我们感谢孝彦松田博士提供pCAG-mCherry建设。陶博馆的支持由从Schweppe基金会事业发展奖“，从白厅基金会的资助。

1。提供制备（没有视频） <ol><li>青霉素/链霉素在1X PBS（PEN /链球菌）准备一个新鲜的1X稀释。不超过50毫升，是必要的。用0.22微米的注射器过滤器过滤。 </li><li>准备注射结合所需的质粒结构结构。到终体积10μL适当的化学计量学在的pCAG标志矢量我们把​​所有的结构。此外，14 pCAG-GFP或pCAG-mCherry应增加用于跟踪电效率和电穿孔细胞。我们用这个实验pCAG-mCherry。最后，在终浓度为0.05％，0.22微米过滤快速的绿色染料到胚胎小鸡midbrains的可视化的质粒DNA混合物注入效率。 </li></ol> 2。蛋制备（视频） <ol><li>为了获得农户11阶段的胚胎，卵需要培养100华氏度（37.8°）约41小时后施肥与25-40％的湿度。地方鸡蛋和他们一边用铅笔划线标记每个顶部。放置托盘中预温，湿的孵化器。慢慢地摇动鸡蛋孵化平台转向功能设置“自动”。“检查湿度水位每24小时。 </li><li>在实验开始前的权利，消除鸡蛋从孵化器，并在室温下离开。这将有助于减缓发展和防止过度开发。 </li><li>每个鸡蛋的表面用70％乙醇的清洁和擦拭一个Kimwipe。 </li><li>以两个4厘米的透明胶带件，并坚持他们端上鸡蛋大矩形比铅笔划线。对鸡蛋顺利磁带。这是将窗口。磁带将防止脱落窗口时切开小块蛋壳。 </li><li>使用10 mL注射器和18½号针头画鸡蛋白蛋白出5毫升。揭开外壳后，针距角从蛋黄，以不打扰或损坏胚胎的同时去除白蛋白。 70％的鸡蛋之间的乙醇清洁的针，但要确保针头刺入下一个蛋之前是干。 </li></ol> 3。玻璃注射针的制备（视频） <ol><li>注射针头使用以下设置火焰/布朗微管普勒（萨特仪器型号P-97）：670热，拉120，VEL 40，时间165。世界精密仪器（API），4英寸1.0毫米薄壁毛细管玻璃（API TW100F-4）是用来使针。 </li><li>使用P10的吸管和长尖毛细管装载枪头（Eppendorf公司5242956.003）加载拉玻璃针。加载5-10微升，取决于多少鸡蛋是被注入。填补玻璃针缓慢，气泡缺席质粒解决方案创建一个连续列。 </li><li>放置显微操作（Narishige立方毫米）和Picospritzer的的第三微量注射器针头持有加载的针。根据dissecti针在显微镜和修剪针，在绿色的液体停止的地步。它有助于使用一个黑暗的背景，以更好地可视化针。适当的针修整与实践。 </li></ol> 4。脑/神经管注射（视频） <ol><li>使用春天剪刀剪卵的直径2-2.5厘米的窗口。开始从以前的针孔窗口绘制白蛋白时提出，在你的手和旋转的鸡蛋，你砍。务必清理与70％的鸡蛋之间的乙醇饱和Kimwipe剪刀。 </li><li>查看夹层显微镜下的鸡胚。它可能是必要培训眼睛。它可以帮助可视化由0.22微米过滤20％的墨汁注射PBS根据鸡胚的胚胎。然而，我们已经找到了这一步，应尽量避免，因为它往往以降低生存。 </li><li>注射效果最好，如果胚胎定位在平行针的路径，头部远离针。慢慢地刺进了45度角，以中脑，后脑交界处的神经管。这是非常关键的，不仅填补无显著扩散到脑和后脑区域代表与质粒DNA和快速的绿色染料脑的神经囊泡。开始注入与该Picospritzer微量注射器的时间设置为25毫秒，这可能需要改变取决于针修整。在一般情况下，使用10-50毫秒之间。 </li></ol> 5。电（视频） <ol><li>检索注射针头。地方3滴在PBS解决胚胎蛋的1X笔/链球菌。 </li><li>检查设置BTX EMC830 electroporator： <table border="1"><tbody><tr><td> 电压： </td><td> 20 V的 </td></tr><tr><td>脉冲长度： </td><td> 25毫秒</td></tr><tr><td> ＃脉冲： </td><td> 3 </td></tr><tr><td>区间： </td><td> 500毫秒</td></tr><tr><td>极性： </td><tD&gt;单极</td></tr></tbody></table></li><li>神经管的底部在同一水平位置放置2毫米长的L形的铂电极，与胚胎脑坐在中间的两个3毫米外电极之间。调整电极与神经管的底部是腹脑高效电的关键。为了实现特定脑，电，我们两个定制的L形铂电极短2毫米长的提示取代原来的A型肉毒毒素电极。这些铂电极直径0.5mm的铂丝（阿法埃莎）。这些铂电极有足够长的一个阶段11个鸡胚，这是约0.9毫米长，没有针对脑或后脑地区，包括中脑。按BTX的脚踏一次。应每个成功电电极周围产生气泡。从鸡蛋小心地取出电极和擦拭ELECTR用70％乙醇覆盖Kimwipe颂小康。将1X笔/链球菌的解决方案，在鸡蛋胚胎，另3滴。 </li><li>覆盖5厘米×5厘米明确包装胶带一块的窗口。务必密封蛋和蛋内容不与磁带接触来。 </li></ol> 6。培育和收获<ol><li>将蛋放回孵化器。确保孵化平台的转折点是OFF。孵化蛋，直到达到所需的农户阶段23。 </li><li>收获生活的胚胎和共同PBS填充培养皿放入每个条件。屏幕收获胚胎荧光解剖显微镜下，保持与mCherry表达只胚胎。 </li><li>转移到个别井24井的菜的胚胎。填写1毫升新鲜的2％多聚甲醛（PFA）每口井，并允许以修复2-3个小时，在4°C。 </li><li>洗三次固定的胚胎在PBS每10分钟。地方胚胎顺序我N 15％和30％的蔗糖，直至平衡。胚胎最初将浮在蔗糖，但会下沉时的平衡。 </li><li>荧光解剖显微镜下检查胚胎，每个胚胎票据mCherry表达模式和水平上。在华侨城中嵌入每个胚胎。这是非常关键的东方胚胎的正常后续分析（ 图1），以产生最佳的脑截面。 </li><li>准备18微米厚的脑部分使用徕卡CM3050S低温恒温器。通过与相应的细胞类型标记免疫分析脑发展和多巴胺神经元分化。 </li></ol> 7。代表结果 如图1所示的电穿孔和分析鸡胚脑图解。在鸡胚注射和电穿孔成功，mCherry表达应该是在中脑区域的进一步发展后可见（ <stronG&gt;图2A，乙，丙类）。假设mCherry与其他共同电穿孔基因表达的相关性，可以被固定参展适当mCherry表达的胚胎和嵌入式脑冰冻切片，以备进一步研究（ 图2D）。一般来说，约50％的中脑腹侧神经祖细胞，可有效地转用上述协议。电穿孔神经祖细胞中的多巴胺的命运规范mCherry合作定位可以通过检查，标志标记基因，多巴胺能神经元的标记，如酪氨酸羟化酶（TH）（ 图3）。腹侧中脑祖域的图案可以由不同的祖域标记的合作immnostaining冰冻切片进行调查。 不足mCherry表达异位基因有可能不能有效地表示。在此条件下的胚胎不应该被用于进一步的研究。同样，胚胎不求生存，以实验过程结束，不能用于数据收集和分析。 <img alt="图1" src="/files/ftp_upload/4017/4017fig1.jpg" /> 图1。 在OVO电检测鸡胚脑的插图。 HH阶段11胚胎鸡胚注射与pCAG-mCherry连同快绿混合可视化（一）注射过程中基因的兴趣。与DNA结构的填充后，midbrains电穿孔方波electroporator。胚胎继续培养，直到达到所需的农户阶段23。然后收获mCherry阳性胚胎，固定的，和嵌入式（乙）。脑冰冻切片与切割白线表示在图1B飞机，准备与脑酪氨酸羟化酶（TH）（三）如标记免疫分析。 <a href="http://www.jove.com/files/ftp_upload/4017/4017fig1large.jpg" target="_blank">点击这里</a>查看大图。 <img alt="图2" src="/files/ftp_upload/4017/4017fig2.jpg" /> 图2。从鸡胚脑冰冻切片的制备。经过孵化41小时，农户阶段23鸡胚表达利益mCherry和基因的收获（，A，B和C），固定与煤灰，与蔗糖处理，并嵌入在OCT为cryosectioning。鸡胚精心为导向，以确保脑节的筹备工作。一个典型的胚胎获取脑部分的形态和切削平面显示（D）， <a href="http://www.jove.com/files/ftp_upload/4017/4017fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4017/4017fig3.jpg" /> 图3。电穿孔中脑部分的分析。从mCherry表达水平高的胚胎冰冻切片（一）编制及与antibod染色IES，识别标志标记基因的利益（B）和中脑多巴胺能神经元标记TH（三）。与pCAG mCherry和pCAG旗空载体作为控制电穿孔胚胎midbrains <a href="http://www.jove.com/files/ftp_upload/4017/4017fig3large.jpg" target="_blank">点击这里查看大图</a> 。

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作者有没有潜在的利益冲突的披露。

在鸡胚脑OVO电提供了一种低成本，快速的替代代转基因或基因敲除动物在中脑多巴胺神经元发育基因的功能进行的体内实验研究。使用短2毫米长L形的铂电极与胚胎脑特异的DNA注入是实现高效的兴趣在中脑多巴胺能神经元祖细胞的基因表达的关键。此外，使用正确的农户阶段11鸡胚是至关重要的。这是因为，第一，在农户阶段11，鸡脑的发展是不够先进等，前脑，中脑，后脑形...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>受精鸡蛋</td><td>查尔斯河实验室</td><td></td><td></td></tr><tr><td>鸡蛋孵化器</td><td> GQF制造</td><td> 1502运动员</td><td></td></tr><tr><td> A型肉毒毒素Electroporator </td><td> A型肉毒毒素哈佛仪器</td><td> ECM830 </td><td></td></tr><tr><td>电极</td><td> A型肉毒毒素哈佛仪器</td><td> 45-0162“L”形genetrodes </td><td> ECM830 electroporator </td></tr><tr><td>铂铱丝</td><td>阿法埃莎</td><td> ＃10056 </td><td> 0.5毫米直径 为了使2毫米长的电极</td></tr><tr><td>玻璃毛细管</td><td>世界精密仪器</td><td> TW100F-4 </td><td></td></tr><tr><td> microloader吸管提示</td><td> Eppendorf公司</td><td> 5242 956.003 </td><td></td></tr><tr><td>墨汁</td><td>斯台普斯</td><td></td><td>过滤器在使用前20％ </td></tr><tr><td>微量注射器</td><td>帕克自动化， Parker Hannifin公司合作</td><td> picospritzer三</td><td></td></tr><tr><td>荧光解剖显微镜</td><td>徕卡</td><td> MZ16F </td><td></td></tr><tr><td>微管拔</td><td>萨特仪器</td><td>的D-97 </td><td></td></tr></tbody></table>

in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

多巴胺能神经元位于中腹脑控制的运动，情绪化的行为，和奖励机制1-3。腹侧中脑多巴胺神经元功能障碍是帕金森氏症，精神分裂症，抑郁症，老年痴呆症1-5牵连。因此，研究中脑多巴胺神经元分化的调节不能只提供重要的洞察到调节脑发育和神经祖命运规范机制，而且还有助于开发治疗多种人类神经系统疾病的新的治疗策略。 多巴胺神经元分化从排队心室胚胎腹侧中脑区的神经祖细胞。神经祖细胞的发展，控制基因表达方案6,7。在这里，我们报告技术利用电汉堡汉密尔顿（HH）阶段11脑基因表达（THirteen体节，42小时）鸡胚8,9。允许在特定的萌芽阶段的实验操作方便，在后来的发展时间点10-13决定的影响，的鸡胚外部发展。鸡胚早期阶段，13（19体节，48小时）超过HH神经管组成的具有多能分化成不同类型的细胞神经系统的神经祖细胞。的pCAG载体，其中包含CMV启动子和小鸡β-actin的增强，使强大的标志或其他抗原标记的鸡胚神经管14结构表达。在这份报告中，我们强调特别措施，以实现区域限制的基因表达，在胚胎中脑多巴胺能神经元祖细胞，包括如何注入DNA构建专门的胚胎中脑区域，以及如何针对小定制电极电。分析C希克在稍后阶段的脑体内质粒载体介导的增益功能和亏损的功能研究脑发展系统提供一个很好的。实验系统的改造，可以延长该法执行的命运映射分析和研究基因表达调控神经系统的其他部分。

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

评估的功能和在发展中脑多巴胺能神经元的基因调控，我们描述了一种方法，涉及<em&gt;在OVO</em&gt;鸡胚腹侧中脑多巴胺能神经元祖细胞质粒DNA结构的电。这种技术可以用来实现感兴趣的基因的高效表达，以研究脑发展和多巴胺神经元分化的不同方面。

在OVO在鸡胚中脑多巴胺神经元发育中的基因功能研究电穿孔

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

in-ovo-electroporation-chick-midbrain-for-studying-gene-function

Research

JoVE Journal

Neuroscience

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

11.3K 次观看.  Children's Hospital of Chicago Research Center. 评估的功能和在发展中脑多巴胺能神经元的基因调控，我们描述了一种方法，涉及在OVO鸡胚腹侧中脑多巴胺能神经元祖细胞质粒DNA结构的电。这种技术可以用来实现感兴趣的基因的高效表达，以研究脑发展和多巴胺神经元分化的不同方面。

视频: In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

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 ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

在微RNA在腹鸡中脑大毛表达

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

从人多能干细胞定向多巴胺能神经元分化

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Studying Brain Function in Children Using Magnetoencephalography

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the human brain. MEG is quiet and less likely to induce claustrophobia compared with magnetic resonance imaging (MRI). It is therefore a promising tool for investigating brain function in young children. However, analysis of MEG data from pediatric populations is often complicated by head movement artefacts which arise as a consequence of the requirement for a spatially-fixed sensor array that is not affixed to the child's head. Minimizing head movements during MEG sessions can be particularly challenging as young children are often unable to remain still during experimental tasks. The protocol presented here aims to reduce head movement artefacts during pediatric MEG scanning. Prior to visiting the MEG laboratory, families are provided with resources that explain the MEG system and the experimental procedures in simple, accessible language. An MEG familiarization session is conducted during which children are acquainted with both the researchers and the MEG procedures. They are then trained to keep their head still whilst lying inside an MEG simulator. To help children feel at ease in the novel MEG environment, all of the procedures are explained through the narrative of a space mission. To minimize head movement due to restlessness, children are trained and assessed using fun and engaging experimental paradigms. In addition, children's residual head movement artefacts are compensated for during the data acquisition session using a real-time head movement tracking system. Implementing these child-friendly procedures is important for improving data quality, minimizing participant attrition rates in longitudinal studies, and ensuring that families have a positive research experience.

This article introduces a child-friendly research protocol designed to improve data quality by reducing head movement during pediatric magnetoencephalography (MEG). We familiarize families with the MEG environment, train children to remain still using an MEG simulator, and correct for residual head movement artefacts using a real-time head movement detection system.

脑磁图在儿童脑功能研究中的应用

Magnetoencephalography (MEG) is a non-invasive neuroimaging technique which directly measures magnetic fields produced by the electrical activity of the ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...