我们要感谢我们实验室的所有成员，他们的意见，有益的讨论和对手稿的评论。我们还要特别感谢玛尔塔·科赫和Bassem哈桑很好的建议，我们直到1.5小时薏仁前等待解剖大脑重塑实验培养。我们也感谢他们的共聚焦显微镜的使用在NHGRI成像设施。 NINDS的院内研究计划，国立卫生研究院（Z01 NS003106）"基本法"神经科学计划支持这项工作。

A.媒体的制备<ol><li>准备施耐德过滤消毒的果蝇媒体10％小牛血清和1％青霉素/链霉素补充完整的媒体。 </li><li>媒体应准备新鲜的每一次。此外，新鲜的预冻媒体等份，​​每次使用前可以解冻。 </li><li>前培养，添加10μg/ ml的胰岛素和2微克/毫升蜕皮媒体。药品准备集中的股票和小等分冻结。不能再冷冻解冻等份。 </li><li>所有的工作方案应保持在25°C。 </li></ol>培养的幼虫脑 1。幼虫大脑的选择和剖析<ol><li>加入500微升至12毫米Millicell小细胞培养插入媒体。解剖大脑将被放置在这些插入方便染色。 </li><li>添加少量的准备媒体到夹层手表的玻璃。 </li><li> ü唱小铲，拿起徘徊第三龄幼虫，并将其放置在手表玻璃。 （我们假设， 体外培养方法也同样适用于第一或第二龄幼虫，但我们还没有测试过这个。） </li><li>控股结束后，幼虫用钳子小心地打开了一个＃5镊子第二对前结束。 </li><li>迅速解剖大脑，保持腹神经索完好。 </li></ol> 2。 前解剖的幼虫脑的体外培养<ol><li>使用预湿枪头，小心脑转移到细胞培养插入，与媒体预填充。 </li><li>转让的大脑，在媒体，孵化器在25°C间，直到达到所需的时间点。此方法适用于文化在48小时有效。 </li><li>在8小时更换介质。除此之外，媒体每5-6小时更换一半。 </li></ol> 3。药理操纵幼虫脑这种体外培养的方法可用于药理证实以前在我们的实验室1所示的遗传相互作用。 <ol><li>新增100微米roscovitine和100μMolomoucine准备的媒体。 </li><li>解剖的大脑传输到该媒体。 </li><li>文化的大脑在25°24小时。 </li><li>在8小时更换介质。除此之外，取代一半的媒体（毒品），每5-6个小时。 </li></ol>培养的蛹脑 1。夹层蛹的选择<ol><li>纪念瓶/瓶白色的蛹。 </li><li>只有选择，是完全白色的蛹。不应包括任何着色蛹。 </li><li>这个阶段是0小时后Puparium组（APF）的。 </li><li>这些蛹将准备用于解剖后1.5小时标记。这一步是至关重要的，以确保发生在体外发育修剪</eM&gt;文化。 </li></ol> 2。剖析蛹<ol><li>加入500微升至12毫米Millicell小细胞培养插入媒体。 （解剖大脑将这些刀片放在方便的染色）。 </li><li>一旦已达到所需的时间点，添加少量的准备媒体到夹层手表的玻璃。 </li><li>用一个小的，湿的锅铲，拿起蛹以前解剖标记，并将其放置在手表玻璃。 </li><li>控股的蛹后端的钳子，小心地打开了＃5镊子第二对蛹的情况下，前结束。 </li><li>迅速解剖出脑，保持完整的腹神经索和连接。 </li></ol> 3， 前蛹脑解剖的体外培养<ol><li>仔细的脑转移到细胞培养插入，与媒体预填充。 </li><li>转移的大脑，在媒体，孵化器在25°C间，直到所需的时间-Point达到。我们用这种方法长达10小时在文化所示的实验，但它可以扩展到较长时期的观察和治疗，如果需要的话。 </li><li>超过8小时，薏仁更长的时间点，每5-6小时更换媒体。 </li></ol>从这一步，该协议仍然不变，幼虫和蛹。 B.固定解剖大脑<ol><li>一旦达到所需的时间点，消除媒体和添加500μl1X PBS 4％甲醛的0.5％的Triton X-100（PBST）30分钟，15分钟后，更换新鲜的甲醛。 </li><li>确保不要让大脑干更换固定液媒体时。 </li><li>洗掉4个10分钟的1X PBS洗涤，0.5％用TritonX-100（PBST）的甲醛。 </li></ol> C.染色固定脑<ol><li>后甲醛已被洪水冲断，阻断大脑中的1X PBS封闭液，用1％正常1小时山羊血清和1％的牛血清白蛋白。 </li><li>加入封闭液的主要抗体。 </li><li>孵育大脑在4℃过夜</li><li>第二天早上，洗掉4个10分钟的PBST清洗的主要抗体。 </li><li>加入封闭液的二次抗体。 </li><li>在室温下离开4个小时。 </li><li>洗掉4个10分钟的PBST清洗二次抗体。 </li></ol> D.安装固定和染色的大脑显微镜<ol><li>平衡1小时在Vectashield安装媒体的大脑。 </li><li>安装使用Vectashield安装媒体上的玻璃幻灯片的大脑。 </li><li>安装前盖玻片，放置在大脑两侧的玻璃碎片。这可以防止从被压扁的大脑，同时保持足够样本薄扫描共聚焦显微镜的焦平面。大脑大约面向后安装，以方便查看感兴趣的功能。必要时，脑图像Imaris软件提供的文件和测量的最佳旋转。 </li><li>封盖单透明指甲油。 </li><li>商店滑离光。 </li></ol> E.代表结果 图1显示面板（一）野生型果蝇三龄幼虫的大脑和（B）果蝇表达显性负的Cdk5的衍生工具（Cdk5DN）（康奈尔-克劳利等，2000）。都表达肌动蛋白GFP（绿色）在MB伽玛神经元。如前所述（Trunova 等 ，2011），有一个地区，在近端野生型伽玛失败积累肌动蛋白GFP的神经元轴突，从而呈现出"明确区"（一个支架）， fasciclin 2蛋白（红色）累计最多只能到本区腹侧边界（白色横线）在轴突。在图1（a）第三小组的虚线表示的MB的立场，强调行为在"清区"和其腹侧边境。在苍蝇表达Cdk5DN，明确的肌动蛋白区是缺席（二）Fasciclin 2（FasII）免疫背侧通过该地区的传播。 （请注意，可变数量的水平跨越的MB FasII阳性轴突项目;这些都是不相关的表型）。比例尺代表20微米。 我们用体外培养的方法，我们对待野生型果蝇的roscovitine和olomoucine的，Cdk5的活性抑制剂的组合， 图2显示了第三龄幼虫（一）大脑的控制面板和药物治疗（二）野生型果蝇。苍蝇表达显性负Cdk5的构造，24小时与Cdk5的抑制剂治疗大脑相似，表明损失的肌动蛋白明确的区域特征的转变和在FasII边界（方括号）。白色线条显示的野生的类型FasII边界的位置。比例尺代表20微米。 figu重3显示了从0-10小时薏仁果蝇蘑菇体的代表系列，膜针对性mCD8-GFP（绿色）标记。括号表示花萼，就是MB的树突状预测。变态之前，我们可以看到MB的轴突后背成腹侧窃喜，在厚厚的捆绑，以及密集的树突状乔木（支架）引起的荧光信号的"阴霾"。面板A显示大脑解剖时表示。请注意在该地区的树突修剪括号表示，这样6-8小时，薏仁，树突状信号的阴霾了（虽然轴突信号不受影响）。在B组的大脑进行解剖0小时薏仁之前，内部蜕皮穗（杜鲁门和Riddiford，2002年），并培养体外所示。这些表明没有树突发育修剪;树突的信号，而持续在10小时薏仁。为了规避这一点，蛹被标记在0小时的薏仁，但在1.5小时APF和培养解剖。窗格升C显示了一个从0-10小时薏仁这些果蝇蘑菇体的代表系列。至于非培养的样品中，树突状信号检测不到8小时薏仁。比例尺代表20微米。

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没有利益冲突的声明。

<html>慢性组织结构和功能，包括转基因的突变和表达的基因操作，通常会产生直接的影响和后期的间接后果，可以是非常难以分开的组合。补充与药理遗传方法，可以方便的时间后面的表型课程的审讯。例如，这一直是一个强大的方法，在不同的细胞骨架成分的贡献在10 果蝇精子解剖。在当前的例子，它使我们有一个Cdk5的持久性要求保持AIS在果蝇的结构。在其他实验中，我?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 终浓度 </td></tr><tr><td>施耐德的果蝇媒体</td><td> Invitrogen公司</td><td> 21720-024 </td><td></td></tr><tr><td>胎牛血清（热灭活） </td><td> Invitrogen公司</td><td> 10082-1​​47 </td><td> 10％ </td></tr><tr><td>青霉素/ </td><td> Invitrogen公司</td><td> 15140-122 </td><td> 1％ </td></tr><tr><td>胰岛素</td><td> Invitrogen公司</td><td> 12585-014 </td><td> 10μg/mL组</td></tr><tr><td>蜕皮</td><td> Sigma-Aldrich公司</td><td> H5142 </td><td>为2μg/ml </td></tr><tr><td> roscovitine </td><td> Sigma-Aldrich公司</td><td> R7772 </td><td> 100μM </td></tr><tr><td> olomoucine </td><td> Sigma-Aldrich公司</td><td> O0886 </td><td> 100μM </td></tr><tr><td>正常山羊血清</td><td> Invitrogen公司</td><td> 50062Z </td><td> 1％ </td></tr><tr><td>牛血清白蛋白</td><td> Fisher Scientific则</td><td> M9501 </td><td> 1％ </td></tr><tr><td>甲醛</td><td> Sigma-Aldrich公司</td><td> D4551 </td><td> 4％ </td></tr><tr><td> TRITON-×100 </td><td> Fisher Scientific则</td><td> BP151 </td><td> 0.5％ </td></tr><tr><td>磷酸盐缓冲液</td><td> Invitrogen公司</td><td> 70011-044 </td><td> 1X </td></tr><tr><td> vectashield </td><td>媒介实验室</td><td> H1000 </TD&gt; <td></td></tr><tr><td> Millicell小细胞培养插入</td><td> Millipore公司</td><td> PI8P01250 </td><td></td></tr><tr><td> multidish，4井</td><td> Thermo Scientific的</td><td> 176740 </td><td></td></tr><tr><td>杜蒙＃5镊子</td><td>罚款科学工具</td><td> 11251-20 </td><td></td></tr><tr><td>显微镜载玻片</td><td> Fisher Scientific则</td><td> 12-544-7 </td><td></td></tr><tr><td>盖玻片</td><td> Fisher Scientific则</td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

我们描述了整个果蝇大脑的体外培养的方法。这可以被用来作为一个调查，让急性药物干预和细胞过程的实时成像的细胞生物学和中央大脑结构的发展对位慢性遗传操作。由于该技术的一个例子，以前的工作，从我们的实验室1显示了先前未知的亚车厢之间的果蝇中央脑的神经轴突轴突和somatodendritic的车厢。结果表明基因，称为轴突起始段（AIS）的2，这个车厢的发展依赖于特定的神经元细胞周期蛋白依赖性激酶，Cdk5的。我们在这里展示，野生型果蝇幼虫大脑Cdk5的具体药理抑制剂roscovitine和olomoucine的3 体外处理导致急性肌动蛋白组织的变化，在细胞表面蛋白Fasciclin 2，模仿，缺少Cdk5的活性基因的突变体中看到了更改的本地化。 一个体外培养技术的第二个例子是提供重构胚胎蘑菇体（MB）的变态从幼虫到成虫期间伽玛神经元的连接。蘑菇体是在飞4嗅觉学习和记忆的中心，这些伽玛神经元的修剪在蛹发展的轴突和树突的分支，然后在稍后的时间点，建立了成人的支配模式5重新扩展分行。 MB的这些神经元修剪已通过6个突起的分支本地变性发生，蜕皮激素，类固醇激素引发的一个机制，在蜕皮激素受体​​B1 7所示，这是依赖于该活动泛素-蛋白酶体系统6。我们对体外培养的方法可以E中审问进一步发展重塑的机制。我们发现，在体外培养的设置，伽玛神经元的MB的概括发展修剪的过程中，同一个时间过程类似体内 。然而，这是必不可少的，之前为了explanting组织细胞的承诺不可逆转的蜕变，等待，直到1.5小时后，形成puparium;导致很少或根本没有文化的变态在对pupariation的发病动物解剖。因此，适当的修改，在体外培养的方法可以应用于研究动态以及中央脑生物学稳态方面。

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

本文介绍了一种方法，由哪一个可以模仿<em&gt;在体内</em&gt;发展<em&gt;果蝇</em&gt;在一个蘑菇体<em&gt;体外</em&gt;文化系统。

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

ex-vivo-culturing-of-whole-developing-drosophila-brains

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Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

13.0K 次观看.  National Institute of Neurological Disorders and Stroke. 本文介绍了一种方法，由哪一个可以模仿在体内发展果蝇在一个蘑菇体体外文化系统。

视频: Ex vivo Culturing of Whole, Developing Drosophila Brains

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

前子宫内胚胎小鼠脑电穿孔和脊髓切片培养迁移 GABAergic 中间神经元的活体成像

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...