这项工作可能要归功于金融支持基金会POUR LA RECHERCHEMédicale（FRM），的米尔顿Safenowitz的博士后研究人员为研究ALS（ALS协会），美国国立卫生研究院的NINDS资助NS05462和NS034382，ANR格兰特HyperMND。

1。第一步麻醉前用药：麻醉诱导前10-15分钟，注射阿托品（0.20毫克/千克）和皮下methylprenidsolone（0.05毫克），以防止唾液分泌和水肿，分别。 2。第二步诱导麻醉：注入戊巴比妥钠（70毫克/千克）或它们的混合物的氯胺酮/甲苯​​噻嗪（100毫克/千克和10毫克/公斤，分别）腹膜内。让鼠标可以得到下，直到没有脚趾掐反射。如果麻醉似乎过轻，补充的剂量的1/4。 3。第三步 *注：这个手术是一个终端程序。 当已达到手术的麻醉，转让的鼠标上提出的温暖的被窝里，在俯卧位。 <ol><li>提供纯净的O 2在100毫升/分钟绕流口罩盖住口鼻部的鼠标。 </li><l我用理发剪，刮背区的颈背基地的尾巴。 </li><li>剃须右后肢。 </li><li>鼠标翻转至仰卧位，但要注意离开氧气面罩。 </li></ol> 4。第四步代替环绕四肢和固定在工作表面的四个角用缝线固定鼠标。 5。第五步将温度探头监控鼠标的核心温度。调整加热毯/灯功率保持核心温度在36°C和38°C之间 6。气管切开术和人工通气<ol><li>使用钝头剪刀，作一个切口，气管，双方拉扯皮肤。 </li><li>使用钝钳，撕开的唾液腺，使两个薄肌（胸骨舌骨），气管暴露。 </li><li>使用钝钳，分开的两个MUSCLES沿其内侧分离，揭示了气管。 </li><li>使用杜蒙7钳，将两个长度为4.0丝线缝合气管。 </li><li>在气管中的两个软骨环之间做一个横向切割，但小心不要完全节的气管。 </li><li>下来的气管，插入气管导管的插入口的两侧，然后将其固定绑在它的缝合线。气管导管连接到鼠标换气装置（SAR-830​​/AP，CWE公司）和一个的的二氧化碳波形（μcapstar，CWE公司）。呼吸机相连，通过合规袋，纯O 2的来源。调整的参数呼吸机（呼吸频率100〜150 BPM，呼气末体积170-310微升），使鼠标不反对的人工通气和呼气末二氧化碳分压是稳定的4％和5％之间。 </li></ol> 7。静脉注射管的放置<ol><li>使用钝性分离，暴露在颈一侧上的颈静脉。在这个层面上，颈内静脉分裂成两个主干，前部和后部的面部静脉。 </li><li>做两次，一组为这些树干下面的步骤： <ol><li>通过使用，杜蒙4或5镊子，仔细分离静脉及其周围的结膜组织。 </li><li>经静脉使用杜蒙7钳，将两个长度为6.0丝线。尽可能地将它们分开沿长度的静脉。 </li><li>放置一个小的血管夹的基端侧的静脉（最接近心脏侧），和配合的前端侧的静脉。 </li><li>使用非常精细虹膜剪，做一个小横切口静脉，特别注意不要第静脉完全。 </li><li>预灌封1FR的导管（premicath，Vygon）插入在所述开口中，最多船只剪辑。 </li><li>保持静脉导管在杜蒙4镊子，小心地取出血管夹，然后按下的导管几个铁道部Ë毫米到静脉。 </li><li>通过把它与插入两侧的两个缝线固定导管。 </li></ol></li><li>一个导管的是，连接到注射器或注射器泵注入补充剂量的麻醉剂在必要时（通常每10-30分钟）。 IV剂量为6毫克/公斤的戊巴比妥钠或1250 40微克/公斤/分钟的氯胺酮/甲苯噻嗪12。其他导管连接到注射泵，一个缓慢静脉输注（50微升/小时）含有的NaHCO 3（1％）和plasmion（14％）的4％葡萄糖溶液。 </li></ol> 8。颈部皮肤针和缝线关闭，并返回鼠标俯卧位 9。后肢肌肉和神经的解剖<ol><li>用剪刀，做一个切口从顶部的大腿跟腱。分开皮肤下面的肌肉，注意不要损伤血管。烧灼根据需要。 </li><l&gt;确定，出现一条白线跑下来的大腿股二头肌的前缘。用剪刀，小心翼翼地打开沿线从膝盖到髋关节骨。分开的肌肉。下股二头肌，坐骨神经是现在可见。 </li><li>仔细解剖股二头肌。烧灼/的结扎的需要，以防止出血。股二头肌可以完全删除，或简单地倾斜，，暴露坐骨神经和小腿三头肌。 </li><li>解剖出腓肠神经。 </li><li>使用8/0丝线，配合腓总神经远端部分，并切结远端。解剖神经的方式，尽量靠近臀部尽可能。 </li><li>确定胫神经，腓总神经和腓肠神经之间。在不同的胫骨神经分支，从树枝上，去更深（以下简称为胫神经）支配的小腿三头肌的分支机构。 </li><li> 8/0丝线线，绑在一起，同时保持完整的分支支配的小腿三头肌，胫骨神经的分支。切远端胫骨神经结和解剖神经尽可能。 </li><li>用生理盐水，以防止熏陶使干燥而进行的下一个步骤，用纱布覆盖整个后肢。 </li></ol> 10。椎板切除术<ol><li>用剪刀沿骨干，做一个切口。分开皮肤下面的肌肉。 </li><li>车型的椎骨分开皮下肌肉的每一侧上的两个切口。然后切成每个腱的肌肉附着在侧的每一侧上的椎骨。 </li><li>使用钝钳和罚款刮匙，除去剩余的肌肉上的背侧的椎骨周围的棘突，清楚地识别每个单独的椎骨。 </li><li>确定椎骨T13和L1。 T13是最后的脊椎肋骨附加。脊柱瓦特生病被固定使用坎宁安脊髓椎夹（Stoelting公司）。将脊髓夹具T13和L1的每一侧上。要小心，不要压迫脊髓，但在纵向轴线放一点点的紧张。检查脊柱固定好，用钳子轻轻按下。 </li><li>使用细咬骨钳，取出脊髓进程，然后薄层T13和L1，从而暴露脊髓。 </li><li>覆盖暴露的脊髓与少量的熏陶用生理盐水，以防止干燥的棉布或spongel。 </li><li>将一个定制的塑料浴缸的背面上，周围的脊髓。在使用4/0丝线固定浴。确保浴防水密封KWIK-CAST密封胶（WPI）。 </li><li> KWIK-主演干燥后，除去覆盖脊髓的棉花，填充矿物油浴。 </li><li>杜蒙5钳子和非常精细虹膜剪，轻轻一拉硬脊膜入ŕ周围的脊髓，并尽可能在两个方向打开。折叠在每边的脊髓硬膜。 </li><li>放下鼠标躺在上，以便保持它暂停椎钳位凸起平台。 </li><li>请使用另一个椎夹具夹紧骶骨部棘突的支持后区的动物。 </li><li>第三个钳位二极管是用来固定右后肢和踝关节，在以90°角在膝盖弯曲。 </li></ol> 11。跟腱解剖<ol><li>在90℃在膝盖和脚踝处的弯曲，将肢体，取出纱布覆盖后肢区域，并解剖无周围组织的跟腱。解剖尽可能多尽可能小腿三头肌，以及从周围组织。 </li><li>切靠近跟骨跖肌肌腱，然后剪了一次完全删除肌腱。 </li><li>使用一个螺纹针，通过第6/0丝线连接Ë跟腱和周围的肌腱，使三结。 </li><li>将力传感器结，切断远端的肌腱，将其连接到力传感器使用的丝线。 </li><li>将两根不锈钢线下筋膜的小腿三头肌。这些导线被连接到细胞外的AC放大器肌电记录。 </li><li>两个双极型钩电极，将腓总神经和胫神经。 </li><li>将小腿三头肌神经的钩电极的阴极上，与阳极接触附近的肌肉。 </li><li>将刺激电极隔离病房。 </li><li>放置球的脊髓背表面电极，连接到外的AC放大器记录脊髓背潜力的。放置一个Ag / AgCl参考电极接触的背面的肌肉。 </li></ol> 12。第十二步刺激小腿三头肌神经使用一个50微秒的方波脉冲的强度的增加在低频率（&lt;1赫兹），直至观察到最大抽搐振幅。慢慢地移动的力换能器拉伸的肌肉，同时监测的抽搐反应的，直到抽搐振幅达到最大振幅。 13。运动神经元细胞内记录从这个角度上，标准的电生理技术用于制备细胞内的电极，穿透脊髓神经元，将其识别为一个运动神经元。 <ol><li>一个玻璃微拉来了〜1微米提示，用吸管拆卸器（P-97微管牵引机，萨特仪器）。填充电极的KCl的3M溶液（电阻电极10-20MΩ）。 </li><li>使用微定位器，驱动微吸管，连接到脊髓内的放大器（Axoclamp 2B，Axon Instruments公司）。监控由电子刺激引起的局部场电位ACH神经定位电机池的小腿三头肌。 </li><li>监测的微电极的电阻的同时，小心地接近推定的运动神经元。当推压的膜，电阻增加。可以使用的“嗡嗡”功能的细胞内放大器的某个时候将促进渗透。 </li></ol> 14。安乐死过程在实验结束时，由过量的戊巴比妥钠（210毫克/千克IV）中，然后用断头动物安乐死。

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没有利益冲突的声明。

这里所描述的制备是首先，允许，在成年小鼠的腰部的运动神经元和其轴突支配肌肉纤维所产生的力的测量，同时细胞内记录。 由于体积小的动物，这个准备所需的手术技巧可以是具有挑战性的收购。然而，一旦这些技能掌握，整个手术可以在三小时内进行，并能根据动物存活长达7个小时的手术过程结束后录音。这项技术的成功，本质上是对麻醉的管理队伍。这是至关重要?...

A correction was made to <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>. There was an error in the name of one author, Marin Manuel. The author's name has been corrected to:
Marin Manuel 
 
instead of: 
 
Manuel Marin

A correction to author list has been made for article <a href="http://www.jove.com/video/4312">Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo</a>.

Erratum: Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In Vivo

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>硫酸阿托品</td><td> Aguettant </td><td></td><td></td></tr><tr><td> Methylprenidsolone </td><td>辉瑞公司</td><td>解决方案Medrol </td><td></td></tr><tr><td>戊巴比妥钠</td><td>赛诺菲 - 安万特</td><td>戊巴比妥</td><td></td></tr><tr><td>氯胺酮</td><td></td><td></td><td></td></tr><tr><td>二甲苯胺噻嗪</td><td></td><td></td><td></td></tr><tr><td>葡萄糖</td><td></td><td></td><td></td></tr><tr><td>血浆扩容</td><td>罗杰·白龙</td><td>胶浆</td><td></td></tr><tr><td>钝头剪刀</td><td> FST </td><td> 14079-10 </td><td></td></tr><tr><td>钝精剪</td><td> FST </td><td> 15025-10 </td><td></td></tr><tr><td> Vannas春风似剪刀</td><td> FST </td><td> 15002-08 </td><td></td></tr><tr><td>精细钳锯齿</td><td> FST </td><td> 11370-32 </td><td></td></tr><tr><td>精细钳锯齿</td><td> FST </td><td> 11370-31 </td><td></td></tr><tr><td>坎宁安脊柱适配器</td><td> Stoelting有限公司</td><td></td><td></td></tr><tr><td> KWIK-CAST密封胶</td><td> WPI </td><td> ＃KWIK-CAST </td><td></td></tr><tr><td>换气扇</td><td> CWE公司</td><td> SAR-830​​/AP </td><td></td></tr><tr><td> Capnograph </td><td> CWE公司</td><td> μcapstar </td><td></td></tr><tr><td>加热毯</td><td>哈佛设备</td><td> 507221F </td><td></td></tr><tr><td>细胞内放大器</td><td>轴突Instrume新界南总区</td><td> Axoclamp 2B </td><td></td></tr><tr><td>用移液管拉马</td><td>萨特仪器</td><td> P-97 </td><td></td></tr><tr><td>氯化钾</td><td> Sigma-Aldrich公司</td><td> P9333-500G </td><td></td></tr></tbody></table>

simultaneous intracellular recording of a lumbar motoneuron and the force produced by its motor unit in the adult mouse in vivo

脊髓运动神经元长期以来一直是良好的模型系统的研究神经功能，因为它是一种中枢神经系统的神经元与独特的性能（1）因此，具有易于识别目标的（肌纤维）和具有非常著名的功能（来控制肌肉收缩），（2）的收敛许多脊髓和下行网络的目标，故名“最后共同通路”;（3）有一个大的胞体，这使得它可以穿透它们用锋利的内电极。此外，当在体内研究，这是能够同时记录的运动神经元的电活动的和由他们的肌肉目标开发的力。因此， 在体内的运动神经元进行细胞内记录将实验者的研究中的独特地位，在同一时间，所有的隔间“机动装置”（名字的运动神经元，其轴突，和它支配的肌纤维1）：撞击的运动神经元的输入，运动神经元的电生理特性，以及这些特性的影响的运动神经元的生理功能， 即其运动单位所产生的力。然而，这种方法是非常具有挑战性的，因为该制剂不能瘫痪，从而减少细胞内记录的机械稳定性。因此，这种实验只被实现在猫和大鼠。然而，这项研究对脊髓运动系统可以使一个强大的飞跃，如果有可能进行类似的实验，在正常和转基因小鼠。 由于技术上的原因，大多是在小鼠的脊髓网络的研究仅限于新生儿在体外的准备工作，是不成熟的运动神经元和脊髓的网络，运动神经元分开，他们的目标，并研究时，莫片toneurons分离大部分来自其输入。直到最近，只有几组2-4体内的运动神经元细胞内记录，包括我们的团队出版了一本新的准备，使我们能够获得非常稳定的运动神经元在成年小鼠体内的录音5,6。然而，这些录音瘫痪的动物， 即没有可以记录这些运动神经元的输出力。在这里，我们提出了一个延伸，这种原始的准备中，我们能够获得的运动神经元的电生理特性和开发他们的运动单位的力量的同时录制。这是一个重要的成就，因为它使我们能够识别不同类型的运动神经元的基础上的力的分布，从而揭示它们的功能。再加上遗传模型干扰脊髓节段电路7-9，或reproducting人disea本身10,11，我们希望这是一个必不可少的工具，为研究脊髓运动系统的技术。

图1显示了如何识别渗透后小腿三头肌组运动神经元。在低的刺激强度，仅可观察到单突触EPSP（ 图1A）。在较高的强度，在EPSP可能会对大到足以触发“顺”尖峰（ 图1B）。在刺激强度更高，出现一个全或无的逆向秒杀，用更短的等待时间比单突触EPSP（ 图1C）。如果有足够的电流被注入通过微电极触发一个尖峰，EMG活动可以记录对肌肉一个短暂的...

Watch this Scientific Journal Video about Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo at JoVE.com

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

这种新方法允许同时细胞内记录一个单一的成年小鼠运动神经元和其肌肉纤维所产生的力的测量。合并后的调查是在正常和转基因动物的运动单位的电气和机械性能的神经肌肉系统的研究的一大突破。

同时细胞内记录的车组在成年小鼠的腰椎运动神经元产生的力<em在体内</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

simultaneous-intracellular-recording-lumbar-motoneuron-force-produced

Research

JoVE Journal

Neuroscience

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

14.6K 次观看.  Northwestern University Feinberg School of Medicine. 这种新方法允许同时细胞内记录一个单一的成年小鼠运动神经元和其肌肉纤维所产生的力的测量。合并后的调查是在正常和转基因动物的运动单位的电气和机械性能的神经肌肉系统的研究的一大突破。

视频: Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

一个在培养皿中的功能电机组：脊髓植和肌肉细胞共培养

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

急性片的成年小鼠前脑的神经细胞迁移的延时成像

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

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

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

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

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

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

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

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.