutions:1/50</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:255px;">Donkey Anti-Goat IgG (Alexa Fluor 488)</td>
			<td style="width:215px;">Abcam</td>
			<td style="width:199px;">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?...

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 ...

an-invasive-method-for-activation-mouse-dentate-gyrus-high-frequency

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.6K 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

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

生物学

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

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 ...

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) ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

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 ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

鼠脑水平海马片

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

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, ...

高频刺激激活小鼠齿状回的侵袭方法

摘要

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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

从成年小鼠海马齿状回的剖析

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Optogenetic 在行为小鼠海马θ振荡中的作用

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

行为小鼠海马空间受限振荡的记录

经颅直流电刺激 (tDCS) 在小鼠中的作用

鼠脑水平海马片

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