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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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

A Novel Approach for Documenting Phosphenes Induced by Transcranial Magnetic Stimulation

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical stimulation of the occipital cortex, but also by non-invasive Transcranial Magnetic Stimulation (TMS)1 of the same cortical regions. The intensity at which a phosphene is induced (phosphene threshold) is a well established measure of visual cortical excitability and is used to study cortico-cortical interactions, functional organization 2, susceptibility to pathology 3,4 and visual processing 5-7. Phosphenes are typically defined by three characteristics: they are observed in the visual hemifield contralateral to stimulation; they are induced when the subject s eyes are open or closed, and their spatial location changes with the direction of gaze 2. Various methods have been used to document phosphenes, but a standardized methodology is lacking. We demonstrate a reliable procedure to obtain phosphene threshold values and introduce a novel system for the documentation and analysis of phosphenes. We developed the Laser Tracking and Painting system (LTaP), a low cost, easily built and operated system that records the location and size of perceived phosphenes in real-time. The LTaP system provides a stable and customizable environment for quantification and analysis of phosphenes.

Phosphenes are transient percepts of light that can be induced by applying Transcranial Magnetic Stimulation (TMS) to visually sensitive regions of cortex. We demonstrate a standard protocol for determining the phosphene threshold value and introduce a novel method for quantifying and analyzing perceived phosphenes.

一个新颖的方法记录经颅磁刺激诱发幻视

Stimulation of the human visual cortex produces a transient perception of light, known as a phosphene. Phosphenes are induced by invasive electrical ...

科研

神经科学

High Density Event-related Potential  Data Acquisition in Cognitive Neuroscience

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part because fMRI data acquisition and analysis techniques are readily available. Because fMRI has excellent spatial resolution but poor temporal resolution, this method can only be used to identify the spatial location of brain activity associated with a given cognitive process (and reveals virtually nothing about the time course of brain activity). By contrast, event-related potential (ERP) recording, a method that is used much less frequently than fMRI, has excellent temporal resolution and thus can track rapid temporal modulations in neural activity. Unfortunately, ERPs are under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and low density ERP recording has poor spatial resolution. In an effort to foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition. Critically, high density ERPs offer the promise of excellent temporal resolution and good spatial resolution (or excellent spatial resolution if coupled with fMRI), which is necessary to capture the spatial-temporal dynamics of human brain function.

Event-related potential (ERP) recording is under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and this method often has poor spatial resolution. To foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition.

高密度事件相关电位在认知神经科学的数据采集

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part ...

Rodent Stereotaxic Surgery and Animal Welfare Outcome Improvements for Behavioral Neuroscience

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to investigate the effects of locally manipulated neurotransmitter and signaling pathways in awake, behaving animals. Moreover, the stereotaxic implantation of electrodes for electrophysiological stimulation and recording studies has been instrumental to our current understanding of neuroplasticity and brain networks in behaving animals. Ever-increasing knowledge about optimizing surgical techniques in rodents1-4, public awareness concerning animal welfare issues and stringent legislation (e.g., the 2010 European Union Directive on the use of laboratory animals5) prompted us to refine these surgical procedures, particularly with respect to implementing new procedures for oxygen supplementation and the continuous monitoring of blood oxygenation and heart rate levels during the surgery as well as introducing a standardized protocol for post-surgical care. Our observations indicate that these modifications resulted in an increased survival rate and an improvement in the general condition of the animals after surgery (e.g. less weight loss and a more active animal). This video presentation will show the general procedures involved in this type of stereotaxic surgery with special attention to our several modifications. We will illustrate these surgical procedures in rats, but it is also possible to perform this type of surgery in mice or other small laboratory animals by using special adaptors for the stereotaxic apparatus6.

Stereotaxic surgery on rodents allows for targeted administration of drugs or electrical stimulation and recordings in awake, behaving animals. In this video presentation we will demonstrate recent procedural refinements to this long-standing procedure that successfully improved survival rate and reduced post-surgical weight loss.

啮齿动物的立体定向手术和动物福利成果改进行为神经科学

Stereotaxic surgery for the implantation of cannulae into specific brain regions has for many decades been a very successful experimental technique to ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

一种新的方法，省去了处理的研究侵略和"失败者"的影响<em&gt;果蝇</em

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

Endoscopic Endonasal Trans-sphenoidal Approach: Minimally Invasive Surgery for Pituitary Adenomas

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary lesions. Refinements in surgical techniques, technological advancements, and incorporation of neuronavigation have rendered this surgery minimally invasive. The complication rates of this surgery are very low while excellent results are consistently obtained through this approach.
This paper focuses on the step-by-step surgical approach to pituitary adenomas, which is based on personal experience, and details the results obtained with this minimally invasive surgery.

The aim of this paper is to describe the different steps of the endoscopic endonasal approach to the sella turcica.

内镜下鼻跨蝶入路: 垂体腺瘤的微创手术

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

立体定向激光捕获创伤性脑区显微解剖

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...