数据由部分由美国国立卫生研究院和尼泊尔国立卫生研究院神经科学研究蓝图的16个NIH研究机构和中心资助的人联系项目（WU Minmin Consortium）（主要研究员:David Van Essen和Kamil Ugurbil; 1U54MH091657）提供;和麦克唐纳华盛顿大学系统神经科学中心。图2A和2D经Rhoton集合57许可（http://rhoton.ineurodb.org/?page=21899）复制。

死者在这里被列为人口，虽然死者不是技术上的人类受试者;人类科目由45 CF 46定义为"活人15,16 "。 1.准备样品<ol><li>检查5个福尔马林固定的大脑（10个半球）和2个全人脑。 </li><li>根据Klingler的方法17将样品固定在10％福尔马林溶液中至少2个月。 </li><li>按照克林格方法17将所有标本在-16℃冷冻2周。 </li><li>在自来水下解冻样品。 </li><li> 在尸体头部进行扩展性额颞开颅手术以暴露大脑。 <ol><li>将尸体头置于三针颅骨夹（ Material Table ）中。 </li><li>用手术刀做一个额叶皮肤切口。</li><li>使用手术刀，镊子和剪刀去除皮肤和肌肉。 </li><li>在头骨中形成一个或多个钻孔，直到达到硬脑膜;使用紧凑型减速器的钻头和79,000 rpm的材料表 （ 材料表 ）的14 mm颅骨穿孔附件。 </li><li>切割骨瓣，并使用2 mm x 15.6 mm带槽铣刀的铣刀打开颅骨，钻孔速度为80,000 rpm（ 材料表 ）为2.1 mm针形毛刺附件。 </li></ol></li><li>使用显微镜在6X至40X放大倍率5,18（ 材料表 ）下，用硬度计去除硬脑膜，蛛网膜和皮肤，并用显微镜切片。 </li></ol>纤维解剖技术注意:在手术显微镜下进行6X至40X放大倍数的所有解剖。 <ol><li> 在每个半球上逐步进行纤维解剖从外侧到内侧和内侧到外侧。 <ol><li>使用panfield解剖器（ 材料表 ）分解大脑皮层，并去除所有额叶皮质组织，以暴露短连接纤维束，这些纤维束是相互连接的回路5,13的U型纤维或间叶纤维。 </li><li>通过在显微镜（ 材料表 ）下轻轻修剪，用panfield解剖器和手术微型钩子去除短结缔组织纤维，以达到并暴露长相关纤维，这种纤维连接在同一半球的远处。 </li><li>深入长关联纤维，以使用手术微型钩和panfield解剖器去除表面缔合纤维;在显微镜（ 材料表 ）下取出每个纤维束，以露出投影连合纤维。 </li><li>查看SMA复合体的每个连接根据之前在文献2,8,18,19,20,21中定义的地形解剖结构。 </li></ol></li><li>将解剖期间在10％甲醛溶液（ 材料表 ）中进行解剖时使用的所有标本（全脑和脑）保留。 </li></ol> 3D摄影技术<ol><li>在拍摄标本期间使用黑色平台。 </li><li> 遵循3D摄影技术22 。 <ol><li>将每个样品放在设计的黑色平台上。 </li><li>选择具有样品全幅正视图的场景，并将相机聚焦在样品上任何点上，靠近相机屏幕上的中心点（仪器选项卡）乐）。使用18至55 mm f / 3.5-5.6 SLR镜头或100 mm f / 2.8L微距镜头，并将光圈设置为F29，ISO 100。 </li><li>将照相机稍微向左旋转，直到相机屏幕上最右侧的点与上述对焦点相同。将相机向右滑动，直到屏幕上的中点与样品上的原始对焦点重叠。将相机聚焦在这一点上，再拍摄一次。 </li><li>将照相机的距离和轴线保持恒定值拍摄的样本。 </li></ol></li><li> 通过使用3D图像生成器程序（材料表）创建3D图像。 <ol><li>打开3D软件程序。 </li><li>选择"从文件打开立体声图像"。 </li><li>选择两个图像（左和右），并确保左图像在左侧插槽，右侧图像在右侧插槽中。 </li><li>选择"半色拼图RL / 2"选项，并以jpeg格式生成浮雕。 </li></OL&gt; </li></ol> DTI技术<ol><li>通过从参考网站下载Human Connectome Project扩散数据23获取预处理的扩散数据。 注意:数据下载预处理并由以下过程组成:扩散数据是在正常志愿者中使用修改的3 T MRI设备（仪表）采用自旋回波回波平面成像（EPI）带图像加速24,25,26,27,28。相关序列参数包括:TR = 5,520 ms; TE = 89.5 ms; FOV = 210×180mm;矩阵= 168×144;切片厚度= 1.25mm（体素大小1.25×1.25×1.25mm）;多频段因子= 3;和b值= 1,000s / mm 2 （95个方向），2,000s / mm 2 （96个方向）和3,000s / mm2 （97个方向）。然后使用FreeSurfer 29和FSL 30处理数据 ;该过程包括涡流校正，运动校正，b0强度归一化，磁敏度失真校正和梯度非线性校正28,31,32,33。相应的T1加权MP-RAGE图像也包含在下载包中。人工互联网项目程序手册23中记录了程序 。 </li><li> 使用扩散光谱成像（DSI）Studio 34对扩散数据进行后处理，以使用广义q采样成像（GQI）算法35产生估计的体素扩散取向分布函数（ODF）。 <ol><li>通过sel将下载的数据集加载到软件中ecting"STEP1:开源图像"，并选择data.nii.gz文件。 </li><li>选择"STEP2:重建"按钮。验证脑屏蔽后，进入"步骤2"，选择"GQI"作为重建方法。选择"长度比"为"1.0"的"r ^ 2加权"。将剩余的选择保留为默认值。 </li><li>选择"运行重建"。 </li></ol></li><li> 为感兴趣的地区放置适当的种子以简化光纤跟踪。 <ol><li>在"区域窗口"中，单击"Atlas"按钮，为上级纵向（SLF）放置种子I.选择"Brodmann"并添加"区域6"和"区域7"。在区域窗口中，将"区域6"类型设置为"种子"，将"区域7"类型设置为"包含区域"（ROI）。 <ol><li>在区域窗口中选择"新建区域"，并手动绘制ROI在冠状面上的额上回的最后方面。执行光纤跟踪，如步骤4.4所述。 </li></ol></li><li>通过在区域窗口中使用"新区域"以类似的方式放置种子，在冠状面中画出中前额叶白质后部的"种子"区域。使用"Atlas"（如步骤4.3.1）和Brodmann区域9，10，46，39和19选择ROI。按照步骤4.4中所述执行光纤跟踪。 </li><li>在"区域"窗口中使用"Atlas"（如步骤4.3.1）将"SLF III"种子放置在"种子"区域，并选择"Brodmann地图册"的"40区"，"区域40"中的"Atlas ..." "和"区域44."执行光纤跟踪，如步骤4.4所述。 </li><li>在区域窗口中使用"新区域"放置胼al体纤维的种子，并在包含第三个矢状面的矢状平面上画一个"种子"胼e体。执行光纤跟踪，如步骤4.4所述。 </li><li>在区域窗口中使用"新区域"放置用于扣带纤维的种子，并在冠状视图中在扣带回中间画一个"种子"区域。使用"新区域"绘制两个ROI，一个在较前面的扣带和一个在冠状回的后扣带回。执行光纤跟踪，如步骤4.4所述。 </li><li>在区域窗口中使用"新区域"放置种子，使用"Atlas ..."功能在电晕辐射中使用ROI进行"种子"绘制。选择图集为"JHU-WhiteMatter-labels-1mm"。 <ol><li>选择并添加"Anterior_corona_radiata"，"Posterior_corona_radiata"和"Superior_corona_radiata"。对于所有纤维，通过使用"新区域"的轴向平面中的低于层级的平面的所有纤维绘制避开区域"在区域窗口中执行光纤跟踪，如步骤4.4中所述。 </li></ol></li><li>使用区域窗口中"Atlas ..."功能的"种子"放置皮质脊髓束种子。选择"JHU-WhiteMatter-labels-1mm"，并添加"Corticospinal_tract"区域。执行光纤跟踪，如步骤4.4所述。 </li><li>使用区域窗口中"Atlas ..."功能的"种子"区域为正面坡道（FAT）种植种子，并选择"区域44"和"区域45"中的布罗德曼图谱和"区域6"ROI。 "执行光纤跟踪，如步骤4.4所述。 </li><li>使用"Atlas ..."功能将"F区域"种子放在"Region 6"区域的"种子"中。在"HarvardOxfordSub"图集中的"尾状物"，"壳核"和"苍白球"中插入新区域，并将区域窗口中的类型设置为"结束"。" 注意:FST的纤维跟踪将通过选择第6区种子和每个跟踪会话中仅一个皮质下种子（ 即第6区和尾状，其次是区域6和壳核，最后是区域6和球）进行苍白球）。 <ol><li>对于每个组合，执行步骤4.4中所述的光纤跟踪。 </li></ol></li></ol></li><li> 执行以上每种组合的光纤跟踪。 <ol><li>在"选项"窗口中，将跟踪参数设置为:qa终止指数为0.08，角度阈值为75，步长为0.675，平滑为0.2，最小长度为20 mm，最大长度为200 mm。将种子方向选择为"全部"，将种子位置选为"子体素"，并将种子随机播种为"开"。使用流线型（Euler）跟踪算法进行三线性方向插值。对于上述区域的每个组合，在＆＃中选择"运行跟踪"34;纤维束"窗口。 注意:由于跟踪的随机性质，确定了明确的"假纤维"并选择性地移除，手工绘制的区域作为"新区域"。 </li></ol></li><li> Affine使用DSI-Studio的"Slices - &gt; Insert T1 / T2 Images"功能，将Human Connectome Project数据集中提供的大脑提取的T1加权3D MP-RAGE扫描注册到扩散数据。通过选择"Slices - &gt;添加等值面"来生成大脑的表面渲染。使用"阈值"665。 </li></ol>

作者声称没有竞争的经济利益，也没有资金来源和支持，包括任何设备和药物。

白色通道的重要性和研究技术大脑皮层被认为是与人类生命250万年相关的主要神经结构。基于形态学和细胞规格，大约有200亿个神经元分成了各个部分。这些皮质部分的结构已经在功能上分组，如感觉运动，感觉运动，情感体验和复杂推理。确定灵长类动物中的所有行为都是通过独特的解剖功能连接和通过神经系统的皮层和皮质下区域拓扑分布的区域形成的。虽然大脑皮质已经...

在Brodmann划定的14个正面区域中，位于中心运动皮质前面的前驱和前额叶区长期被认为是一个无声模块，尽管额叶在认知，行为，学习中起着重要作用，和语音处理。除了前SMA和SMA正面（Brodmann Area; BA 6）组成的辅助电机区（SMA）复合体之外，前动脉/前额模块还包括背侧前额叶（BA 46,8，和9），前额叶（BA 10）和腹外侧前额叶（BA 47）皮质，以及脑1,2侧表面的部分眶额叶皮层（BA 11）。 SMA复合体是由其功能及其连接定义的重要解剖区域。该区域的切除和损伤导致称为SMA的显着的临床缺陷综合征。 SMA综合征是包含SMA复合物3的前额神经胶质瘤病例中特别观察到的重要临床病症。 SMA复合体与边缘系统，基底神经节，小脑，丘脑，对侧SMA，上顶叶以及通过纤维束的额叶的部分具有连接。对这些白质连接的损伤的临床效果可能比皮质更严重。这是因为由于高皮质可塑性4，5，6，7，8，9，10，11，12 ，皮质损伤的后果可以随着时间的推移而改善。因此，SMA区域解剖学和白质通路应该是深层次的特别理解胶质瘤手术。 全面了解白质通道的解剖学对于神经外科病变的广谱治疗是重要的。最近对显微手术中获得的解剖结果的三维文献的研究被用于更好地了解脑白质通路13,14的形态解剖学和相互关系。因此，本研究的目的是使用纤维解剖技术对尸体标本和磁共振成像（MRI）动物图谱的组合来检查SMA复合物（SMA前SMA和SMA）的白质连接，并解释所有方法和这两种技术的原理及其详细的文件。 研究规划与策略在进行实验之前，一升对纤维夹层的基本原理进行了检索，进行了解剖前和解剖过程中需要应用于标本的手术，以及已经用剥离和DTI显示的SMA区域之间的所有连接。以前关于前SMA和SMA适合区域的解剖定位和分离以及其连接的地形解剖学的研究进行了综述。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>%4 Paraformaldehyde Solution</td>
			<td>AFFYMETRIX, Inc.&#160;</td>
			<td>2046C208</td>
			<td>used to fixation</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>INSIGNA</td>
			<td>NS-CZ70WH6</td>
			<td>used to freez</td>
		</tr>
		<tr>
			<td>Panfield Dissector</td>
			<td>AESCULAP</td>
			<td>FD305</td>
			<td>used to dissection</td>
		</tr>
		<tr>
			<td>Surgical Micro Scissor</td>
			<td>W. Lorenz</td>
			<td>&#160;04-4238</td>
			<td>used to miscrodissection</td>
		</tr>
		<tr>
			<td>Surgical Micro Hook</td>
			<td>V. Mueller&#160;</td>
			<td>NL3785-009</td>
			<td>used to miscrodissection</td>
		</tr>
		<tr>
			<td>MICRO VESSEL STRETCHER/DILATOR</td>
			<td>W. Lorenz</td>
			<td>&#160;04-4324</td>
			<td>used to miscrodissection</td>
		</tr>
		<tr>
			<td>Emax2 SC 2000 Electric Console</td>
			<td>Anspach Companies</td>
			<td>SC2102</td>
			<td>used to craniatomy</td>
		</tr>
		<tr>
			<td>Drill Set</td>
			<td>Anspach Companies</td>
			<td>NS-CZ70WH6</td>
			<td>used to craniatomy</td>
		</tr>
		<tr>
			<td>20-1000 operating microscope</td>
			<td>Moeller-Wedel,Germany</td>
			<td>FS 4-20</td>
			<td>used to miscrodissection</td>
		</tr>
		<tr>
			<td>Canon EOS 550D 18 MP CMOS APS-C Digital SLR Camera</td>
			<td>Canon Inc.</td>
			<td>DS126271</td>
			<td>used to take photos</td>
		</tr>
		<tr>
			<td>EF 100mm f/2.8L IS USM Macro Lens</td>
			<td>Canon Inc.</td>
			<td>4657A006</td>
			<td>used to take photos</td>
		</tr>
		<tr>
			<td>MR-14EX II Macro Ring Lite (Flash)</td>
			<td>Canon Inc.</td>
			<td>9389B002</td>
			<td>used to take photos</td>
		</tr>
		<tr>
			<td>Tripod</td>
			<td>Lino Manfrotto</td>
			<td>322RC2</td>
			<td>used to take photos</td>
		</tr>
		<tr>
			<td>MAYFIELD Infinity Skull Clamp</td>
			<td>Integra Inc.</td>
			<td>A0077</td>
			<td>used to fix the head</td>
		</tr>
		<tr>
			<td>Modified Skrya 3T &#34;Connectome&#34; Scanner</td>
			<td>Siemens Company, Inc.</td>
			<td>&#160;A911IM-MR-15773-P1-4A00</td>
			<td>used to scan DTI</td>
		</tr>
		<tr>
			<td>XstereO Player</td>
			<td>Yury Golubinsky</td>
			<td>Version 3.6(22)</td>
			<td>used to create anaglyphs</td>
		</tr>
		<tr>
			<td>EF-S 18-55mm f/3.5-5.6 IS II SLR Lens</td>
			<td>Canon Inc.</td>
			<td>2042B002</td>
			<td>used to take photos</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>6B INVENT&#160;</td>
			<td>7-104-L</td>
			<td>used to make incision</td>
		</tr>
		<tr>
			<td>Compact&#160; Speed Reducer&#160;</td>
			<td>Anspach Companies</td>
			<td>CSR60</td>
			<td>used to make burr hole&#160;</td>
		</tr>
		<tr>
			<td>14 mm Cranial Perforator&#160;</td>
			<td>Anspach Companies</td>
			<td>CPERF-14-11-3F</td>
			<td>used to make burr hole&#160;</td>
		</tr>
		<tr>
			<td>2 mm x 15.6 mm Fluted Router&#160;</td>
			<td>Anspach Companies</td>
			<td>A-CRN-M</td>
			<td>used to make craniotomy</td>
		</tr>
		<tr>
			<td>2.1 mm Pin-shaped Burrs</td>
			<td>Anspach Companies</td>
			<td>03.000.130S</td>
			<td>used to make craniotomy</td>
		</tr>
	</tbody>
</table>

fiber connections of the supplementary motor area revisited: methodology of fiber dissection, dti, and three dimensional documentation

本研究的目的是显示使用纤维解剖技术对尸体标本和磁共振（MR）的组合来检查辅助运动区（SMA）复合体（SMA前SMA和SMA）的白质连接的方法）造影。该方案还将描述人脑白质解剖的程序，扩散张量成像和三维文档。人脑和3D文件的纤维解剖在明尼苏达大学，神经外科系显微外科和神经解剖学实验室进行。根据Klingler的方法制备了五个死后人脑标本和两个整体头。在手术显微镜下，从外侧到内侧和外侧逐步解剖脑半球，并在每个阶段捕获3D图像。所有解剖​​结果均受扩散张量支持成像。根据Meynert纤维分类，包括关联纤维（短，优越的纵向筋膜I和前额叶），投影纤维（皮质脊髓，幽闭皮质，关节和前额皮带）和连合纤维（胼al体纤维）的连接的调查分别为也进行了。

SMA复合体位于额上回的后部。 SMA复合体的边界是前中央沟，下侧上髁，下侧有扣带沟18 。 SMA复合体由两部分组成:前SMA前侧和SMA正侧18 。这两个部分18 （ 图1A和B ）之间的白质连接和功能方面存在差异。我们使用纤维解剖和DTI技术研究了这两个部分的皮质和皮质下连接，并将其显示在3D图像中?...

Watch this Scientific Journal Video about Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation at JoVE.com

Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

本研究的目的是展示纤维解剖技术在人类尸体大脑上的每一步骤，这些解剖的3D文件以及解剖解剖纤维通路的扩散张量成像。

补充电机区的光纤连接重新审视:光纤解剖，DTI和三维文档的方法

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex ...

fiber-connections-supplementary-motor-area-revisited-methodology

Research

JoVE Journal

Neuroscience

11.2K 次观看.  University of Minnesota. 本研究的目的是展示纤维解剖技术在人类尸体大脑上的每一步骤，这些解剖的3D文件以及解剖解剖纤维通路的扩散张量成像。 

视频: Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

从巨纤维通路的电生理记录 D。果蝇</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

科研

神经科学

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra2. The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or &beta;III tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.

Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.

小鼠多巴胺和三维胶原基质检测纹状体外植体的解剖和文化

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum1. ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

小龙虾的游泳足系统:实用指南为神经线及电机模式外记录的解剖

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...