Name: optogenetic entrainment of hippocampal theta oscillations in behaving mice
Uploaded: 2018-06-29T13:30:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice at JoVE.com

我们要感谢玛丽亚 Gorbati 的专家帮助与数据分析和珍妮弗 Kupferman 评论的手稿。这项工作得到德意志 Forschungsgemeinschaft (DFG) 的支持;激动 257 NeuroCure、TK 和 AP;优先方案1665、1799/1-1 (2)、海森堡方案、1799/2-1、AP)、德国-以色列科研开发基金会 (GIF;I-1326-421.13/2015, TK) 和人类前沿科学计划 (HFSP;RGY0076/2012, TK)。

使用了59, 10-25 周大的光伏撞击雄性小鼠。老鼠被安置在动物设施的标准条件下, 并保持在12小时的光/暗循环。所有程序均按照国家和国际准则执行, 并经当地卫生当局批准 (Landesamt Natur、Umwelt 和 Verbraucherschutz、Nordrhein Westfalen)。
1. 病毒注射
<ol><li>在整个过程中, 遵循生物安全准则60。穿上实验室大衣、手术面罩、罩和两副手套。</li>
	<li>用无菌手术刀或剪刀将油管切开至约1米长, 将其插入泵的注射器座块中, 并加以修复。<ol>
<li>使用注射器完全用硅油填充油管。</li>
		<li>检查油管内是否出现气泡。如果观察到气泡, 再用油重新填充油管。</li>
		<li>将柱塞固定在推板上。</li>
		<li>慢慢地向前推进对注射器支架块, 直到顶端的活塞接触油管。</li>
		<li>将柱塞的尖端插入油管中, 然后通过在命令窗口中选择 "注入" 和低输液率 (例如, 500 nL/分钟), 直到到达注射器座块, 以低速的速度自动移动推板块。</li>
	</ol>
</li>
	<li>准备注射针 (34G)。<ol>
<li>使用连接到切削盘的精密钻头/磨床, 用清晰的切口拆卸针毂。如有必要, 使用针头从刚切割的表面去除金属残渣。</li>
		<li>螺纹毛细管 (3-4 厘米长) 通过针。</li>
		<li>用超级胶水将针头粘在油管上。</li>
		<li>将注射针连接到油管的末端, 连接到微量泵泵。</li>
	</ol>
</li>
	<li>将针连接到立体定向支架上。</li>
	<li>取出空气直到针上透明管上的气泡可见。</li>
	<li>麻醉 1.5-3% 异氟烷在氧中的老鼠。等待约2-3 分钟, 然后确认鼠标完全麻醉, 评估其对脚趾捏的反应。在整个手术监测动物的呼吸和调整异氟醚浓度, 如果需要的话。</li>
	<li>将鼠标放在立体定向的框架内, 使用无创伤的耳座。</li>
	<li>用脂质凝胶保护老鼠的眼睛。</li>
	<li>用0.01-1 毫升注射器和26G 注射套管在头部皮肤下注射0.1 毫升利多卡因 intradermally。</li>
	<li>用乙醇溶液对老鼠的头部进行剃须和消毒。然后用乙醇替代三乘以2% 的氯己定消毒手术部位。</li>
	<li>用锋利的剪刀从耳朵的后到眼睛的水平进行中线切口, 使 bregma 和 lambda 可见。</li>
	<li>用注射器涂上大约50µL 的氯化钠, 用纸巾和粉扑擦干, 清洁头骨。</li>
	<li>通过调整鼻钳的 dorso 腹位来定位鼠标头, 使 bregma 和 lambda 在相同的 dorso 腹面上 (0.3 毫米)。</li>
	<li>在参考 bregma 的情况下, 在 MS (AP 0.98 和 L 0.5 毫米) 上方钻孔。</li>
	<li>在这一点上, 解冻病毒的整除 (应该包含至少2µL 的AAV2/1.ChR2. tdTomato) 约5分钟室温 (RT)。</li>
	<li>离心机整除在大约 4000 x g 在 RT 为1分钟。</li>
	<li>将病毒的2µL 移到一块 Parafilm 上;使用以前保护的边。</li>
	<li>将注射针头的尖端浸入液体中, 并在观察液体水平的同时, 小心地提取约 500 nL/分钟。为防止空气吸痰, 在病毒完全被占去之前停止撤退。根据溶液黏度调整提取率;更快的速度可以帮助提取更高粘度的病毒。</li>
	<li>用纸巾清洁针头。</li>
	<li>检查病毒是否包含在管中, 病毒和油被气泡隔开。标记病毒在管上的水平, 以控制在注射过程中是否成功注入病毒。</li>
	<li>将针头放在开颅术上方, 在第一个注射点 (AP 0.98、L 0.5、V-5.2、5.5°侧) 慢慢插入大脑。</li>
	<li>注射 450 nl 的病毒, 速度为 100-150 nl/分钟. 等待10分钟. 小心地将针向上移动0.1 毫米, 再等待5分钟。<ol>
<li>将针头移动到第二个注射点 (AP 0.98, L 0.5, V-4.6), 然后在 100-150 nl/分钟内再注入 450 nl。</li>
		<li>在将针向上移动0.1 毫米前再等待10分钟. 在拔针前再等5分钟。</li>
	</ol>
</li>
	<li>用丝黑色编织缝线缝合切口, 使用方结。用红灯加热动物以加速恢复。管理抗生素 (0.3 毫升 Erycinum (1:4 不育氯化钠)) 和卡洛芬每天2-3 天的手术后。</li>
	<li>在指示病毒水平的标记上方切开管。该管可用于进一步注射。每次注射前都要准备一个新鲜的注射针。 注意: 这个手术大约需要 1-1 5 小时。动物通常在手术后5分钟内苏醒。在进行立体定向植入前, 等待6周的最短时间, 但不能超过5月, 因为在注射病毒后, 表达水平开始下降约6月。</li>
</ol>2. 光纤的制备 (图 1A)
<ol><li>采用多模光纤 (105 µm 芯, 玻璃包覆硅芯, 0.22 NA)。用微脱衣舞女将纤维芯的125µm 包覆, 而纤维仍附着在纤维线轴上。</li>
	<li>用金刚石刀将纤维切成大约2-3 厘米的长度。</li>
	<li>将纤维插入氧化锆陶瓷棒箍 (ID: 126 µm)。大约 0.5-1 毫米的光纤应该突出从凸边的箍。</li>
	<li>使用针头, 将一滴环氧树脂胶涂在套圈两端, 而不是插在套圈的两侧。或者, 使用超级胶水。</li>
	<li>允许胶水干至少30分钟。</li>
	<li>用金刚石研磨纤维抛光膜 (3 µm 磨粒) 抛光套圈凸边。</li>
	<li>使用光功率计测试光传输的保真度。<ol>
<li>将功率计上的波长设置为与所使用的激光器相同的波长。</li>
		<li>将修补程序线与传感器的中心位置对着。对激光进行通电, 并读取功率计的光输出。记录该值。</li>
		<li>通过联接套筒将光纤连接到贴片线上, 并将其定位到传感器中心所面对的光纤尖端。对激光进行通电, 并读取功率计的光输出。记录该值。</li>
		<li>计算传输速率: 将第二个值除以第一个值。如果传输速率低于 0.5, 则丢弃纤维, 否则将其用于植入。</li>
		<li>在植入前测试每种纤维的传输速率。</li>
		<li>为以后的实验, 调整激光的光强输出到光纤的传输速率: 设置从贴片线尖端到5-15 除以传输速率的光输出, 实现从光纤尖端的5-15 兆瓦的最终光输出。 注: 参阅参考61为光纤的制备。</li>
	</ol>
</li>
</ol>3. LFP 记录用钨线阵列的制备 (图 1B)
<ol><li>胶水几个 (例如 6) 100 µm 硅管导轨平行的粘着的一面胶带。切割一条, 大约4-6 毫米, 为一个导线阵列装配。</li>
	<li>螺纹 formvar 绝缘45µm 钨导线通过导管使用镊子。</li>
	<li>用手术刀刮掉两端的绝缘, 六层搪瓷绝缘的细铜线 (约5毫米长) 和接地线 (大约2-3 厘米长)。把它们焊到 nanoconnector 的针脚上。</li>
	<li>分别用一滴银导电漆将每根粘合线连接到一根钨丝。让干燥至少30分钟。</li>
	<li>用最小量的水泥盖住电线。不要将水泥涂在钨线上 , 这将入脑组织或 nanoconnector 的上部。让水泥干至少30分钟。</li>
	<li>使用钝式不锈钢剪刀进行钨线的角切口 (5-20°), 使其能可靠地在靠近地层 pyramidale 的区域腹部的下方或上方植入导线, 那里的θ振幅过低, 无法估计夹带逼真度。</li>
	<li>用手术刀刮掉绝缘物, Deinsulate (大约2毫米) 的地线。用通量和 presolder 治疗。</li>
	<li>使用数字万用表检查电极之间潜在的交叉对话。为此, 请将连接器针脚连接到万用表, 必须将其设置为电阻测量模式。检查通道的配对组合;在 5 MΩ以下的万用表上的读数表明了重要的交叉谈话。</li>
	<li>用阻抗计检查盐水中每根电极的阻抗。典型的阻抗值低于 100 kΩ。</li>
	<li>为了便于植入, 将一根光纤粘附在线阵列上, 使光纤尖端处于最短导线的水平, 纤维尖端靠近, 但不接触钨线。保持纤维的角度尽可能小, 以防止组织损伤在植入过程中。 注: 另见参考62 , 用于制作钨线阵列。</li>
</ol>4. 立体定向植入
<ol><li>执行步骤 1.6-1.13 中所述的准备工作。</li>
	<li>从头骨顶部取出结缔组织, 并将颈部肌肉彻底推至大约2毫米, 以防止在录音过程中出现肌肉制品。</li>
	<li>使用棉尖涂抹剂和生理盐水清洁头骨, 并钻4个孔 (2 在前面和2以上小脑, 0.8 毫米直径) 放置骨不锈钢螺钉 (00-96x1/16) 的地面和稳定的种植体 (图 1D)。将地面螺钉定位在小脑上方, 连接到一根铜线 (大约2-3 厘米长)。</li>
	<li>完全用水泥覆盖地面螺钉, 以防止肌肉制品在电生理记录中。建立一个连接所有螺钉的水泥环 (图 1E)。</li>
	<li>在植入侧 (海马, AP-1.94, L 1.4, V 1.4 参考 bregma) 上执行开颅手术。在脑组织表面应用大约5µL 不育氯化钠。</li>
	<li>用导航外科手术在开颅手术中慢慢降低线阵列。对于单一的录音, 植入硅胶探头而不是线阵列63;为了防止光电光工件, 在海马体中单独植入光纤, 而不直接面对探头的光纤尖端 (图 1C -I)。为研究两半球间夹带的协调性, 在对侧海马 CA1 区植入附加的光纤。</li>
	<li>应用大约5µL 的温液体蜡/石蜡油, 预热在70摄氏度, 用注射器在植入部位, 以保护脑组织。</li>
	<li>在线阵周围涂上水泥, 用水泥盖住头骨。</li>
	<li>将一滴通量应用于 presoldered 地线/参考线和与地面螺钉连接的 presoldered 线, 例如使用针, 并用焊锡机将导线熔断。</li>
	<li>用水泥覆盖整根地线。</li>
	<li>术后0.3 毫升 Erycinum (1:4 不育氯化钠) 和卡洛芬 (5 毫克/毫升), 至少两天以下。老鼠通常在手术后15分钟内醒来。用红灯加热动物以加速恢复。</li>
	<li>在手术后的第一周监视鼠标的重量, 或直到体重稳定。体重下降不应超过10% 的鼠标重量记录手术前。为了加速体重的稳定, 在手术后的第一天, 给老鼠提供湿食物和炼乳。</li>
	<li>在夹带过程中记录海马细胞活动, 在海马中植入一个硅胶探针 (AP-1.94, L 1.4, V 1, 随后降低), 如 ref 62所述 (图 1C -I)。将光纤植入海马的 3, L 1.4, V 1.6, 39°尾延髓。如果需要刺激细胞胞体, 则在 MS 中植入额外的光纤 (AP + 0.98, L 1, V 3.9, 外侧)。</li>
</ol>5. Optogenetic 刺激和电生理数据采集
<ol><li>将鼠标习惯到录制设置 (例如, 15 分钟的会话, 每天1-2 个会话3天)。在第一次实验开始前先检查动物的行为。如果鼠标在室内移动, 探索环境, 嗅探, 执行 rearings等, 启动第一个实验会话。</li>
	<li>把鼠标放在一个熟悉的房间里, 因为在同一间屋子里没有其他动物。</li>
	<li>用胶带将 LED 贴在头上, 以跟踪动物的位置。在开始实验之前, 请确保相机在整个周期内捕捉到了 LED 灯。记录在黑暗中, 以跟踪 LED 灯。在录音室上方放置一个摄像头。</li>
	<li>检查修补程序线的光源输出。根据光纤注入的传输速率, 估计连接后光纤尖端的光输出。确保从光纤尖端的光输出在5-15 兆瓦之间, 以实现可靠的夹带。</li>
	<li>将 headstage 前置放大器连接到长期植入的连接器。将纤条线连接到长期植入的海马纤维进行 optogenetic 刺激实验。在控制光刺激实验中, 将光纤连接到与耳机连接的虚拟插接。</li>
	<li>将鼠标放在录音室中。</li>
	<li>打开软件控制刺激生成器, 生成激励协议。</li>
	<li>选择控制 473 nm DPSS 激光器的通道。在第一行输入 3000 mV (第一栏), 时间30毫秒 (第二列), 值 0 mV (第三列), 时间112毫秒 (第四列), 行重复840和组重复 1, 以生成一个协议2分钟7赫兹刺激与 30 ms 长脉冲。如果需要其他频率或不同持续时间的刺激, 请调整第四列中的时间长短和行重复次数。在第二行选择0兆瓦 (第一栏), 时间 500 h (第二列), 行重复1和组重复 1, 以确保激光被关闭后, 刺激协议终止。</li>
	<li>单击 "文件" > "另存为", 并使用所需的名称保存文件。</li>
	<li>确保触发激光的刺激器 TTL 输出连接到数字山猫模拟输入板, 以同步获得的电生理和 optogenetic 数据。</li>
	<li>或者, 为了参数化地调节θ振荡频率的变异性, 在不同的脉冲间隔内应用光脉冲的训练, 并在高斯分布之后使用句点。修改不同协议之间脉冲间隔的色散,例如从步骤3.2 到15.1 毫秒2。应用这些协议生成θ频率的不同可变性的θ纪元 (图 6)。</li>
	<li>打开录音系统的软件。点击 "ACQ" 获取和 "录制" 记录。在启动光刺激之前等待记录基线行为 (例如, 2 分钟检索基线速度或30分钟提取基线位置字段)。</li>
	<li>打开软件来控制刺激发生器。单击 "文件" > "打开", 然后选择要选择的协议文件。点击 "下载并开始" 启动光刺激。 注: 实验可以进行调查, 并通过遥控启动刺激;这排除了实验者在场对动物行为的影响。根据研究的目的, 刺激可以在特定的行为过程中启动和终止。</li>
</ol>6. Optogenetic 夹带和投射特异抑制海马输出的组合方法
<ol><li>ChR2 GABAergic 细胞的表达, 如1节所述。</li>
	<li>此外, 共注入2.4 µL 的 CamKIIα依赖 halorhodopsin (eNpHR3.0, AAV2/1.CamKIIa eNpHR3.0 EYFP. 生长激素) 在两个背海马半球 (AP-1.7;L 1.05;V-2.05 和-1.4 毫米;AP-1.7;L 1.7;V-2.05 和-1.55 毫米;AP-2.3;L 1.5;V-2.2 和-1.3 毫米;AP-2.3;L 2.2;V-1.65 和-2.45 毫米)。</li>
	<li>允许6周的表达时间。</li>
	<li>在海马 CA1 区植入一根钨线阵列, 如4节所述。另外, 在横隔膜中植入双侧光纤 (LS、AP 0.1、L 0.25、V-2.25 毫米和 ap 0.5、l-0.3、V-2.7 毫米)。</li>
	<li>执行 optogenetic θ夹带实验, 如步骤 5.4-5.5 所述。<ol>
<li>为了同时抑制海马输出到 LS, 生成一个协议刺激生成:例如, 触发连接到 593 nm DPSS 激光器的输出通道的开始, 连续脉冲持续在四十五年代, 在触发之前脉冲输出到 473 nm DPSS 激光器。</li>
		<li>通过用多模光纤耦合器将 LS 中的两根光纤连接到 593 nm DPSS 激光器。</li>
		<li>启动录音并下载并触发协议以控制刺激生成。</li>
	</ol>
</li>
</ol>7. 数据处理
<ol><li>转换电生理信号并将跟踪数据与神经数据 (ND) 管理器定位到. dat 和. pos 格式, 分别为64。</li>
	<li>利用 ND 管理器66对宽带信号进行低通滤波和下采样, 获得 1250 Hz 的 LFP。</li>
	<li>在每个记录中, 选择具有θ振荡最大振幅 (使用 Neuroscope)19的信道。</li>
	<li>用阈值检测算法 (使用 MATLAB 函数 findpeaks 或相似) 检测激光脉冲的时间戳和激发时代的时间.11。</li>
	<li>要在多通道数据分析软件中导入. dat 文件, 请单击 "文件 > 导入", 选择 "二进制文件" 作为数据类型, 然后选择. dat 文件。在 "配置" 对话框中, 输入正确的通道数和采样速率 1250 Hz, 单击 "确定", 然后另存为. smr 文件。通过选择 "分析 > 新结果视图 > PowerSpectrum" 来绘制功率谱。在设置中, 选择具有最高θ振幅和 16384 FFT 大小的信道, 单击 "新建", 定义为 "开始时间" 刺激时代的开始, 并作为 "结束时间" 结束的刺激时代, 并单击 "进程"。</li>
	<li>计算夹带保真度为累计功率谱密度 (PSD) 在 optogenetic 刺激频率范围 (激励频率 0.5 Hz), 到θ (5-12 Hz) 带的累计 PSD 与多窗口方法 (NW = 3,窗口大小 8192) 为十年代世纪 (例如, 工具箱 < http://chronux.org/>)。</li>
	<li>排除从分析 (非θ时代) 占主导地位的 PSD 峰值为≤ 5 Hz 的记录时代 (使用 MATLAB 函数查找. m)。</li>
	<li>根据计算的夹带逼真度 (使用 MATLAB 函数 sortrows 和 pcolor), 绘制所有记录的纪元的 LFP 功率谱的光栅。通过单击 "文件 > 打开" 来加载功率谱和夹带逼真度, 存储变量 in. 类型电源 = sortrows (在, 1);pcolor (力量 (:, 2: 末端))。</li>
</ol>

作者没有什么可透露的。

在这里, 我们提出了一个广泛的方法, 以 entrain 和诱发海马θ振荡的行为动物。该方法对于研究θ节律在信息处理和行为中的作用具有一定的参考价值。该方法的关键方面包括: (1) 视蛋白的选择和靶向 ChR2 在海马中 MS 细胞的轴突, (2) 植入式光纤线阵列组件的鲁棒光学和电学特性, 以确保持续的刺激和 LFP记录在行为小鼠, (3) 应用最佳的光量在θ频率, (4) 后的夹带保真度的特殊量化, (5) 控制光电?...

哺乳动物的神经元活动由网络振荡协调, 这有助于大脑区域内和脑区之间的信息传递1、2、3、4。大脑节奏包括从非常慢 (< 0.8 赫兹) 到超快 (> 200 赫兹) 频率的振荡。大量的证据支持网络振荡参与不同的大脑功能, 包括认知5,6,7,8,9,10, 先天行为11,12以及精神疾病, 如帕金森病和癫痫13,14,15。因此, 对网络振荡进行实验性操作的选择性和世俗的精确方法, 对于发展生理上可行的同步模型和建立与行为的因果关系是至关重要的。
网络同步由不同的生物基质和过程介导, 从离子通道的分子识别和动力学到调节兴奋性和网络连通性。韵律发生器16的生物设计已经被揭示为许多大脑节律, 不同的方面 (例如, 频率, 振幅) 往往是由不同的细胞类型和网络的动态带来。例如, 抑制中间神经元靶向胞体的主要细胞是最重要的球员跨频段和脑区17,18, 包括θ19,20, 伽玛20,21, 波纹 (140-200 赫兹)22振荡。同时, 利用金字塔细胞的鲁棒前馈信号来保证远距离细胞的相位同步, 从而重置中间神经元的发射。振荡的一个关键参数, 即同步神经元种群的大小, 与测量的 LFP 振荡振幅密切相关, 至少对于快速振荡, 取决于兴奋性驱动到中间神经元2。相比之下, 较慢的振荡, 如三角洲和θ节律, 是由长距离折返循环产生的, 由皮质丘脑23,24和海马内侧间隔预测25组成, 分别为 26、27。在参与单元28、29、30中, 信号传播延迟、兴奋响应和频率偏好的相互作用带来了这种电路中的振荡,31,32. GABAergic parvalbumin (PV) 阳性细胞 (MS) 对海马25、33、海马旁区域和嗅皮质26的抑制性预测为中间神经元在颞叶中形成θ振荡的必要条件。因此, 利用光遗传学实时精度, 可以对网络振荡和神经元同步的生理机制进行操作。
细胞类型特异的 optogenetic 操作已被应用于研究海马和皮层振荡的体外34,35,36,37,38和在体内30,39,40,41,42,43,44,45, 包括功能调查伽玛5,12,36,46,47,48,49,50, 51,52和波纹振荡40,53,54和睡眠纺锤55,56。最近, 我们表达了一项依赖于 ChR2 病毒的 MS, 这是一个关键地区的海马θ节律的生成, 光伏的小鼠。利用该制剂, 海马θ振荡 (频率和时间稳定性) 的特征通过 optogenetic 刺激海马11中 MS 的抑制投影来控制。此外, θ频率 optogenetic 刺激抑制 septo-海马投射诱发θ节律在清醒静止。optogenetically 的θ节律显示了在 LFP 和神经元活动水平的小鼠自发θ振荡的性质。
该协议的主要特点包括: (1) 在生理上对自发θ振荡有重要影响的抑制通路的利用, 同时避免对海马兴奋性的不特异效应;(2) 轴突,即投射特异刺激, 以尽量减少对非海马 efferents MS 的直接影响;(3) 局部θ-韵律光刺激, 确保最小直接干扰的θ韵律 septo-海马动力学和全球双边夹带的θ振荡;(4) θ振荡频率和规律性的参数控制;(5) 利用 LFP 对具有高时间分辨率的夹带保真度进行量化, 以使行为动物的定量因果分析。由于这一准备基本上是利用 septo-海马抑制在θ25,30, 它的一个众所周知的作用, 它使得鲁棒控制的几个参数的θ振荡的行为小鼠。septo - 海马电路的其他研究较少的通路和细胞类型的研究纵了38,39,47,49,50,51,52,53,54,55,56,57,58揭示θ节奏的进一步机制。

<table><tbody><tr><td>PV-Cre mice</td><td>The Jackson Laboratory</td><td>B6;129P2-Pvalbtm1(cre)Arbr/J</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Surgery&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Stereotaxis</td><td>David Kopf Instruments, Tujunga, CA, USA</td><td>Model 963</td><td>Ultra Precise Small Animal Stereotaxic Instrument</td></tr><tr><td>Drill bits, 0.8 mm</td><td>Bijoutil, Allschwil, Switzerland</td><td>49080HM</td><td></td></tr><tr><td>0.01-1 ml syringe</td><td>Braun, Melsungen, Germany</td><td>9161406V</td><td></td></tr><tr><td>Sterican cannulas</td><td>Braun</td><td>26 G, 0.45x25 mm BL/LB</td><td></td></tr><tr><td>Fine and sharp scissors</td><td>Fine Science Tools Inc., Vancouver, Canada</td><td>14060-09</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools Inc.</td><td>11210-10</td><td>Dumont AA - Epoxy Coated Forceps</td></tr><tr><td>Blunt stainless steel scissors</td><td>Fine Science Tools Inc.</td><td>14018-14</td><td></td></tr><tr><td>Soldering station</td><td>Weller Tools GmbH, Besigheim, Germany</td><td>WSD 81</td><td></td></tr><tr><td>Erythromycin</td><td>Rotexmedica GmbH, Trittau, Germany</td><td>PZN: 10823932</td><td>1g Powder for Solution for Infusion</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Optogenetics&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Hamilton pump</td><td>PHD Ultra, Harvard Apparatus, Holliston, MA, USA</td><td>model 703008</td><td>PHD Ultra Syringe Pump with push/pull mechanism</td></tr><tr><td>Hamilton 5 &amp;micro;L Syringe, 26 gauge</td><td>PHD Ultra, Harvard Apparatus</td><td>Model 75 RN SYR</td><td></td></tr><tr><td>Hamilton 5 &amp;micro;L Plunger</td><td>PHD Ultra, Harvard Apparatus</td><td>Model 75 RN SYR</td><td></td></tr><tr><td>Tubing</td><td>Fisher Scientific, Pittsburgh, USA</td><td>PE 20</td><td>Inner diameter 0.38 mm (.015&quot;), Outer diameter 1.09 mm (.043&quot;)</td></tr><tr><td>Sterican cannulas</td><td>Braun, Melsungen, Germany</td><td>27 G, 25x0.40 mm, blunt</td><td></td></tr><tr><td>Precision drill/grinder</td><td>Proxxon, Wecker, Luxemburg</td><td>fbs 240/e</td><td></td></tr><tr><td>Cutting disks</td><td>Proxxon</td><td>NO 28812</td><td></td></tr><tr><td>Cre dependent channelrhodopsin</td><td>Penn Vector Core, Philadelphia, PA, USA</td><td>AV-1-18917P</td><td>Contruct name: AAV2/1.CAGGS.flex.ChR2.tdTomato, titer: 1.42x1013 vg/ml</td></tr><tr><td>Cam kinase dependent halorhodopsin</td><td>Penn Vector Core</td><td>AV-1-26971P</td><td>Construct name: eNpHR3.0, AAV2/1.CamKIIa.eNpHR3.0-EYFP.WPRE.hGH, titer: 2.08_1012 vg/ml</td></tr><tr><td>Multimode optic fiber</td><td>ThorLabs, Dachau, Germany</td><td>FG105LCA</td><td>0.22 NA, Low-OH, &amp;Oslash;105 &amp;micro;m Core, 400 - 2400 nm</td></tr><tr><td>Ceramic stick ferrule</td><td>Precision Fiber Products, Milpitas, CA, USA</td><td>CFLC126</td><td>Ceramic LC MM Ferrule, ID 126um</td></tr><tr><td>Polishing paper</td><td>Thorlabs</td><td>LF3D</td><td>6&quot; x 6&quot; Diamond Lapping (Polishing) Sheet</td></tr><tr><td>Power meter</td><td>Thorlabs</td><td>PM100D</td><td>Compact Power and Energy Meter Console, Digital 4&quot; LCD</td></tr><tr><td>Multimode fiber optic coupler</td><td>Thorlabs</td><td>FCMM50-50A-FC</td><td>1x2 MM Coupler, 50:50 Split Ratio, 50 &amp;micro;m GI Fibers, FC/PC</td></tr><tr><td>Fiberoptic patch cord</td><td>Thorlabs</td><td>FG105LCA CUSTOM-MUC</td><td>custom made, 3 m long, with protective tubing, Tubing: FT030, Connector 1: FC/PC, Connector 2: 1.25mm (LC) Ceramic Ferrule</td></tr><tr><td>Sleeve</td><td>Precision Fiber Products, Milpitas, CA, USA</td><td>ADAL1</td><td>Ceramic Split Mating Sleeve for &amp;Oslash;1.25 mm (LC/PC) Ferrules</td></tr><tr><td>473 nm DPSS laser</td><td>Laserglow Technologies, Toronto, ON, Canada</td><td>R471005FX</td><td>LRS-0473 Series</td></tr><tr><td>593 nm DPSS laser</td><td>Laserglow Technologies</td><td>R591005FX</td><td>LRS-0594 Series</td></tr><tr><td>MC_Stimulus II</td><td>Multichannel Systems, Reutlingen, Germany</td><td>STG 4004</td><td></td></tr><tr><td>Impedance conditioning module</td><td>Neural microTargeting worldwide, Bowdoin, USA</td><td>ICM</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Electrophysiology&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Tungsten wires</td><td>California Fine Wire Company, Grover Beach, CA, USA</td><td>CFW0010954</td><td>40 &amp;micro;m, 99.95%</td></tr><tr><td>Capillary tubing</td><td>Optronics</td><td>1068150020</td><td>ID: 100.4 &amp;micro;m</td></tr><tr><td>Omnetics nanoconnector</td><td>Omnetics Connector Corporation, Minneapolis, USA</td><td>A79038-001</td><td></td></tr><tr><td>Screws</td><td>Bilaney, D&amp;uuml;sseldorf, Germany</td><td>00-96x1/16</td><td>stainless-steel</td></tr><tr><td>Silicone probe</td><td>NeuroNexus Technologies, Ann Arbor, MI, USA</td><td>B32</td><td></td></tr><tr><td>Headstage</td><td>Neuralynx, Bozeman, Montana USA</td><td>HS-8</td><td>miniature headstage unity gain preamplifiers</td></tr><tr><td>Silver conductive paint</td><td>Conrad electronics, Germany</td><td>530042</td><td></td></tr><tr><td>Liquid flux</td><td>Felder GMBH L&amp;ouml;ttechnik, Oberhausen, Germany</td><td>L&amp;ouml;t&amp;ouml;l ST</td><td>DIN EN 29454.1, 3.2.2.A (F-SW 11)</td></tr><tr><td>LED</td><td>Neuralynx</td><td>HS-LED-Red-omni-10V</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>Mathworks, Natick, MA, USA</td><td></td><td></td></tr><tr><td>MC_Stimulus software</td><td>Multichannel, Systems</td><td></td><td></td></tr><tr><td>Neurophysiological Data Manager</td><td></td><td></td><td>NDManager, http://neurosuite.sourceforge.net</td></tr><tr><td>Klusters</td><td></td><td></td><td>http://neurosuite.sourceforge.net, Hazan et al., 2006</td></tr><tr><td>Software of the recording system</td><td>Neuralynx</td><td>Cheetah</td><td>https://neuralynx.com/software/cheetah</td></tr><tr><td>Multi-channel data analysis software</td><td>Cambridge Electronic Design Limited, Cambridge, GB</td><td>Spike2</td><td></td></tr></tbody></table>

optogenetic entrainment of hippocampal theta oscillations in behaving mice

广泛的数据, 神经网络振荡行为和组织神经元放电的大脑区域要求新的工具, 有选择地操纵大脑节律。在这里, 我们描述了一种方法, 结合投影特异光遗传学与细胞外电生理学的高保真度控制海马θ振荡 (5-10 Hz) 的行为小鼠。optogenetic 夹带的特异性是通过靶向 channelrhodopsin-2 (ChR2) 到内侧间隔细胞 GABAergic 的人群中实现的, 关键的是海马θ振荡的产生, 以及局部同步活化海马中抑制性间隔传入的子集。optogenetic 节律控制的功效通过同时监测 CA1 区和/或神经元放电的局部场电位 (LFP) 进行验证。利用这一易于实施的准备, 我们显示了各种 optogenetic 刺激协议的有效性, 以诱导θ振荡和操纵他们的频率和规律性。最后, 将θ节律控制与投影特异抑制相结合, 解决了传出区域海马同步的特定方面的读数。

如图 2A所示, ChR2 在 MS 中的靶向 GABAergic 细胞, 如第1节所述。Optogenetic GABAergic 细胞在背海马中的轴突通过一根被植入 CA1 区的光纤, 拽在同侧 (图 2B) 和对侧的刺激频率上的θ振荡。半球 (图 2C)。θ振荡可以或多或少有效的 optogenetic 刺激 (图 3A), 其效能, 计算为每个记录时代作为一个相对...

Watch this Scientific Journal Video about Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice at JoVE.com

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

我们描述使用光遗传学和电生理记录的选择操作的海马θ振荡 (5-10 赫兹) 在行为小鼠。利用局部磁场电位监测节律夹带的效果。光和遗传药理学抑制的结合解决了海马同步的传出读数。

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 ...

optogenetic-entrainment-hippocampal-theta-oscillations-behaving

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.6K 次观看.  Heinrich Heine University Düsseldorf. 我们描述使用光遗传学和电生理记录的选择操作的海马θ振荡 (5-10 赫兹) 在行为小鼠。利用局部磁场电位监测节律夹带的效果。光和遗传药理学抑制的结合解决了海马同步的传出读数。

视频: Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Automatic Detection of Highly Organized Theta Oscillations in the Murine EEG

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable electroencephalography (EEG) radiotelemetry or tether system approaches. Pharmacologically, hippocampal theta is heterogeneous (see dualistic theory) and can be differentiated into type I and type II theta. These individual EEG subtypes are related to specific cognitive and behavioral states, such as arousal, exploration, learning and memory, higher integrative functions, etc. In neurodegenerative diseases such as Alzheimer&#39;s, structural and functional alterations of the septohippocampal system can result in impaired theta activity/oscillations. A standard quantitative analysis of the hippocampal EEG includes a Fast-Fourier-Transformation (FFT)-based frequency analysis. However, this procedure does not provide details about theta activity in general and highly-organized theta oscillations in particular. In order to obtain detailed information on highly-organized theta oscillations in the hippocampus, we have developed a new analytical approach. This approach allows for time- and cost-effective quantification of the duration of highly-organized theta oscillations and their frequency characteristics.

Theta activity in the hippocampus is related to specific cognitive and behavioral stages. Here, we describe an analytical method to detect highly-organized theta oscillations within the hippocampus using a time-frequency (i.e., wavelet analysis)-based approach.

高度组织的Theta振荡在鼠脑的自动检测

Theta activity is generated in the septohippocampal system and can be recorded using deep intrahippocampal electrodes and implantable ...

科研

行为学

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

神经活性的光遗传学操作，调节自由移动小鼠的行为

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Generation of Local CA1 &#947; Oscillations by Tetanic Stimulation

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A protocol is provided to generate a model for studying CA1 &#947; oscillations (20 - 80 Hz). These &#947; oscillations are stable for at least 30 min&#160;and depend upon excitatory and inhibitory synaptic activity in addition to activation of pacemaker currents. Tetanically stimulated oscillations have a number of reproducible and easily quantifiable characteristics including spike count, oscillation duration, latency and frequency that report upon the network state. The advantages of the electrically stimulated oscillations include stability, reproducibility and episodic acquisition enabling robust characterization of network function. This model of CA1 &#947; oscillations can be used to study cellular mechanisms and to systematically investigate how neuronal network activity is altered in disease and by drugs. Disease state pharmacology can be readily incorporated by the use of brain slices from genetically modified or interventional animal models to enable selection of drugs that specifically target disease mechanisms.

Oscillations are fundamental network properties and are modulated by disease and drugs. Studying brain-slice oscillations allows characterization of isolated networks under controlled conditions. Protocols are provided for the preparation of acute brain slices for evoking CA1 &#947; oscillations.

本地CA1γ振荡通过强直刺激生成

Neuronal network oscillations are important features of brain activity in health and disease and can be modulated by a range of clinically used drugs. A ...

神经科学

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

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 ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Selective Manipulation of Theta Oscillations in a Mouse Model

Source: Bender, F. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/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 ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Implanting an Electrode Array and Optic Fiber into the Mouse Hippocampus

Source: Bender, F., et. al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57349">Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice.</a> J. Vis. Exp. (2018)
This video demonstrates the stereotactic implantation of a wire electrode array and an optic fiber in the hippocampus of a mouse model.

将电极阵列和光纤植入小鼠海马体

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Optogenetic Control of Hippocampal Network Dynamics

Source: Manseau, F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55851">Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit.</a> J. Vis. Exp. (2017)
This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.

海马网络动力学的光遗传学控制

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

	Optogenetic control in the ...