Name: adaptable angled stereotactic approach for versatile neuroscience techniques
Uploaded: 2020-05-07T13:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques at JoVE.com

这项工作得到了国家糖尿病、消化和肾脏疾病研究所（NIDDK）的支持，该研究所授予F31-DK-113673（C.L.F.）、T32-GM-095421（C.L.F.）、DK-089056（G.J..M） #1-19-IBS-192授予G.J.M）和NIDDK资助的营养肥胖研究中心（DK-035816）、糖尿病研究中心（DK-017047）和糖尿病， 华盛顿大学肥胖与代谢培训助学金 T32 DK0007247 （T.H.M） 。

所有程序均根据国家卫生研究院、动物护理和使用指南获得批准，并得到机构动物护理和使用委员会（IACUC）和华盛顿大学环境健康与安全部的批准。
1. 角度坐标的计算
<ol><li>使用日冕脑图集，标记一个右三角形，使低度通过感兴趣的目标区域。在具有代表性的例子（图1）中，下丘脑腹腔核（VMN）以日冕中线15°角为目标。 注: 图1 中描述的旋转轴的位置（因此，侧C的长度）是任意的，可以修改为针对任何大脑区域。虽然这似乎有悖常理，但协议中的后一步将调整头部在 z 轴中的位置，使此点与旋转的立体定向中心对齐（参见第 6 节）。但是，由于头部支架装置的物理约束，建议不要超过 15° 的日冕旋转角度。</li>
	<li>建立所需的角度（a）和估计的B侧长度，并使用三角学来计算侧A和C的长度。此步骤对于在旋转过程中正确定位头部非常重要。 注:在 图1的例子中，地图集网格线用于近似B侧的长度，产生7.576毫米的长度。此信息用于计算 A 侧的长度: <img alt="Equation 1" src="/files/ftp_upload/60965/60965equ01.jpg"> 在此示例中，2.03 mm 表示当头部旋转 15°时，纤维光管进入大脑的中线的 R/L 距离。<ol>
<li>可选计算侧 C 的长度以接近 D/V 坐标: <img alt="Equation 2" src="/files/ftp_upload/60965/60965equ02.jpg"> 注:1） 下垂 （C） 的长度不表示注射深度，但将有助于确定 D/V 坐标，可能需要调整以适应直入注射的长度增加与 B 侧。因此，建议进行测试注射以优化 D/V 坐标。2） 在此示例中针对 VMN， 获得两组坐标:一组用于非角度的微投射（A/P = -1.4，R/L = 0.4 在 0°，D/V = -5.7），另一组用于角纤维植入（A/P = -1.4，R/L = 0.0 在 15°，D/V = -5.4）。</li>
	</ol>
</li>
</ol>2. 为角度程序准备立体定型法
<ol><li>确认立体定向帧和微操纵器已校准（请参阅 Kopf 手册以了解完整协议）。</li>
	<li>将中心高度计放入头架基板的插座中。</li>
	<li>将中心范围固定在工具架中，然后向下观察范围。调整微动器的位置，直到十字线对齐并聚焦于仪表十字线。 注:在此步骤中，示波器被定位到头部支架旋转中心的焦点平面上。一旦建立，微操纵器不应在剩余步骤中移动。</li>
	<li>将耳塞放入支架中，使其中心，使两侧的指示线为 0（图 3A）。</li>
	<li>使用头架上的中端和前后旋钮（图 2）将耳杆在中心高度计十字线上方的 x 平面和 y 平面上对齐（图 3A）。</li>
	<li>要对齐 z 轴中的耳栏位置，请从支架上取下耳条并移除中心高度计。更换耳塞，并在 0 点再次集中耳塞。</li>
	<li>向下看范围。分别使用垂直移位旋钮（图 3B）和日冕倾斜旋钮来降低和旋转耳杆，直到整个日冕旋转期间，范围十字线始终集中在耳杆之间。</li>
	<li>立体定版现在已校准并准备就绪。不要对头持有人的位置作任何进一步的调整。</li>
</ol>3. 准备注射/植入材料
<ol><li>确保所有仪器、手术工具和材料都经过消毒，并放置在立体定型塔旁边的无菌手术场中。</li>
	<li>根据其生物安全水平和相关机构生物安全监管要求处理和存储病毒结构。</li>
	<li>将病毒放入注射器中，小心使用适当的处理方法和个人防护设备。</li>
</ol>4. 麻醉
<ol><li>手术前记录鼠标的体重。</li>
	<li>使用异黄素对鼠标进行深度麻醉。</li>
	<li>通过执行脚趾捏测试确保鼠标深度麻醉，直到没有退缩反应。如果动物继续表现出强烈的反射，增加麻醉的浓度和/或持续时间。</li>
	<li>将眼药膏涂在每只眼睛上，使其在手术过程中保持湿润。</li>
	<li>用剪发器将头皮从耳朵后面剃到眼睛后面。</li>
	<li>为鼠标提供 IACUC 批准的镇痛剂。</li>
	<li>在整个外科手术过程中持续监测动物，并提供热支持。</li>
</ol>5. 外科手术
<ol><li>将上切口放入咬杆的缝隙中，将头部放入头部支架，确保舌头在咬杆以下。</li>
	<li>通过轻轻地将耳塞插入外部听觉肉中，将耳塞固定在耳塞的头部，确保耳塞对称放置（成人鼠标通常为三到四个耳塞）。此步骤对于确保头部稳定且以旋转为中心至关重要。</li>
	<li>用三个交替磨砂的βdine和酒精拭子，或用其他机构批准的手术部位准备，无所适从地准备剃须切口区域。</li>
	<li>在动物上铺上手术窗帘，以维持无菌手术场，并降低术后感染的风险。</li>
	<li>沿着头皮的下垂中线切口，露出头骨。轻轻刮擦头骨表面，去除任何筋膜并露出缝合线。 注:如果缝合线难以可视化，则使用无菌棉尖施用器将过氧化氢应用于头骨，以改善缝合线可视化。</li>
	<li>将中心范围放入支架中，将十字线放在布雷格玛（图4，左面板）上。微操纵器为零。</li>
	<li>将十字线刻薄地移动到兰姆达，注意到布雷格玛-兰姆达（B-L）的距离。 注:如果缝合线不沿着中线走直线，建议使用"最适合线"通过布雷格玛和兰布达建立中线。但是，如果遵循上述步骤，范围视网的初始位置应位于耳杆之间的一半，并接近 B-L 中线缝合线。</li>
	<li>如果 B-L 距离明显小于或大于 4.21 mm，则逐步调整分配的布雷格玛，以获得 4.21 mm ± 0.2 mm 的 B-L 距离。</li>
	<li>用对齐指示器替换中心示波器。将探头放在兰姆达和布雷格玛上，将头架上的正点倾斜旋钮调整为下垂平面（鼻子朝上或向下），然后使用中心范围重新分配胸针。</li>
	<li>使用对齐指示器，使用日冕倾斜旋钮在日冕平面上调平。测量整个轮状/囊轴的多个点，以解释头骨的表面变形。</li>
	<li>注意日冕倾斜旋钮表盘上的位置，因为这是 0° 旋转位置。</li>
</ol>6. 调整旋转的中心轴以调整角度坐标
<ol><li>将中心范围固定在工具支架中，并将微操纵器从第 1 节中定位到计算的坐标。请注意，角度植入的 R/L 坐标对应于侧 A 的长度。<ol>
<li>在 图 1中，针对 VMN 的光纤位置的倾斜坐标为 （A/P = -1.4， R/L = [2.03] 在 0° 日冕旋转， [0.00] 在 15° 日冕旋转， D/V = -5.4） 。</li>
	</ol>
</li>
	<li>向下观察范围并标记此坐标（R/L 2.03 mm 从 VMN 示例的中线开始; 图4，中间面板）。此标记表示一旦头部旋转，管子将进入大脑的点。</li>
	<li>将微操纵器重新定位到中线（R/L = 0.00）。使用日冕倾斜旋钮将头部旋转到第 1 节计算的角度。<ol>
<li>如果范围十字线已经与标记一致，则继续到第 7 节。</li>
		<li>如果示波交叉线与参考标记不一致，则使用垂直移位旋钮（图 2）调整 z 轴中的头部位置，直到十字线尽可能靠近标记。</li>
	</ol>
</li>
	<li>将头部旋转回 0° 日冕位置。如果垂直移位在步骤 6.3 中调整，则使用中心范围重新分配布雷格玛。</li>
	<li>重复步骤 6.3 和 6.4，直到转动头部时十字线始终达到参考标记（图 4C）。</li>
	<li>此时，第 1 节中确定的任意旋转点现在应与旋转的立体定向中心对齐。</li>
</ol>7. 微投射
<ol><li>将立体战术钻头放在支架中，并将微操纵器操纵到第一个注塑坐标。<ol>
<li>以 VMN 为目标，在头部水平时在 A/P = -1.4 和 R/L = 0.4 进行钻头。</li>
	</ol>
</li>
	<li>降低钻头，直到位就在头骨上方。打开钻头，轻轻降低，直到位刚刚钻过头骨（不是杜拉）。</li>
	<li>重复上述注射部位。</li>
	<li>使用无菌针驱动程序将 90° 弯度引入 27-30G 针头（例如无菌 0.5 mL 胰岛素注射器），并使用弯曲针轻轻戳穿 dura 母体。</li>
	<li>注意:如果发生出血，使用无菌棉尖施用器施加压力，用无菌水清洁，直到出血停止。</li>
	<li>准备注射时，小心地将充满汉密尔顿注射器的注射器放入立体定向支架中。 注:微操纵器上的坐标在切换到新工具后不再适用。使用毛刺孔的中心作为新的注射目标。</li>
	<li>小心地将针头放在毛刺孔上方。</li>
	<li>放下针头，直到它稍微触及毛刺孔中心内的杜拉。关键:仅在 z 轴中零微操纵器，从而保持立体定向中心范围和钻头的微操纵器上的坐标。</li>
	<li>慢慢地将针头放入大脑，仔细观察，以确保针头不会偏转在毛刺孔的边缘。继续降低，直到0.05毫米的通风口到D/V注射坐标，等待1分钟。这个额外的步骤创建一个小的"口袋"，以尽量减少病毒回流的针去除。</li>
	<li>慢慢地将针头举到 D/V 坐标，然后开始注射。 注:流量和体积会因目标区域和实验设计而异。对于 VMN 神经元的光遗传沉默，需要足够的覆盖，因此以 1 nL/s 的速度注射 200 nL 病毒。</li>
	<li>微注射后，在注射部位等待10分钟，以尽量减少取出过程中的病毒流出。</li>
	<li>以大约1毫米/分钟的速度慢慢从大脑中取出微管。</li>
	<li>一旦针头清除头骨，就会弹出少量病毒，以确保针头不会堵塞血液或组织。在继续使用无菌棉尖施用器以去除病毒之前。</li>
	<li>重复步骤 7.6000 7.12 为反向侧。</li>
	<li>用骨蜡密封微注射毛刺孔，以改善愈合（图5B）。</li>
</ol>8. 纤维植入
注:病毒注射后，按每节1个计算角度植入双面纤维素罐。请注意，这些坐标应该已经从第 6 节的头骨上标记。
<ol><li>对于倾斜坐标，重复步骤 7.1 00 7.4。</li>
	<li>将头部返回到 0° 级别位置。</li>
	<li>接下来，使用手钻为骨螺钉再生成四个孔:两个应提前放置，两个后应放置（图 5A）。这些将作为锚，将纤维光子贴在头骨上（图5D）。 注意:请务必将孔间隔到离角坐标毛刺孔足够远的地方，以适应位于头骨上方的纤维光的铁质部分。</li>
	<li>尽可能轻轻地，使用小平头螺丝刀设置骨螺丝，使他们坚定地坐在头骨，但不穿透大脑。</li>
	<li>将纤维光管夹入管架中，并将其放入立体定向支架中。</li>
	<li>将头部旋转到计算的角度，再次指出微操纵器上的坐标不适用于新工具。使用角毛刺孔的中心作为植入目标。</li>
	<li>降低纤维光，直到它只是触及毛刺孔中心的杜拉（图5C）。零 z 轴中的微操纵器，然后将光纤光速缓慢减慢到 D/V 角度坐标（VMN 示例中的 5.4）。</li>
	<li>使用氰酸丙烯酸酯凝胶将纤维光杉与虹膜锚螺钉连接起来，然后用微管尖（图5D）应用加速器。</li>
	<li>一旦氰丙烯酸酯凝胶完全硬化，轻轻松开管架，并提高，直到清除纤维光铁杉。</li>
	<li>重复步骤 8.5008.9 用于反向角度坐标，然后平住头部。为了获得额外的安全性，在两个角度的纤维素罐与氰丙烯酸酯凝胶和加速剂之间进行额外的连接（图5D）。</li>
	<li>准备少量、相对稀薄的牙科水泥。涂抹在头骨表面，确保彻底覆盖抛锚螺丝和纤维素罐底。留下足够的铁杉清洁，以便随后与光纤补丁电缆交配。</li>
	<li>水泥干涸后，将鼠标从立体定向装置中取出。</li>
	<li>将鼠标放在具有热支持的回收笼中。允许它恢复，并转移到家庭笼子，一旦它出现警报，移动，并正在梳理。</li>
</ol>9. 手术后护理
<ol><li>每天监测动物术后3天的行为、姿势、活动和梳妆，并记录食物摄入量和体重。</li>
	<li>如果动物表现出任何疼痛或健康状况不佳的一般指标，请咨询兽医服务。</li>
	<li>在开始行为研究之前，让小鼠至少2周恢复和病毒表达。</li>
</ol>10. 光遗传学
<ol><li>对于光遗传学研究的表现，请参阅西多尔等人。</li>
	<li>在完成研究时验证病毒表达和纤维位置。</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+customizable+procedure+for+angled+stereotaxic+implantation+and+microinjection+in+the+rodent+brain.">A customizable procedure for angled stereotaxic implantation and microinjection in the rodent brain.</a> Kopf Carrier. 96, (2019).</li><li>Correia, P., Matias, S., Mainen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+Adeno-associated+Virus+Injection+and+Cannula+Implantation+in+the+Dorsal+Raphe+Nucleus+of+Mice.">Stereotaxic Adeno-associated Virus Injection and Cannula Implantation in the Dorsal Raphe Nucleus of Mice.</a> Bio-Protocol. 7, 2549 (2017).</li><li>Cardozo Pinto, D. F., Lammel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+topic+in+optogenetics:+new+implications+of+in+vivo+tissue+heating.">Hot topic in optogenetics: new implications of in vivo tissue heating.</a> Nature Neuroscience. 22, 1039-1041 (2019).</li></ol>

作者没有什么可透露的。

神经科学的最新进展支持了对大脑神经导路的活动和功能的深入洞察和理解。这包括应用光遗传学和化学遗传学技术来激活或静音离散神经元群及其在体内的投影位点。最近，这包括开发基因编码钙指标（如GCaMP，RCaMP）和其他荧光生物传感器（如多巴胺，去甲肾上腺素），用于在自由移动的动物中在定义细胞类型中活体记录神经元活动。然而，这些技术的有效应用依赖于成功的立体定向手术来瞄...

行为、喂养和新陈代谢的神经控制涉及高度复杂、综合和冗余神经导路的协调。神经科学领域的一个驱动目标是剖析神经元回路结构和功能之间的关系。虽然经典的神经科学工具（即病变、局部药理注射和电刺激）已经发现了关于控制行为和新陈代谢的特定大脑区域的作用的重要知识，但这些工具由于缺乏特异性和可逆性而受到限制。
神经科学领域的最新进展大大提高了以具有高空间分辨率的细胞类型特定方式询问和操纵电路功能的能力。例如，光遗传学2 和化学遗传学3 方法允许快速和可逆地操纵基因定义的自由移动动物细胞类型的活动。光遗传学涉及使用光敏离子通道，称为通道红花素，以控制神经元活动。这项技术的关键是通道红霉素的基因传递和光源来激活蛋白。基因传递的常见策略是通过1）基因工程小鼠在离散神经元中表达Cre-重组的组合，以及2）对Cre依赖性病毒载体编码通道罗多普辛的组合。
虽然光遗传学提供了一个优雅，高度精确的手段来控制神经元活动，该方法取决于病毒载体和纤维光定位到一个定义的大脑区域的成功立体战术微投射。虽然立体定向程序在现代神经科学实验室中是司空见惯的（并且有几个优秀的协议描述这个程序）4，5，6，能够一致和可重复地瞄准中线沿线的离散大脑区域（即平庸的下丘脑，一个大脑区域，对调节家居功能至关重要7）提出了额外的挑战。这些挑战包括避免优越的下垂鼻窦，第三心室，和相邻的下丘脑核。此外，抑制研究所需的双边硬件植入存在重大空间限制。考虑到这些挑战，此协议提出了一个可修改的程序，通过角度立体定向方法定位离散的大脑区域。

<table><tbody><tr><td>Fiberoptic Cannulae</td><td>Doric Lenses</td><td>MFC_200/230-0.57_###_MF1.25_FLT</td><td>Customizable</td></tr><tr><td>Kopf Model 1900 Stereotaxic Alignment System</td><td>Kopf</td><td>Model 1900</td><td></td></tr><tr><td>Kopf Model 1900-51 Center Height Gauge</td><td>Kopf</td><td>Model 1900-51</td><td></td></tr><tr><td>Kopf Model 1905 Alignment Indicator</td><td>Kopf</td><td>Model 1905</td><td></td></tr><tr><td>Kopf Model 1911 Stereotaxic Drill</td><td>Kopf</td><td>Model 1911</td><td></td></tr><tr><td>Kopf Model 1915 Centering Scope</td><td>Kopf</td><td>Model 1915</td><td></td></tr><tr><td>Kopf Model 1922 60-Degree Non-Rupture Ear Bars</td><td>Kopf</td><td>Model 1922</td><td></td></tr><tr><td>Kopf Model 1923-B Mouse Gas Anesthesia Head Holder</td><td>Kopf</td><td>Model 1923-B</td><td></td></tr><tr><td>Kopf Model 1940 Micro Manipulator</td><td>Kopf</td><td>Model 1940</td><td></td></tr><tr><td>Micro4 Microinjection System</td><td>World Precision Instruments</td><td>--</td><td></td></tr><tr><td>Mouse bone screws</td><td>Plastics One</td><td>00-96 X 1/16</td><td></td></tr><tr><td>Stereotaxic Cannula Holder, 1.25mm ferrule</td><td>Thor Labs</td><td>XCL</td><td></td></tr><tr><td>Surgical Drill</td><td>Cell Point Scientific</td><td>Ideal Micro Drill</td><td></td></tr></tbody></table>

adaptable angled stereotactic approach for versatile neuroscience techniques

立体手术是现代神经科学实验室的重要工具。然而，精确和准确地瞄准难以到达的大脑区域的能力仍然面临挑战，尤其是在瞄准中线沿线的大脑结构时。这些挑战包括避免优越的下垂鼻窦和第三心室，以及持续瞄准选择性和离散性脑核的能力。此外，更先进的神经科学技术（例如光遗传学、纤维光子光子测量和双光子成像）依赖于有针对性地将重要的硬件植入大脑，而空间限制是一个常见的障碍。这里介绍了一个可修改的协议，用于使用角冠状方法对啮齿动物大脑结构进行立体定向定位。它可以适应1）小鼠或老鼠模型，2）各种神经科学技术，和3）多个大脑区域。作为一个具有代表性的例子，它包括用于针对小鼠下丘脑腹腔核（VMN）的立体定向坐标的计算，用于光遗传抑制实验。此过程从腺相关病毒 （AAV） 的双边微注射开始，将光敏氯化物通道 （SwiChR+） 编码为依赖克雷的鼠标模型，然后对光纤冠状腺进行倾斜的双边植入。使用这种方法，研究结果显示，对胰岛素引起的低血糖进行完整的葡萄糖反调节反应需要激活一组VMN神经元。

本方案描述了进行光遗传学研究的外科手术程序，以询问下丘脑VMN神经元在血糖控制9中的作用。首先使用的是一种标准（非角度）立体定向方法，用于将抑制性通道罗多普辛病毒的双边微射到VMN。虽然角度方法也合适，但选择标准（非角度）方法，是因为它足以定位感兴趣的大脑区域，并且是一种简单、可靠和一致的方法。然而，鉴于VMN靠近中线，空间限制不允许无角度植入双...

Watch this Scientific Journal Video about Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques at JoVE.com

Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

这里描述的是一个立体定向程序，可以使用角冠状动脉方法瞄准具有挑战性和难以到达的大脑区域（由于空间限制）。该协议适用于小鼠和大鼠模型，可应用于多种神经科学应用，包括冠状植入和病毒结构的微射。

多功能神经科学技术的适应性角度立体定向方法

Stereotactic surgery is an essential tool in the modern neuroscience lab. However, the ability to precisely and accurately target difficult-to-reach brain ...

adaptable-angled-stereotactic-approach-for-versatile-neuroscience

Research

JoVE Journal

Neuroscience

4.9K 次观看.  University of Washington. 这里描述的是一个立体定向程序，可以使用角冠状动脉方法瞄准具有挑战性和难以到达的大脑区域（由于空间限制）。该协议适用于小鼠和大鼠模型，可应用于多种神经科学应用，包括冠状植入和病毒结构的微射。

视频: Adaptable Angled Stereotactic Approach for Versatile Neuroscience Techniques

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

科研

行为学

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer scale. However, current methods mostly rely on tethered cable recordings or only use unidirectional systems, allowing either recording or stimulation of neural activity, but not at the same time or same target. Here, a new wireless, bidirectional device for simultaneous multichannel recording and stimulation of neural activity in freely behaving rats is described. The system operates through a single portable head stage that both transmits recorded activity and can be targeted in real-time for brain stimulation using a telemetry-based multichannel software. The head stage is equipped with a preamplifier and a rechargeable battery, allowing stable long-term recordings or stimulation for up to 1 h. Importantly, the head stage is compact, weighs 12 g (including battery) and thus has minimal impact on the animal&#180;s behavioral repertoire, making the method applicable to a broad set of behavioral tasks. Moreover, the method has the major advantage that the effect of brain stimulation on neural activity and behavior can be measured simultaneously, providing a tool to assess the causal relationships between specific brain activation patterns and behavior. This feature makes the method particularly valuable for the field of deep brain stimulation, allowing precise assessment, monitoring, and adjustment of stimulation parameters during long-term behavioral experiments. The applicability of the system has been validated using the inferior colliculus as a model structure.

A Wireless, Bidirectional Interface for In Vivo Recording and Stimulation of Neural Activity in Freely Behaving Rats

A wireless, bidirectional system for multi-channel neural recordings and stimulation in freely behaving rats is introduced. The system is light and compact, thus having minimal impact on the animal&#180;s behavioral repertoire. Moreover, this bidirectional system provides a sophisticated tool to assess causal relationships between brain activation patterns and behavior.

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

In vivo electrophysiology is a powerful technique to investigate the relationship between brain activity and behavior at a millisecond and micrometer ...

Building An Open-source Robotic Stereotaxic Instrument

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for around $1,000 (excluding a drill), using industry standard stepper motors and CNC controlling software. Each axis has variable speed control and may be operated simultaneously or independently. The robot&#39;s flexibility and open coding system (g-code) make it capable of performing custom tasks that are not supported by commercial systems. Its applications include, but are not limited to, drilling holes, sharp edge craniotomies, skull thinning, and lowering electrodes or cannula. In order to expedite the writing of g-coding for simple surgeries, we have developed custom scripts that allow individuals to design a surgery with no knowledge of programming. However, for users to get the most out of the motorized stereotax, it would be beneficial to be knowledgeable in mathematical programming and G-Coding (simple programming for CNC machining).
The recommended drill speed is greater than 40,000 rpm. The stepper motor resolution is 1.8&#176;/Step, geared to 0.346&#176;/Step. A standard stereotax has a resolution of 2.88 &#956;m/step. The maximum recommended cutting speed is 500 &#956;m/sec. The maximum recommended jogging speed is 3,500 &#956;m/sec. The maximum recommended drill bit size is HP 2.

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (computer numeric controlled; CNC) stereotaxic instrument for around $1,000 (excluding a drill).

建设一个开放源码的机器人立体定位仪

This protocol includes the designs and software necessary to upgrade an existing stereotaxic instrument to a robotic (CNC) stereotaxic instrument for ...

神经科学

Operative Technique and Nuances for the Stereoelectroencephalographic (SEEG) Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy surgery. Recently, the application of a robotic stereotactic guidance system for implantation of SEEG electrodes has become more popular in many epilepsy centers. The technique for the use of the robot requires extreme precision in the pre-surgical planning phase and then the technique is streamlined during the operative portion of the methodology, as the robot and surgeon work in concert to implant the electrodes. Herein is detailed precise operative methodology of using the robot to guide implantation of SEEG electrodes. A major limitation of the procedure, namely its heavy reliance on the ability to register the patient to a preoperative volumetric magnetic resonance image (MRI), is also discussed. Overall, this procedure has been shown to have a low morbidity rate and an extremely low mortality rate. The use of a robotic stereotactic guidance system for the implantation of SEEG electrodes is an efficient, fast, safe, and accurate alternative to conventional manual implantation strategies.

Operative Technique and Nuances for the Stereoelectroencephalographic SEEG Methodology Utilizing a Robotic Stereotactic Guidance System

The SEEG methodology is simplified and made faster with a stereotactic robot. Careful attention must be paid to the registration of the preoperative volumetric MRI to the patient prior to use of the robot in the OR. The robot streamlines the procedure, leading to decreased operative times and accurate implantations.

利用机器人立体定向引导系统的立体脑电图（SEEG）方法的操作技术和细微差别

The SEEG methodology has gained favor in North America over the last decade as a means of localizing the epileptogenic zone (EZ) prior to epilepsy ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

调查深皮层的功​​能和皮层下结构采用立体定向脑电图:由前扣带皮层的经验教训

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a physiological barrier preventing large molecules (&#62;500 Da) from entering the brain tissue. Microbubble-mediated focused ultrasound (FUS) can be used to induce a transient local BBB opening, allowing larger drugs to enter the brain parenchyma.
In addition to large-scale clinical devices for clinical translation, preclinical research for therapy response assessment of drug candidates requires dedicated small animal ultrasound setups for targeted BBB opening. Preferably, these systems allow high-throughput workflows with both high-spatial precision as well as integrated cavitation monitoring, while still being cost effective in both initial investment and running costs.
Here, we present a bioluminescence and X-ray guided stereotactic small animal FUS system that is based on commercially available components and fulfills the aforementioned requirements. A particular emphasis has been placed on a high degree of automation facilitating the challenges typically encountered in high-volume preclinical drug evaluation studies. Examples of these challenges are the need for standardization in order to ensure data reproducibility, reduce intra-group variability, reduce sample size and thus comply with ethical requirements and decrease unnecessary workload. The proposed BBB system has been validated in the scope of BBB opening facilitated drug delivery trials on patient-derived xenograft models of glioblastoma multiforme and diffuse midline glioma.

The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...

使用微棱镜对头部固定和自由移动的动物进行多层成像

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

作者聚焦:清醒和自由移动状态下神经活动的比较成像

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

多功能神经科学技术的适应性角度立体定向方法

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此视频中的章节

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

用于体内的无线双向接口记录和刺激自由行为大鼠的神经活动

建设一个开放源码的机器人立体定位仪

利用机器人立体定向引导系统的立体脑电图（SEEG）方法的操作技术和细微差别

调查深皮层的功能和皮层下结构采用立体定向脑电图:由前扣带皮层的经验教训

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

立体定向病毒注射和梯度指数晶状体植入术用于深部脑体内钙成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

多功能神经科学技术的适应性角度立体定向方法

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建设一个开放源码的机器人立体定位仪

利用机器人立体定向引导系统的立体脑电图（SEEG）方法的操作技术和细微差别

调查深皮层的功​​能和皮层下结构采用立体定向脑电图:由前扣带皮层的经验教训

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

调查深皮层的功能和皮层下结构采用立体定向脑电图:由前扣带皮层的经验教训

立体定向病毒注射和梯度指数晶状体植入术用于深部脑体内钙成像