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&#945;-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 &#216;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&#39;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>Software</td>
			<td>Company</td>
			<td>Version</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Intan Recording Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuroexplorer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plexon Offline Spike Sorter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACSF Composition:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>oxygenated ACSF with 95% Oxygen/5%CO2 constantly being bubbled through the ACSF (ACSF; in mM 125 NaCl, 25 NaHCO3, 3 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2 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

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

完整的犁鼻器和副嗅球的体外制剂

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

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

此视频中的章节

浦肯野细胞树突形态的器官切片培养分析

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

在黑质和腹侧被盖区多巴胺调控的综合性能分析

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

完整的犁鼻器和副嗅球的体外制剂

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

有条件的遗传跨突触标记在胚胎小鼠脑

同时

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

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

此视频中的章节

浦肯野细胞树突形态的器官切片培养分析

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

在黑质和腹侧被盖区多巴胺调控的综合性能分析

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

完整的犁鼻器和副嗅球的体外制剂

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

有条件的遗传跨突触标记在胚胎小鼠脑

同时

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合