第1部分。准备手术区<ol><li>如果与生物危险性试剂（如逆转录病毒载体）的工作，手术应进行第二生物安全柜（BSC）内。超声背向散射显微镜（UBM）应坐在旁边的BSC，与换能器内的BSC，并在举行一个特别设计的手术阶段由电机控制支持。建构在视频显示的阶段所需的组件列上线。这份清单，连同阶段的图像，从多个角度（应要求提供），应允许建设。 </li><li>开腹的开放和关闭（在腹腔被剖开，以允许访问内部器官的外科手术）通常是塑料底板的吸水垫。手术前的所有必要的工具（如剃须刀，手术剪，钳，装载autoclip应用方等），材料（如PBS中，缝线，麻醉，鼠标持有者和覆板，等）应进行消毒，并放置在手术区。手术成功的机会最大化，限制切口开放的时间是非常重要的。所有动物的处理应与MB - 10解决方案，或相当于已消毒手套。应进行反复消毒，根据需要，以确保无菌手套。 </li></ol>第2部分。鼠标麻醉<ol><li>填写与麻醉剂含有1部分戊巴比妥（苯巴比妥50毫克/毫升），4个部分，过滤消毒25毫克/毫升的PBS硫酸镁溶液1毫升注射器。这将导致在一个解决方案，是10毫克/毫升的苯巴比妥和20毫克/毫升硫酸镁。应单独加权每只小鼠注射巴比妥每公斤体重90毫克（例如，一个28 GM鼠标将被注入250μL）。 </li><li>穿透皮肤和腹部肌肉的腹部怀孕小鼠在胚胎一天（五）9.5（腹腔，IP）（如需要可以使用其他年龄段）注入。允许8-12分钟鼠标无响应。足够的麻醉应确认的动物悠然自得，和缺乏运动尾巴和脚趾捏。轻轻之间的指甲掐住是非常敏感的，缺乏任何如捏的反应，表明动物有足够的麻醉。注意甚至完全反应迟钝，尾部和脚趾捏的动物，可能在最初的手术切口，有时抽搐略有。然而，这是自反的反应，并不表示不足麻醉。应适用于以石油为基础的眼科唇膏鼠标，以避免在手术过程中干燥的眼睛。 </li><li>手术区和超声波机的设置，而等待的鼠标成为麻醉。由于使用的逆转录病毒或慢病毒需要BSL2以上的病毒注射所述的遏制，应在生物安全II内阁。此外，接触到病毒的所有解决方案和一次性材料应与10％的漂白粉溶液进行消毒。应视为手术工具和超声波设备，杀菌解决方案，如MB - 10 <a href="http://www.quiplabs.com/mb10.htm">（</a> http://www.quiplabs.com/mb10.htm），或由有关设施使用等效。 </li></ol>第3部分。暴露的胚胎<ol><li> （取决于给定的动物设施和可用空间的限制）如果可能的话，它是首选手术准备区有一个单独的位置从手术区。这将最大限度地减少动物之间的交叉污染的机会。要启动的手术，它的后面放置到动物无菌吸水的表面，并用70％乙醇冲洗腹部，彻底。可以更广泛的术前治疗（例如，连续两轮优碘和70％乙醇洗涤），但在我们的经验，这是这种类型的啮齿类动物的生存手术没有必要。采用双边缘剃刀，刮脸从腹部面积约1英寸宽，1.5英寸长的头发。使用剃刀短期快速移动，在大约45 °角。重要的是适当治疗，手术区，但不会受到过度湿润，因为这可能导致不必要的令人心寒的动物，甚至可能体温过低。 </li><li>使用精细的手术剪，先通过皮肤1"纵形切口，然后通过腹部肌肉的动物（切割通过结缔组织的皮肤肌肉，将有助于与第二个切口）。注意，整个过程护理采取保证手术工具保持无菌的，并应根据需要进行治疗用消毒液（即如果该工具提示接触与非STE激怒材料）。 </li><li>使用镊子小心吸取子宫角，并记录每边的胚胎数量。同时留下两个相邻的胚胎暴露（选择其中两个将取决于每边的配置），子宫角到动物。在此背景下，两个胚胎将被暴露在某一特定时间和沐浴用PBS，而她的母亲仍然在她的背上。 </li><li>放入有机玻璃持有人的动物在它的后面，将手术过程中（Figure1A）*.下一步，降低10厘米组织培养皿**（应消毒，使用的MB - 10前）在母亲，对通过绿色硅橡胶膜（使用L RTV硅橡胶基地，并略有放置一个镊子固化剂;陶氏康宁公司的产品编号分别为3142761和2156946）。正如你降低动物板，使用镊子夹住要注入的两个胚胎在子宫组织，并通过膜轻轻一拉。与此同时，该板块下调到持有人完全放下，图钉推举行的地方。预热（至37 ° C），然后加入PBS是菜完全覆盖的胚胎。 </li><li>将下Figure1A）创建通过暴露的胚胎在子宫壁上（Figure1B）实时成像的超声探头（含动物和暴露的胚胎注射阶段。然后探头降低到PBS浴（使用机动控制）直接对胚胎进行成像。图片可当探头远离胚胎约2-3毫米。这是非常重要，以确保探头不打胚胎，因为它来回振荡采集图像。 </li></ol> *母亲在手术过程中所掌握的持有人可以很容易地由一个机加工车间。它应包括广泛的有机玻璃基地，有两个有机玻璃双方粘在上面，并在它们之间的空间（足够宽的鼠标，而在它的后面，以适应躺在）。双方应的谷地上，然后填充黑色蜡（例如，从解剖托盘）。这蜡作为融入其中图钉将按下按住板，胚胎沐浴在注射过程中的PBS材料。 **10厘米细菌菜应该有一个洞（约直径2cm）砸出底部中心（这可以用重型便携式打孔和第一个13/16英寸的圆形模具<a href="http://roperwhitney.com/punching/2-1011.cfm">:</a> http://roperwhitney <a href="http://roperwhitney.com/punching/2-1011.cfm">.com/punching/2-1011.cfm</a> ）。此外，两方之一的中心孔（约1.5cm的距离）的小孔应与火焰加热的针（或他们可以当然演练）烧毁。真空润滑脂，将持有该板块到持有人的图钉推到黑蜡通过这些小孔，然后覆盖。真空润滑脂，以防止泄漏出来的PBS。 第4部分。装入一个显微注射针的病毒<ol><li>倒角为1mm（内径）一个时尚显微注射针拉玻璃毛细管。针的清晰度是至关重要的，不够锋利的针，可以严重危及子宫壁的渗透和amnionic囊，可导致实验失败。削尖的针，然后放入吸管持有人，这是通过油管安装手动泵输液/提款（可以使用其他设备来调节的装载和放电针，但是这就是25μLmicrosyringe连接在视频显示）。最佳的响应（以避免空气中弥漫系统的扩展和压缩），microsyringe，管，针头应填写与矿物油。 </li><li>装货前针，确保有一路针尖从注射器系统无气泡。然后绘制一个小气泡（1-2毫米长）进针开始之前加载病毒。这气囊有助于保持病毒和矿物油之间的距离。被注入病毒的股票应包含在一个终浓度为0.08毫克/毫升的聚凝胺（病毒生产的更多信息，请参阅参考文献1，2）。注意集中逆转录病毒的股票一般都含有大量的颗粒物，其中包含了大多数的传染性物质，因此不能被过滤掉了没有5 - 10倍滴度下降。另外，根据编制后，病毒的股票可能会裂解包装细胞的基因组DNA（尤其如此，如果使用293细胞生成MLV / VSV的假型病毒）的粘性。如果病毒等分是粘稠的，少量的DNA酶，我可以和应该被添加到放松的股票，或加载中针会是非常困难的。一<html>病毒的股票（8-10μL）液滴应放在一个封口膜无菌针加载准备。然后，使用撤出泵，谨慎地装载针重视，以避免堵塞。加载针放入显微注射区的位置，不小心打破针，对胚胎或超声探头。请注意，如果由于某种原因针注射前发生明显加载，它可以帮助吸引到一角，开始注射前可以出院了少量空气。这个气囊可以防止病毒在针尖（几乎总是呈现，装针无用的发生）干燥的股票。 </li></ol>第5部分。超声引导下的病毒注射液<ol><li>超声探头的实时图像创建在视频监视器上显示，并可以用来指导胚胎的显微注射针。可以使用这个系统的病毒注入羊膜囊E8.5或羊膜囊或从E9.5 - E11.5（Figure1B）脑室系统。这是程序的最困难的部分，并要求一举手一掌握（5-20​​小时取决于研究者的顺序）的大量经验。针尖在超声图像作为屏幕上的亮点是显而易见的，应按照调整探头位置，使用机动控制在Y和Z平面（移动，并在X手动控制平面）。探头的振荡方向平行的针的长度，这是极为重要的。这将保证针尖保持在运动图像平面位置注入胚胎。 </li><li>应定位于胚胎，这样的目标区域（在这种情况下，脑脑室）是随时可见，子宫材料量至少在注射路径针。此外，关键是不通过胎盘注入。一旦目标的大脑区域是内衬针进步的方向（使用的显微操纵器上的旋钮），针应只接触子宫壁，然后一个短期的快速申诉委员会提出应渗透组织。类似的方法是用来穿透amnionic囊，然后大脑。在某些情况下，可以打，经验丰富的研究员在一项议案心室，尤其是当针是非常尖锐的。移动探头，针和胚胎（移动舞台上的母亲）的机制，需要动手实践掌握。 </li><li>一旦所需的胚胎数已注入5.0丝线缝合关闭腹壁。这种类型的手术缝合机制不特定。最后，使用兽医autoclips超过主食皮肤腹部肌肉针。因为鼠标会咀嚼缝线缝合皮肤，不建议。镇痛应适用于切口面积，以减少疼痛的金额，由经验丰富的鼠标，因为它唤醒。这是建议，注入0.25％布比卡因溶液（0.8毫升/公斤，或2毫克/公斤），皮下沿切口部位的每一个几毫米。 </li><li>当鼠标轻轻复苏奠定吸水纸含有凝胶包（便于饲养和水化）在一个干净的笼子里的动物。笼放置到温暖的一个幻灯片设置在37 ° C，保持体温。惊醒时，放入一个干净的笼子的鼠标和凝胶包，并返回到动物机架。 </li><li>次日检查鼠标，任何阴道出血，腹部收缩，和/或整体心疼的样子，所有这一切都表明流产。如果是这种情况，立即安乐死的鼠标。头发以及培养和整体健康的外观显示手术成功恢复。注射的胚胎可以牺牲embryonically或出生后，作进一步的分析。如果病毒对记者如人类碱性磷酸酶（PLAP）或GFP基因，染色揭示阳性病毒感染（Figure1C）的地区。 </li></ol>代表性的成果注射的胚胎可以牺牲后1-2天注射到成年的方式。如果病毒对记者如PLAP或GFP的基因，然后记者表达用来识别病毒感染的细胞和这些细胞的克隆。例如，在图1C所示是一个产后的大脑是在子宫内感染在E9.5与逆转录病毒载体表达PLAP的组织切片。紫色集群组织化学染色后观察感染的细胞。此外，以确定病毒感染的细胞，记者表达允许一个研究细胞的形态和位置，以及基因表达状态。 NT"&gt; <img src="/files/ftp_upload/2047/2047fig1.jpg" alt="图1" /> 图1。病毒注射到E9.5小鼠脑脑室。 A.麻醉动物被放置在注射阶段。两个胚胎会被揭露和定位下指示的位置（箭头）的超声探头。位于附近的胚胎显微注射针充满病毒和内衬超声探头。 B.屏幕捕捉的实时超声图像从E9.5小鼠胚胎。五，心室; AS，羊膜囊，威斯康星大学，子宫壁。 C.胚胎在E10.5 PLAP表达病毒注射。组织化学染色PLAP揭示病毒感染事件的位置。

<ol>
	<li>Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+high-titer+pseudotyped+retroviral+vectors+with+very+broad+host+range.">Generation of high-titer pseudotyped retroviral vectors with very broad host range.</a> Methods Cell Biol. 43, 99-112 (1994).</li><li>Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+rapid+gain-of-function+studies+in+the+mouse+embryonic+nervous+system.">A method for rapid gain-of-function studies in the mouse embryonic nervous system.</a> Nat Neurosci. 2, 812-819 (1999).</li></ol>

使用超声引导下的病毒注射液可进行E8.5 - E11.5，其中只有羊膜囊，可以在E8.5注射，而两个羊膜囊和脑室系统，可以从E9.5 - E11.5注入。正确执行时，病毒颗粒注入脑室系统使用超声引导下进入脑室系统的上皮细胞，因此，可以感染前脑（大脑皮层，基底节，间脑，眼等），中脑的各种结构和后脑（Figure1C）。注入羊膜囊病毒进入细胞胚胎的外部。除了病毒，还可以用于超声引导下注入到发育中的胚胎细胞。因此，这种技术提供了多种选择，从设计实验时，选择（一段时间的发展，结构，病毒/细胞）。 至关重要的是，注射的病毒或细胞表达病毒/注射细胞易于识别的记者。从历史上看，人类胎盘碱性磷酸酶（PLAP）或GFP已被使用。从以往的经验，我们发现PLAP可以是一个非常有用的报告基因，因为PLAP组织化学和免疫组织化学染色产生一个尖锐而鲜明的单细胞染色，让来分析细胞的形态和位置，并进行克隆分析。由于病毒GFP的表达可以是淡淡的，GFP的记者可能不理想的染色组织，但有用的，如果想获得单个细胞，通过荧光激活细胞分选（FACS）。

"以下的超声背向散射显微镜可以用于此过程中，虽然在视频范式医疗P40，P40是不再市售，并已取代P60的，这是同样的价格，可以用于同一个应用程序。，分期建设中描述的其他材料与范式医疗设备的使用（S）。VEVO 2100自带小动物成像设备。 <ol><li> P60的超声生物显微镜 （UBM）从范式医疗 （ <a href="http://www.paradigm-medical.com/ubms.html">http://www.paradigm-medical.com/ubms.html</a> ）。 </li><li>从VisualSonics 2100 VEVO（ <a href="http://www.visualsonics.com/">http://www.visualsonics.com/</a> ） </li></ol>麻醉和镇痛可从Bulter施恩动物卫生戊巴比妥溶液（50毫克/毫升注射戊巴比妥钠;雅培生产）。请注意，这是一个时间表第二类受控物质，并要求获得一个缉毒局（DEA）许可证。 可从分销商的数量，包括摩尔医疗布比卡因溶液（0.25％; Hospira公司，公司，其中包括生产）。

ultrasound-guided microinjection into the mouse forebrain in utero at e9.5

小鼠的存活在宫内手术，可在开发过程中基因表达的分子操纵。然而，由于子宫壁是不透明的，在早期胚胎发育，极大地限制的能力为目标的特定部位的显微注射胚胎。幸运的是，高频率的超声波成像许可证生成可用于实时图像引导胚胎地区利益的一个显微注射针。在这里，我们描述了利用这种成像引导胚胎一天鼠标的前脑的脑室系统（E）9.5逆转录病毒载体注射。这个方法使用一个允许访问的子宫角，和一个特别设计的板，允许主机的胚胎，而他们的成像和注射生理盐水中沐浴的剖腹探查。成功的手术，往往会造成大部分或全部注入的胚胎存活到任何利益随后的时间点（embryonically或产后）。这里所描述的原则，可以稍作修改用于执行注射到E8.5胚胎羊水（从而允许尚未闭合的神经管，后沿前程度的感染），或进入脑室系统大脑在E10.5/11.5。此外，在中期源性年龄（〜E13.5），超声显像可用于直接注射到病毒感染或细胞移植特定的大脑区域。使用超声显像在小鼠子宫内注射，以指导是一个非常强大的技术许可证，否则将异常艰难，如果不是不可能的方式操纵分子和细胞的小鼠胚胎。

Watch this Scientific Journal Video about Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5 at JoVE.com

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

小鼠的存活在宫内手术，可在开发过程中基因表达的分子操纵。在这里，我们描述了使用高频率的超声波成像引导逆转录病毒载体注射到鼠脑在胚胎一天（五）9.5。

超声引导下显微注射到小鼠前脑在子宫内在E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

18.0K 次观看.  Johns Hopkins University School of Medicine. 小鼠的存活在宫内手术，可在开发过程中基因表达的分子操纵。在这里，我们描述了使用高频率的超声波成像引导逆转录病毒载体注射到鼠脑在胚胎一天（五）9.5。

视频: Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

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 ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

超声引导下显微注射到小鼠前脑在子宫内在E9.5

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