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) 控制光电工件。
第一点的主要告诫是安全的病毒注射, 内侧定位静脉丛。这一步骤的手术执行的次优可以降低注射成功率, 并可能延迟获得结果。视蛋白的动力学应考虑, ChR2 (活化时间: 2 毫秒, 失活时间: 9 ms65)。第二个点要求控制在植入前记录电极的光传输保真度和阻抗, 通常会从及时制造额外的植入物中获益。
第三个考虑是常见的光遗传学瞄准大规模的电路操作, 并涉及光源和光互连, 通过100µm 光纤光功率在大脑的5-15 兆瓦。对于每一个鼠标和每个记录之前, 可以执行一个测试记录, 将光输出设置为实验的最佳强度。光输出应该足够高, 以激活足够数量的投影, 使θ振荡的可靠夹带, 但不太高, 以防止热诱发反应和组织损伤。
第四方面是光脉冲和 LFP 数据的同步, 通过同一 AD 转换器采样两种数据类型, 达到了最高的精度。同步时间戳特别需要其他潜在的应用, 目的是研究神经放电和 LFP。夹带效果在刺激时期内和范围内都不同;这很可能发生, 因为节律干扰设置的 optogenetic 刺激与内在和/或感官驱动信号, 由于多发电机的θ的节奏16,66,67,68. 因此, 对这一高度动态的 optogenetic 操作69的瞬时保真度进行量化, 对于因果推断非常有帮助,即揭示效果幅度之间的关系 (例如,、行为的变化) 和 optogenetic 控制的瞬时功效。重要的是, 多振荡参数可能受 optogenetic 刺激的影响,例如, 夹带介导的不仅是频率锁定, 而且还有一个更规则的θ振荡振幅11。
第五, optogenetic 刺激通常与光电工件有关, 在金属电极中诱发光电效应。他们的程度取决于电极材料, 电极尖端接近光纤和轻的力量。在这里所描述的实验中, 通过增加光纤与记录电极之间的距离, 可以避免光电工件的闭合定位, 而对θ振荡的监测并不重要。光电工件在时间和通道之间显示一致的形状, 因此, 在穗排序期间, 它们通常被分组在一个不同的簇中, 并且不与记录的神经元70重叠。同时, 一个小部分的动作电位, 它的波形是由工件改变, 并没有分组的排序算法与其他峰值发射的神经元。使用各种电极配置 (如线阵列 (图 2B) 和线性硅胶探头) 获得的层流 LFP 轮廓, 使工件和真实 LFP 模式的清晰区分, 基于前者的恒定相位和后者的生理层流相抵消。在刺激之前的基线记录能够探索特征θ特征, 如典型的层流相剖面。
Optogenetic 控制的θ振荡导致θ夹带在所有层的海马 CA1 层, 甚至对侧夹带。如果某项试验旨在进行局部刺激 (如果对受限制的分区域或亚人口的影响旨在研究), 则应考虑到这一点。另一方面, 空间有限的刺激, 对 MS PV+细胞的海马预测, 使对θ振荡的确定性操纵,即夹带, 比体细胞 optogenetic 刺激MS PV+细胞最近应用于麻醉小鼠71。后体细胞刺激持续导致海马振荡频率高达40赫兹, 从而代表了高度可靠的节奏性起搏的情况。相比之下, 这里提出的轴突刺激是有效的优先在θ频段 (图 3C), 因此更类似于夹带, 可能通过 Kuramoto 过渡72,即, 工作在频率接近那些当前在刺激开始自发θ振荡。
虽然这里描述的 optogenetic 准备可以控制θ振荡的特征, 但 optogenetic 抑制 MS 抑制神经元的体细胞, 已被用于有效抑制θ振荡在 REM 睡眠73。两种技术结合使用 optogenetic 执行器反对控制神经元兴奋性 (例如, 12) 可以有潜在的双向控制的θ节律在同一动物进一步研究因果网络振荡与行为各方面的联系。

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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgery</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optogenetics</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 &#181;L Syringe, 26 gauge</td>
			<td>PHD Ultra, Harvard Apparatus</td>
			<td>Model 75 RN SYR</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton 5 &#181;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&#34;), Outer diameter 1.09 mm (.043&#34;)</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, &#216;105 &#181;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&#34; x 6&#34; 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&#34; 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 &#181;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 &#216;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Electrophysiology</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 &#181;m, 99.95%</td>
		</tr>
		<tr>
			<td>Capillary tubing</td>
			<td>Optronics</td>
			<td>1068150020</td>
			<td>ID: 100.4 &#181;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&#252;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&#246;ttechnik, Oberhausen, Germany</td>
			<td>L&#246;t&#246;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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θ LFP 功率周围的刺激频率,即,夹带逼真度 (图 3B)。超过0.3 的夹带保真度,即, 高于自发的光关闭录音, 被观察到大约80% 的记录时代。Optostimulation 在非θ频率下的效率较低 (图 3C)。
θ 振荡频率的参数化处理伴随着θ规律性的紧急变化: 随着时代的推移, θ振荡振幅和频率的时间规律性增加, 在高夹带逼真度。振荡的稳定性也可以通过应用光脉冲的训练来进行参数化调节, 而周期跟随不同分散体的高斯分布 (图 4)。
Optogenetic 控制振荡频率消除了θ频率和运行速度的关系, 与频率控制通过 MS 通过上升传入在运动期间 (图 5A)。Optostimulation 在静止时也诱发了θ振荡 (图 5B)。相对于自发θ (图 6), 在假定的锥体细胞和中间神经元的 CA1 区域中记录的优先发射阶段与 optogenetically 的θ振荡不变。
为了研究在θ介导的运动中, 海马对横隔膜通路的贡献, 我们 optogenetically 抑制这一途径。Halorhodopsin (eNpHR3.0) 是双侧表达的海马锥体细胞 (图 7A), 而 ChR2 是表达在 MS GABAergic 细胞如上所述, θ振荡是 optogenetically 的气流 (图 7B)。θ夹带减少了运行速度的变化, 但不是当海马到 LS 通路被抑制 (图 7C)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57349/57349fig1v2.jpg"> 图 1: 光纤、电极和手术的插图.(A) 光纤的插图。(B) 在海马θ振荡的夹带过程中, 用粘在光纤上记录海马 LFP 的线阵列的插图。(C) 为记录海马细胞活动, 在 microdrive 上安装了硅胶探头。(D) 将微型螺钉定位在头骨上。铜线被 presoldered 到地面和参考螺钉, 然后再定位在小脑上方。(E) 水泥用于盖住螺钉并将其连接起来。上蓝色圆圈表示在哪里进行了开颅手术的硅胶探头的植入。下蓝色圆圈表示在海马区内为光纤植入的开颅手术。(F) 将一根光纤植入尾延髓角, 以靶向海马 CA1 区域。如果需要刺激细胞胞体 (可选), 第二纤维可以植入内侧隔膜。(G) 硅胶探头降低到仅在海马 CA1 区域上方。(H) microdrive 和连接器的边界被粘接在植入物和地面上, 并且参照导线是焊接的。(一) 铜网被构造成环绕植入物, 用作法拉第笼。<a href="/files/ftp_upload/57349/57349fig1v2large.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/57349/57349fig2.jpg"> 图 2: optogenetic 海马θ夹带的制备.(A) ChR2 是在 pv+中隔细胞中表达的。明亮的荧光在 MS (1, 2) 确认成功的构造表达在胞体。MS 纤维项目通过穹隆 (f) 和穹隆 (fi) 到海马体 (3-6);前合生面;前部。HDB: 对角带水平肢的核;或: 地层 oriens。将蓝光 optogenetic 刺激的光纤植入海马区 CA1 (下方案) 的锥体层上方。刻度条: 500 µm (图像1、3、4) 和50µm (图像2、5、6)。(B) 海马 LFP 在自发θ振荡 (左) 和7赫兹 (中间) 或10赫兹 (右) optogenetic 夹带。蓝色条纹表示光应用的时间窗口。注意由箭头指示的光脉冲的相位复位。注意伽玛信封在自发和夹带θ, 一个指标的生理θ节律。在夹带过程中还保持了地层 oriens (str) 和地层 radiatum (str) 之间的相反转。(C) 在同侧 (上部地块) 和对侧 (下地块) optogenetic 刺激中, 夹带是可靠的。方案说明了纤维在电极位置方面的位置。示例 LFP 在θ中的痕迹和光脉冲的应用显示在中间。右侧, 海马 LFP 在信息学和对侧刺激时的功率谱根据刺激频率进行颜色编码。此数字已从 ref 11修改。<a href="/files/ftp_upload/57349/57349fig2large.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/57349/57349fig3.jpg"> 图 3: optogenetic 海马θ夹带的保真度.(A) 海马 LFP 在低和高夹带保真度期间的痕迹。(B) 在自发θ期间, 十年代纪元的功率谱密度, 根据前导θ频率 (左) 排列的行, 在7赫兹 (中间) 和10赫兹 (右) optogenetic 刺激下, 按照夹带保真度排列的行。各自例子功率光谱 (由箭头指示) 在上面被绘。注意跨世纪可靠的夹带保真度。在右侧, 显示了θ频率的夹带保真度的累计概率。(C) 夹带需要θ韵律刺激。海马网络活动可以成功地使用频率在6-12 赫兹之间。在较低的频率 (例如, 2 或 4 hz) 或更高的频率 (例如, 20 hz) 夹带是不可靠的。此数字已从 ref 11修改。<a href="/files/ftp_upload/57349/57349fig3large.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/57349/57349fig4.jpg"> 图 4: θ振荡规律的参数化操作.(a) 在高斯分布后, 在θ范围内采用不同频率的刺激, 平均频率为7.8 赫兹。从σ = 3.19 到σ = 15.09 跨协议增加了脉冲间间隔的标准偏差。总共产生和应用了11个协议, 每一个都有1分钟的刺激期的总持续时间。其中, 5 协议的概率分布显示在图的左侧。在应用各自的协议时, 在1-14 赫兹的海马 LFP 范围内的功率谱密度绘制在图的中间。在相应的协议应用中, θ周期的概率在右边说明。(B) 应用间脉冲间隔的方差确定了同期θ周期的方差 (皮尔逊 r = 0.94, p = 0.0002)。(C) θ振幅变异性与脉间间隔之间的关系 (皮尔逊 r = 0.61, p = 0.08)。此数字已从 ref 70修改。<a href="/files/ftp_upload/57349/57349fig4large.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/57349/57349fig5.jpg"> 图 5: Optogenetic θ有节奏的夹带在行为过程中决定海马 LFP.(A) Optogenetic 刺激频率决定了在运动过程中的θ频率。因此, 与速度相关的传入不会影响海马θ频率, 因此, 速度与θ频率 (蓝色) 不相关, 因为它是自发θ (黑色)。数据显示为平均值 s.e.m. (B) 在安静的觉醒, 海马θ可以在没有运动的情况下诱发。在成功的夹带之前和过程中, 海马 LFP 的痕迹显示在上面, 在夹带过程中记录的速度痕迹显示在下面 (红色的痕迹对应于上面描述的海马 LFP 迹)。蓝色条纹标记光刺激脉冲的时间窗口。此数字已从 ref 11修改。<a href="/files/ftp_upload/57349/57349fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57349/57349fig6.jpg"> 图 6: θ夹带过程中的海马细胞活动.(A) 使用硅胶探头 (方案) 记录细胞活动。单个中间神经元和锥体细胞被隔离并根据各自的波形进行识别。这里显示的是一个例子孤立的锥体细胞的平均波形 (中间) 和自动 correlogram。(B) 锥体细胞 (Pyr) 的首选放电阶段在自发 (黑色、n = 29 神经元) 和 optogenetically (在蓝色、n = 30) θ (p = 0.79) 中不是不同的。(C) 这里显示的是自动 correlogram (左) 和优先射击阶段的快速射击 interneuron 在自发和 optogenetically 夹带θ。在相应的海马 LFP 节律下自发 (左) 和夹带 (右) θ。(D) 在自发 (黑色) 和 optogenetically (蓝色, n = 28 神经元) θ (p = 0.97) 中, 快速发射中间神经元的首选放电阶段不是不同的。平均自动 correlogram 显示在左侧。(E) 平均自动 correlogram str oriens 细胞。(F) oriens 中间神经元的最佳放电阶段在自发 (黑色) 和 optogenetically (蓝色, n = 10 神经元) θ (p = 0.56) 期间不是不同的。右侧显示了首选放电相位的直方图。(G) 在锥体细胞中的θ夹带 (p = 0.98)、快速发射中间神经元 (p = 0.96) 或 str oriens 中间神经元 (p = 0.85) 不影响平均射击率。此数字已从 ref 11修改。<a href="/files/ftp_upload/57349/57349fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57349/57349fig7v2.jpg"> 图 7: 海马θ夹带和 optogenetic 抑制海马皮层下输出的 LS.(A) eNpHR3.0 (halorhodopsin) 在海马锥体细胞 (上部方案) 中表达。胞体在海马 (上部图像) 和轴突 (下部图像) 中的明亮荧光证实了该结构的成功表达。光纤被植入双边以上的 LS (较低的方案)。刻度条: 500 µm (左侧图像), 50 µm (右侧图像)。(B) 海马θ在抑制海马到 LS 通路时成功地夹带。这里显示的功率谱密度为9赫兹蓝光刺激在输出抑制。(C) 抑制主要海马皮层下输出通路可防止海马θ夹带对速度的影响。这里显示是减退在速度变异性在 optogenetic 夹带 (白色酒吧与蓝色边界), 与是缺席的在同时抑制海马对 LS 通路 (黄色酒吧与蓝色边界)。各自的平均基线速度显示在左侧。此数字已从 ref 11修改。<a href="/files/ftp_upload/57349/57349fig7v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

科研

神经科学

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

从产前小鼠海马神经元的分离和培养

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

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 ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

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 ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

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 ...