这项工作是由哥伦比亚广播公司桥接赠款授予OOM资助。OOM、PAA 和 AS 设计了这项研究并进行了实验。AS 和 PAA 分析了结果。欧姆和PAA准备了手稿。我们感谢卡尔·迪塞罗斯博士（斯坦福大学）提供AAV供我们使用。

所有实验和动物处理程序都得到了路易斯安那州立大学兽医学院机构动物护理和使用委员会（IACUC）的批准。
1. 实验动物
<ol><li>使用5至6周大的小鼠。</li>
	<li>在12小时交替光和暗循环的标准条件下，每笼有3至5只动物。食物和水应该提供。</li>
</ol>2. 颅骨切除术和动物制备
注:本节描述了小鼠颅骨切除术的术前和术前手术程序。使用标准立体定向仪器和适当的坐标（前哨:AP、医疗:ML 和多索文特:DV）。参考小鼠大脑地图集，以确定感兴趣的大脑区域的坐标。
<ol><li>通过腹内氯胺酮（100毫克/千克）/西拉津（10毫克/千克）鸡尾酒注射麻醉小鼠。进行脚趾捏测试，以确保手术开始前没有感觉。 注:另外，伊索夫卢拉内麻醉与适当的鼻腔也可以用于这一步骤。</li>
	<li>轻轻地将动物的头部固定在立体定向仪器上。 注意:在此过程中，谨慎处理动物非常重要。此外，在手术前检查呼吸速率和其他生命体征。让动物休息7分钟，以减轻压力。<ol>
<li>在立体声框架上放置一个加热垫，使动物的身体躺在它身上。这将有助于保持体温和保持动物温暖，虽然出程序。保留加热垫，用于术后护理和恢复。</li>
	</ol>
</li>
	<li>使用剪子去除头皮上的头发，并用碘溶液清洁皮肤。<ol>
<li>应用局部利多卡因来阻止头皮上的感觉。</li>
		<li>使用手术刀使中线头皮切口从正面延伸到腹膜区域。</li>
		<li>用碘溶液清洁切口区域，然后暴露钙化物。 注:少量的3%过氧化氢溶液可用于从钙化物中去除腹膜。这将提高地标（布雷格玛和兰姆达）和缝合线的能见度。</li>
		<li>使用鼠标大脑立体战术地图集，确定AP和ML坐标相对于布雷格玛。</li>
		<li>对于 VTA 注射，将超细钝点针注射器（例如神经注射器）放置在坐标 AP: -3.08 mm/ML: 相对于布雷格玛 0.5 mm。</li>
		<li>使用钻孔工具在标有 AP/ML 坐标的头骨上钻一个 1 mm 孔（颅骨切除术）。 注:此步骤需要高度谨慎。在钻探时应施加最小的压力，以防止钻头压碎脑组织。在目前的研究中，钻头为 0.8 mm，钻头速度设定为 15，000 rpm。 警告:如果在清洁切口或钙化物时使用过氧化氢，请确保完全去除溶液或允许在钻探前完全干燥。</li>
	</ol>
</li>
</ol>3. AAV 鸡尾酒注射
注:本节描述了AAV注射到成年C57BL/6小鼠VTA（23×27克）的过程。上述方法可用于使用标准立体战术装置和适当的坐标将 AAV 注射到任何大脑区域。为了证明这个协议，eYFP和hChR2在VTA谷氨酸神经元中使用AV5介质的转染在CaMKII®促进器下表达（图1）。Cre-lox重组也可用于此步骤。
<ol><li>将带超细钝点针的注射器（32 G;5 μL 容量，100 nL 精度）安装在喷油器上。将注射器支架固定在微操纵器上。 注:对于此过程，使用了具有 1.6 nL 校准的手动注射器支架。还可使用自动喷油器。</li>
	<li>将注射器装满双蒸馏水，以清洁和测试液体的流动。</li>
	<li>在冰上解冻 AAV （血清型 5） 鸡尾酒的酒。AAV 最好存储在 -80 °C 下，以保持病毒滴答声以获得良好的表达。</li>
	<li>将安装的注射器填充 1，000 nL 的 AAV 溶液（磷酸盐缓冲盐水 （PBS） 10 mM，pH 7.4）。在将针头放入注射部位之前，分配 AAV 溶液的 10 nL 以确认液体的流动。</li>
	<li>使用微操纵器将针头立体定向降低到所需的深度（DV 坐标）。将针头降低到所需的深度后，让注射器在注射前保持原位 10 分钟。 注:对于此程序，针头从大脑的皮层表面以 4.5 mm 的深度 （DV） 降低到 VTA 中。对于其他大脑区域，使用适当的多索文特坐标（指小鼠大脑地图集）。</li>
	<li>向 VTA 注入 600 至 800 nL 的 AAV。注射量可根据目标站点的大小进行调整。<ol>
<li>以 60 nL/min（3 分钟间隔）的速度交付 AAV 解决方案。</li>
		<li>要在 VTA 谷氨酸神经元和投影中表达 eYFP 和 hChR2，注射 AAV-卡姆基®-ChR2-eYFP。 注 :Cre-lox 重组方法也可以根据实验目标使用。</li>
		<li>在 AAV 注射后，让针头保持原位 15 分钟。这将减少扩散和回流。</li>
	</ol>
</li>
	<li>取下注射器针头，然后缝合切口以关闭伤口。</li>
	<li>作为术后护理的一部分，使用抗生素和镇痛剂。将鼠标放在一个温暖的填充平台上，并监视动物直到醒来。 注:注射3周后，通过在小鼠大脑部分检测记者蛋白（eYFP）的荧光，可以观察到AAV表达。此外，光刺激实验可以在3周后进行（图2）。</li>
</ol>4. 设置为带光遗传学的体内神经记录
注:本节描述了急性神经记录的步骤与大脑回路的光遗传学跟踪（VTA谷氨酸神经元投影到CA1）。如有必要，请在开始此步骤之前检查系统（放大器和连接）是否有电气噪声和接地问题。在法拉第笼子中执行此步骤有助于消除录音中的电气噪音。
<ol><li>在 AAV 注射后 3 周，通过腹膜尿素注射（0.2 毫克/千克）麻醉小鼠。 警告:尿素是致癌的，必须谨慎处理，而穿着防护装备。此外，在这个浓度下进行尿素麻醉是非生存的。</li>
	<li>将动物的头部贴在如前所述的立体刻画框架上（第 2.2 步， 图 3A）。进行颅骨切除术，以暴露杜拉（图3A）。使用钻孔工具（位大小:0.8 mm，速度:15，000 rpm）去除部分骨骼。<ol>
<li>颅骨切除术应约3毫米×4毫米宽。将人工脑脊液 （aCSF） 滴到此区域，以防止干燥。</li>
	</ol>
</li>
	<li>在解剖（数字）显微镜下，使用弯曲的针尖（27 G）切除暴露的杜拉。小心不要拉开这个区域细腻的皮叶覆盖物和皮质组织。</li>
	<li>在腹骨上钻一个洞（位大小:0.8毫米，速度:10，000转/分）抱住地面螺丝（平头菲利普斯螺丝:M0.6 x 2.0 毫米）。</li>
	<li>连接不锈钢接线。 注:其他类型的电线也可以使用（图3b）。</li>
	<li>对于合并光刺激 （VTA） 和神经记录 （CA1），请使用配备超细（10 μm 或 1 μm） 微操纵器的多轨立体定型处理设备。分别在 10 μm 范围和 1 μm 范围微操纵器上安装光纤并记录神经探头。<ol>
<li>如有必要，请使用头部舞台探针适配器（图3c）。 注:在当前协议中，32 通道头台装有适配器，可容纳 4 通道录制电极（图3d）。不锈钢接地线焊接到适配器的地面连接端口。</li>
	</ol>
</li>
	<li>在协调 AP: -3.08 mm ML: 0.5 毫米， 降低 400 μm 直径光纤到 VTA.<ol>
<li>在降低光纤之前，将铁杉连接到与光纤耦合 LED 源相连的光纤电缆（不锈钢或陶瓷铁杉）。 注意:确保光线强度和焦点随需设置。LED 光源的选择应与目标操作相对应。在这里，一个470纳米（蓝光）LED驱动器用于hChR2光激活（图4a）。</li>
		<li>要将光脉冲与神经记录同步，将 LED 驱动程序和录制控制器数字 IN 端口连接到晶体管晶体管逻辑 （TTL） 脉冲器 （图 4b）。这可以通过 BNC 拆分器实现。 注:使用TTL脉冲器可灵活地以数字方式设置所需的脉冲列车频率和持续时间。在当前协议中，10 ms 光脉冲在 50 Hz18触发。对于 LED 驱动程序的 TTL 控制，将驱动程序切换为"触发器"。在此模式下，光强度可以通过转动"旋钮"来调节。刺激频率可以调整以适应实验变量，范围从 1 到 100 Hz。对于神经回路追踪，建议刺激频率为 >20 Hz。</li>
		<li>调整旋钮，以确定有效强度，可以在不产生光电伪影的情况下产生响应。如有必要，重新定位光纤以消除人工制品19。 注:当前协议中使用的 LED 驱动程序产生的光功率为 ±21.8 mW。</li>
	</ol>
</li>
</ol>5. 神经记录
<ol><li>使用 15°50 μm 厚柄的急性神经探针，长度至少为 5 毫米。 注:从深层大脑结构进行记录需要用更长的刀柄进行神经探针。在目前的研究中，使用了一个20毫米柄长的探针。</li>
	<li>通过串行外设接口 （SPI） 电缆将预放大器头阶段连接到录制控制器。检查录制控制器端口上的 LED 颜色照明。绿色和黄色 LED 表示连接放大器板上的适当电压。红色 LED 表示工作软件头级控制（图 5b）。</li>
	<li>使用微操纵器，将电极接触点放置在 CA1 的金字塔细胞层中（AP: -1.94mm，ML:+1.0mm，DV:+1.1 至 1.2mm）。 注:光纤和电极可以放置在其他所需的大脑区域进行光刺激和记录。</li>
</ol>6. 放大器和滤光片设置
<ol><li>通过 USB 3.0 端口将录制控制器放大器系统连接到计算机。使用 SPI 电缆将头部阶段连接到放大器。启动控制器软件。如果连接不当，系统将显示"找不到设备"消息。</li>
	<li>单击 "运行" 查看频道上的活动。每个波形被绘制为电压（y轴）与时间（x轴）。根据感兴趣区域，调整电压和时间尺度以调整波形显示。</li>
	<li>如果只有少数通道处于活动状态，则禁用未使用的通道以减少存储盘上的文件大小。</li>
	<li>通过编辑采集软件上的带宽参数来设置采样速率和截止频率。对于单单元录制，将上下截止频率分别设置为 300 Hz 和 5，000 Hz。将放大器采样速率更改为 30 kS/s。 注:选择高抽样率将产生更大的文件大小。</li>
	<li>在录制之前，请通过测量 1，000 Hz 的阻抗性来检查电极通道的完整性。这可以通过在用户界面（控制器软件）中启动功能来完成。工作通道的阻抗值范围应为 0.1 至 5 MΩ。</li>
	<li>如果音频输出（扬声器）连接到 模拟输出 端口，请调整 增益 和 消音器 以获得最佳峰值声音。</li>
	<li>要在峰值记录中显示 TTL 脉冲列车标记，可启用 数字 IN 标记的显示。要记录脉冲列车时间戳，请在主实验前启用 数字 IN 通道显示。 注:通常有多个"数字"通道（1 或 2）。确保来自 TTL 脉冲器的 BNC 电缆连接到选定用于录制控制器软件中显示的"数字 IN"端口。</li>
	<li>要实时查看峰值波形，请打开峰值示波窗口，并使用鼠标单击设置阈值。</li>
	<li>通过监控根平均方块（μV 中的 RMS）来检查噪声水平。对于干净的峰值记录，首选神经峰值阈值至少为 5 倍 RMS。</li>
	<li>设置完成后，选择文件输出格式。 注:在这里，CA1神经尖峰被记录在.rhd格式。还可以选择其他文件格式（.rhs 和 .dat）来匹配分析软件允许的文件格式。</li>
</ol>7. 在体内光遗传学记录和设置
<ol><li>打开脉冲控制软件 （TTL），显示各种可调脉冲参数。在软件上选择适当的 COM 端口。通过调整 列车 和 组 设置设置脉冲参数。 注:脉搏条件应通过考虑实验变量和设计来确定。</li>
	<li>在放大器控制器软件上，单击 "运行" 以检测海马区或所需大脑区域的神经尖峰。如有必要，调整电极深度以检测可行和活跃的神经元。</li>
	<li>一旦检测到细胞外电压活动，观察基线尖峰15分钟，并监测动物的生命体征。此外，测试光脉冲以检测CA1或所需的解剖目标区域内反应灵敏的神经元。 注意:检查光电器件，并通过调整光强度或光纤位置消除。</li>
	<li>在以所需的频率触发光脉冲之前，将基线活动记录为 ±10 分钟（图 5a，b）。这将允许比较有照明和没有照明的射击或爆裂率。 注:在目前的研究中，基线活动被记录为10分钟没有照明，其次是照明在50赫兹（电影1）。</li>
</ol>8. 分析
<ol><li>将 .rhd 文件转换为 Nex5 输出格式。</li>
	<li>处理文件名。Nex5 文件在离线分拣软件中检测单个单元拉斯特列车和波形 （图 5a×b）。<ol>
<li>从下拉窗口中选择所需的通道。 注:可在 OFSS 的单独窗口上查看连续录制的峰值数据。时间尺度也可以调整。如果使用四重奏，可以设置 OFS 将 4 个通道排序为四重奏。</li>
		<li>设置阈值交叉波形提取的电压级别。 警告:使用至少 5 倍 RMS 进行适当的峰值检测。</li>
		<li>检测波形，然后使用寻谷（自动）或 K 形（半自动）方法执行峰值排序。必要时，合并占据 3D 主组件分析 （PCA） 空间类似区域的单位。</li>
		<li>出口分类。Nex5 文件供进一步分析 （图 5c×d）。 注:对间尖间隔、发射速率和爆裂率的分析结果可导出到其他分析平台。</li>
	</ol>
</li>
</ol>9. AAV 表达物的荧光检测
<ol><li>录音后，在异黄素室对动物实施安乐死。</li>
	<li>用 10 mM PBS （+10 mL） 进行跨心灌注，然后用 4% 磷酸盐缓冲的副甲醛 （PB-PFA， +10 mL） 进行。</li>
	<li>将整个大脑完整地取出，并将其固定在 4% PB-PFA 中，保持 48 小时。</li>
	<li>将固定的大脑转移到 4% PB-PFA 中，其中含有 30% 蔗糖，在 4 °C 下进行低温保存。 72小时后，将大脑切成低温，并将切片安装在明胶涂层的幻灯片上。 注:在当前协议中，选择包含海马封闭部分（星体）的部分，以演示 DG、CA3 和 CA1 中的 AAV 标记终端。</li>
</ol>

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作者没有什么可透露的。

在过去的十年中，AAV结构的设计有了显著的进步。因此，更多的神经元特异性促进剂已被纳入AAV血清型阵列，以改善转染特异性14。通过结合荧光蛋白、运输器、受体和离子通道的基因，AAV 的库现在用于成像、神经调节和突触活动检测。在商业上可用的AAV结构中，基因编码的荧光和离子通道（opsin）的组合允许神经解剖学和电生理学的神经回路14，18，19...

在过去的十年中，开发了一系列遗传工具，以提高神经元类型调制的特异性，并绘制复杂的神经网络图谱1。值得注意的是，神经元细胞中具有内在感染和复制能力的神经毒性病毒已被部署用于表达或消融神经元亚类型的特定蛋白质。当含有荧光蛋白或基因编码突触活性指标时，转染AAV载体标签，并划出大脑区域2、3的神经网络。AAV 结构中的发起人选择指导具有一定特定性的神经元类型（促进者依赖表达）的载体表达。然而，通过Cre-lox重组，AAV结构被部署在神经元标记4，5，6，7的更大特异性。值得注意的是，在AAV载体中包装的光活化微生物蛋白和荧光蛋白可以用各种神经元亚型8来表达，非常适合成像、神经元型电路追踪和光调制9、10。
AAV 构建立体注射到大脑区域（或细胞核）中，驱动报告人蛋白质在索马、树突和轴突终端中的表达。AAV的神经表达含有记者基因（eYFP），有助于神经元细胞体的标签和分析跟踪投影到和从其他大脑区域11，12，13，14。AAV-eYFP结构携带光控制蛋白（例如hChR2），可以作为成像6，15和基于刺激的生理跟踪神经投影的工具，以目标大脑区域在体内16。根据 AAV 血清类型，神经元标记的方向可能是机前或逆行11，12。先前的研究已经证实，AAV5在神经元12中会异常旅行。因此，表达hChR2的细胞体的光刺激在大脑（目标）17的其他地方产生先天效应。
在这里，AAV（血清型5）与CaMKII®促进器被用来表达eYFP（记者）和hChR2（蛋白）在VTA谷氨酸神经元和轴向投影。这项研究的结果证明了VTA-谷氨酸早产终端在CA1、CA3和DG海马区域的层依赖分布。此外，与基线值相比，VTA 谷氨酸神经元的光刺激增加了 CA1 多单元和单单元活体中的发射速率。该协议使用经济实惠的工具和商用软件，可以提高从神经回路跟踪实验获得的数据质量。

<table><tbody><tr><td>3% Hydrogen peroxide</td><td>Fisher chemical</td><td>H312</td><td></td></tr><tr><td>AAV-CaMKII&amp;alpha;-ChR2-eGYP</td><td>Addgene</td><td>Plasmid #26969</td><td></td></tr><tr><td>BNC cable</td><td>Amazon</td><td></td><td></td></tr><tr><td>BNC Splitter</td><td>Amazon</td><td></td><td></td></tr><tr><td>Ceramic Split Mating Sleeve for &amp;Oslash;1.25mm Ferrules.</td><td>Thorlabs</td><td>ADAL1-5</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>LR 39098</td><td></td></tr><tr><td>Gelatin coated slides</td><td>Fisher scientific</td><td>OBSLD01CS</td><td></td></tr><tr><td>Hamilton&#039;s syringe (Neuros)</td><td>WPI Inc.</td><td>06H</td><td></td></tr><tr><td>Head stage adapter</td><td>Neuronexus</td><td>Adpt-Q4-OM32</td><td></td></tr><tr><td>High impedance silicon probe</td><td>Neuronexus</td><td>Q1x1-tet-5mm-121-CQ4</td><td></td></tr><tr><td>INTAN 512ch Recording Controller</td><td>INTAN</td><td>RHD2000</td><td></td></tr><tr><td>Iodine solution</td><td>Dynarex</td><td>1425</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td>NDC 66794-017-25</td><td></td></tr><tr><td>Ketamine</td><td>Spectrum</td><td>K1068</td><td></td></tr><tr><td>LED Driver</td><td>Thorlabs</td><td>LEDD1B</td><td></td></tr><tr><td>LED light source (470 nm)-blue light</td><td>Thorlabs</td><td>M470F3</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>M0-203</td><td></td></tr><tr><td>Optic fiber</td><td>Thorlabs</td><td>CFMLC14L05</td><td></td></tr><tr><td>Pan head philips screw (M0.6 X 2mm)</td><td>Amazon</td><td>M0.6 X 2mm</td><td></td></tr><tr><td>Pre-amplifier headstage (32 Channel)</td><td>INTAN</td><td>C3314</td><td></td></tr><tr><td>Stereotaxic frame</td><td>Kopf</td><td>1530</td><td></td></tr><tr><td>TTL pulser</td><td>Prizmatix</td><td>4031</td><td></td></tr><tr><td>Urethane</td><td>Sigma</td><td>U2500</td><td></td></tr><tr><td>Xylazine</td><td>Alfa Aesar</td><td>J61430</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Version&lt;/strong&gt;</td><td></td></tr><tr><td>Graphpad Prism</td><td>GraphPad Software</td><td>version 8</td><td></td></tr><tr><td>Intan Recording Controller</td><td>Intantech</td><td>version 2.07</td><td></td></tr><tr><td>Neuroexplorer</td><td>Nex Technologies</td><td>version 5.215</td><td></td></tr><tr><td>Plexon Offline Spike Sorter</td><td>Plexon Inc</td><td>version 4.5</td><td></td></tr><tr><td>&lt;strong&gt;ACSF Composition:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>oxygenated ACSF with 95% Oxygen/5%CO&lt;sub&gt;2&lt;/sub&gt; constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO&lt;sub&gt;3&lt;/sub&gt;, 3 KCl, 1.25 NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 1 MgCl&lt;sub&gt;2&lt;/sub&gt;, 2 CaCl&lt;sub&gt;2&lt;/sub&gt; and 25 Glucose).</td><td></td><td></td><td></td></tr></tbody></table>

combined in vivo anatomical and functional tracing of ventral tegmental area glutamate terminals in the hippocampus

大脑中神经元亚群的光遗传调节使研究人员能够解剖体内和前体内的神经回路。这为确定神经元类型在神经回路中的作用及其在信息编码中相对于学习的意义提供了前提。同样，该方法也可用于测试清醒和麻醉动物中两个或两个以上相连的大脑区域的生理意义。目前的研究展示了VTA谷氨酸神经元如何调节麻醉小鼠CA1（海马）中假定金字塔神经元的发射速率。该协议采用腺相关病毒 （AAV） 依赖 VTA 谷氨酸神经元的标签，用于追踪海马层中的 VTA 早熟谷氨酸终端。AAV载体所蕴藏的光控制蛋白（通道罗多普辛;hChR2）和荧光蛋白（eYFP）的表达允许对VTA谷氨酸终端进行机前追踪，并可对VTA谷氨酸神经元细胞体（在VTA中）进行光刺激。高阻抗急性硅电极被放置在CA1中，以检测体内对VTA光刺激的多单元和单单元反应。这项研究的结果表明，在海马体（CA1，CA3和DG）中，早熟VTA谷氨酸终端的层依赖分布。此外，VTA谷氨酸神经元的光刺激增加了假定CA1金字塔单位在体内的发射和爆发率。

羚生追踪
AAV表达通过C57BL/6小鼠注射后21天（图2）的记者蛋白（eYFP）免疫荧光成像进行验证。在海马区成功标记了早产VTA谷氨酸投影，也通过在DG、CA3和CA1层的eYFP检测（图6a+d:电影2和3）。
VTA 谷氨酸预测海马调节 CA1 活动
VTA谷氨酸神经元的光刺激增加?...

Watch this Scientific Journal Video about Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus at JoVE.com

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

目前的协议演示了一个简单的方法来跟踪腹腔四分位区域 （VTA） 谷氨酸投影到海马。VTA谷氨酸神经元的光刺激与CA1记录相结合，以演示VTA谷氨酸终端如何调节CA1假定金字塔发射率在体内。

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

combined-vivo-anatomical-functional-tracing-ventral-tegmental-area

Research

JoVE Journal

Neuroscience

3.8K 次观看.  Louisiana State University School of Veterinary Medicine. 目前的协议演示了一个简单的方法来跟踪腹腔四分位区域 （VTA） 谷氨酸投影到海马。VTA谷氨酸神经元的光刺激与CA1记录相结合，以演示VTA谷氨酸终端如何调节CA1假定金字塔发射率在体内。

视频: Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

科研

行为学

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

<meta charset="utf8"></head><body>使用放射性标记的 L-[3H]-谷氨酸测量从啮齿动物切除的海马体获得的急性横切片中的谷氨酸摄取

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

使用放射性标记的 L-[3H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生物化学

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

高分辨率<em&gt;在体内</em&gt;使用3T磁共振成像手动分割协议为人类海马子字段

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

神经科学

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 ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

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 ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

摘要

探索更多视频

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

使用放射性标记的 L-[³H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

使用放射性标记的 L-[³H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

高分辨率

树突分支中的海马区对小鼠的齿状回评

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

体内小鼠海马体中小胶质细胞的慢性双光子成像

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

摘要

探索更多视频

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

使用放射性标记的 L-[3H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

使用放射性标记的 L-[3H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

高分辨率

树突分支中的海马区对小鼠的齿状回评

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

体内 小鼠海马体中小胶质细胞的慢性双光子成像

使用放射性标记的 L-[³H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

使用放射性标记的 L-[³H]-谷氨酸测量从啮齿动物切除的海马获得的急性横切中谷氨酸摄取

体内小鼠海马体中小胶质细胞的慢性双光子成像