我们要感谢Drs。 Leslayann Schecterson，Yuan Wang，Andres Barria和Ximena Optiz-Araya太太，为协议设置和提供质粒提供初步帮助。这项工作得到NIH / NIDCD授权DC013841（JTS）的支持。

所有程序均经西北大学机构动物护理和使用委员会批准，并按照国家卫生研究院"实验动物护理和使用指南"进行。 蛋处理<ol><li>从当地的供应商处购买受精卵。在孵育前不要超过5天，将冰箱中的鸡蛋在13°C储存。 1周后胚胎活力明显下降。 </li><li>用70％乙醇清洗每个鸡蛋，然后放入培养箱中。 </li><li>在温度设定在38°C〜50％湿度的孵化器中，在其侧面设置1-2打鸡蛋。这确保了电穿孔的适当胚胎定位和最佳的生存能力。 注意:孵化48-52小时后，鸡蛋将在汉堡汉密尔顿（HH）阶段〜12-13，并准备进行电穿孔。 </li></ol>准备<ol><li> 质粒米兴<ol><li>每7μLDNA混合物加入1μL0.1％快速绿色（0.1％在18MΩ去离子和蒸馏水中[ddH 2 O]）。 </li><li>对于多重质粒的共电穿孔，以1:1的比例混合质粒。最终DNA浓度应为5-8μg/μL。 </li></ol></li><li> 注射移液器 <ol><li>将移液器拉到微量吸管拔出器上。使用28G注射器针头，用1-2μL质粒填充移液管。 </li><li>使用镊子将移液管吸头切断至10 - 20μm。使用显微镜来帮助确定破碎的移液器吸头的尺寸。将移液器连接到喷雾枪的移液器支架上。 </li></ol></li></ol>开窗注意:窗口过程已在13之前发布。有关其他视觉信息，请参阅参考13。 <ol><li>擦拭所有仪器和工作区域70％乙醇。 </li><li>将所需数量的鸡蛋从孵化器中取出（6-10），离开室温，再次用70％乙醇单独取蛋。除去培养箱后，鸡蛋保持至少2小时。 </li><li>将鸡蛋放在光照射器的顶部，以确定胚胎定位。用铅笔圈住黑暗中心（ 即胚胎）。 </li><li>使用一根19G的针头，在鸡蛋的钝末端戳一个洞。 </li><li>在鸡蛋的尖端旁边绘制圆圈附近的另一个洞。用19G针去除一小块外蛋壳（约16 mm 2 ），然后用镊子取出一小块内蛋壳（约8 mm 2 ）。 </li><li>用配有19G针头的3mL注射器，通过钝头孔从鸡蛋中取出1.5-2.5mL白蛋白。确保将针角向下倾斜45°。 </li><li>用一小块胶带（1厘米×1厘米）覆盖钝头孔。覆盖绘制的圆形d带第二个孔（2.5厘米x 4厘米）。 </li><li>用弯曲的剪刀（切边= 2.5厘米），在圆圈内切一个窗口。剪刀应平行于蛋壳，窗口直径应小于2厘米。 </li></ol> 4.质粒注射<ol><li>打开picospritzer。将压力设置为18 psi，持续时间设置为5μs。打开气囊和灯泡用于解剖显微镜。 </li><li> 将30mL购买印度墨水的1:10稀释的储备溶液混合在无菌磷酸盐缓冲盐水（PBS）中。储存于4°C。注射印度油墨是获得鸡胚更好的可视化的重要一步。 <ol><li>用稀释的印度墨水溶液填充1 mL注射器。安装27G针头并排出注射器中存在的任何气泡。 </li><li>将胚胎插入距离胚胎约2-3毫米的蛋黄膜下，并在胚胎下轻轻注入〜0.2毫升印度墨水。做不会注射超过0.5毫升的印度墨水，因为它可能会降低胚胎存活。 </li></ol></li><li>将窗口蛋放在夹层显微镜下的鸡蛋架上。使用最高放大倍率进行质粒注射部位的最佳可视化。 </li><li>使用镊子，去除注射区域上的膜。听觉脑干感兴趣区域（ 即 NM和NL）来自Rhombomeres 5和6（R5 / R6）。 R5 / R6位于邻近于作为R5 / R6质粒注射的标志的耳朵（发育成内耳的脊椎动物中的胚胎结构）。 </li><li>将DNA填充的移液管放置在覆盖R5 / R6区域的神经管和使用显微操纵器的耳朵之间。理想放置的示意图如图1A所示 。 </li><li>从picospritzer施加空气压力（18 psi，5μs持续时间），以在神经管中排出DNA。 </li></ol>电穿孔<ol><li>打开t他电流/电压刺激器。 </li><li>用PBS注入3 mL注射器，并连接注射器过滤器。在胚胎上加入1-2滴PBS。 </li><li>使用显微操纵器将双极电极降低至胚胎，负极位于注射部位（内侧）上方，正电极位于R5 / R6侧面（ 图1A ）。避免与胚胎直接接触。使用双极电极，其中电极材料是铂铱。使用镊子将尖端之间的间距调整为0.5 mm。 </li><li>使用电流/电压刺激器，以1秒的间隔在50 V脉冲20次脉冲1 ms。 </li><li>电穿孔后，在暴露的区域添加一滴PBS。轻轻取出电极，并用实验室组织和70％乙醇清洗。关闭窗口，用胶带暴露胚胎。 </li><li>标记蛋壳与注入的质粒类型和孵化日期。 </li><li>将鸡蛋放回孵化器，窗口朝上。在38℃下孵育50℃湿度达到所需的发育阶段。 </li><li>如果电穿孔对tet基因载体进行基因表达的时间控制，则每24小时应用多西环素（Dox）以诱导基因表达（参见第6节）。 </li></ol> 6.基因表达时间控制的Tet-on系统<ol><li> 使用以下质粒: pCAGGS-T2TP:表达转座酶的质粒。 pT2K-CAGGS-rtTA-M2:稳定表达Dox结合蛋白的质粒。 pT2K-BI-TRE-EGFP:含有双向tet-on启动子（TRE）的质粒，其表达EGFP报告基因和第二种感兴趣的基因。 注意:上述三种质粒需要以1:1:1的比率共同电穿孔。 </li><li> 储备溶液制备: <ol><li>将Dox溶于无菌PBS中以产生1mg / mL储备溶液。储存于-20°C。 </li></ol></li><li> Dox应用程序: <ol><li>诱导表达n在某些发育阶段的感兴趣的基因，每24小时应用Dox。 </li><li>当应用Dox时，取蛋，打开窗户，将50μLDox吸取到绒毛膜蛋白膜上，关上窗户，将鸡蛋放回培养箱。 </li></ol></li></ol> 7.脑干切片解剖区的准备注意:以下章节（7 - 9）和程序已在14之前发布。有关其他视觉信息，请参阅这些参考。 <ol><li>制备琼脂溶液（40 mg / mL琼脂糖， 即 ddH 2 O中为 4％）。将琼脂溶液倒入培养皿中，使其在室温下固化。将冷藏的琼脂板存放在冰箱中，以便将来在组织切片过程中使用。 </li><li>持续发泡95％O 2和5％CO 2 （pH 7.2-7.4和渗透压:295-310 mOsm / L）的人造脑脊髓液（ACSF）。</li><li>用70％乙醇清洁工作区域和振膜，并用蒸馏水冲洗刀片。 </li><li>使用适当的解剖工具将干净的吸液垫放在工作区域上。 </li><li>将琼脂板从冰箱中取出，切成小琼脂立方体（10mm×10mm×8mm）。使用市售的超级胶将琼脂块胶粘到vibratome切片室的阶段。 </li></ol> 8.分离鸡听觉脑干<ol><li>在录音窗口打开鸡蛋。用手术刀穿刺膜囊并清除鸡胚的头部。 </li><li>用锋利的剪刀剁碎鸡肉。 </li><li>将剃刀刀片尖端稍微靠近眼睛。在头尾到尾部中线切开头骨。根据胚胎的年龄施加轻微的压力。较老的胚胎需要更多的压力。 </li><li>轻轻推开皮肤和羽毛，露出头骨，并验证中线切口。 </li><li>申请压力很大，用剃刀刀片切割头骨的头部。立即将刀片放置在眼睛后面，切割整个头骨和脑组织。 </li><li>使用剪刀在头骨的尾部区域，头部两侧的颈部肌肉稍微前方进行中线到外侧的切口，通过拔除头骨和多余的组织来暴露脑和小脑。 </li><li>使用剪刀将组织切割到脑干上，并清除脑部，自由脱离头骨。 </li></ol> 9.准备脑干切片用于体内电生理或成像<ol><li>通过视觉图形来降低脑干。 </li><li>用剪刀切割花梗去除小脑，暴露第四脑室的地板。 </li><li>使用镊子，从脑干表面去除任何膜组织和血管。 </li><li>要阻止脑干，在四分之一的最底端的水平切割第三脑室，只是尾皮到皮层。使横切通过视网膜垂直，以分离脑干。 </li><li>在琼脂块前面的vibratome舞台上放少量超级胶水。 </li><li>使用镊子提升脊髓的脑干，并将其放置在超级胶水上，朝向侧面向下和背侧朝向颤振片。用实验室纸巾或滤纸去除多余的胶水。 </li><li>将氧合的ACSF倒入振动台。将vibratome刀片设置在20-22°角度。以最大振幅振荡开始振动。将舞台向上移动到刀片，使得组织的顶部与刀片平行。 </li><li>迅速将刀片移动到脑干组织。在刀片与组织接触之前，大大减缓刀片。 </li><li>切片组织具有最慢的前进速度。一旦叶片通过整个冠状组织切片，使用移液管轻轻取出切片/ LI&gt; <li>放下200-300微米，再次切片。重复，直到听觉核的解剖标志变得可见，例如，NL的独特的背侧/腹侧神经纤维区域和NM相对于NL的内侧位置。 </li><li>仔细维护ACFS中的切片。这可以使用温水浴在室温（22°C）或接近生理条件（〜41°C）下进行。注意切片的顺序。第一个切片对应于组织的最尾部区域，最后一个切片对应于最多的分布区域。这粗略地代表了从低频编码区域到高频编码区域的听觉脑干的tonotopic组织。 </li></ol>

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ovo电穿孔是一种表达或敲低感兴趣的基因以分析体内基因功能的方法8,9 。在鸡胚中，将质粒编码的基因表达到不同的听觉脑干区域中是一种创新的方法。为了确保最佳表达，需要几个关键步骤。首先，只注射其囊肿清晰可见的胚胎。如果卵巢不可见，则胚胎不能在电穿孔的正确发育阶段。第二，较小的移液管尖端使得更容易穿透神经管并导致更少的组织损伤。然?...

声音的快速神经编码对于正常的听觉功能至关重要。这些包括声音定位能力1 ，噪音识别2中的语音，以及其他与行为相关的通信信号3的理解。鸟类和哺乳动物的听觉脑干中的类似神经元是高度专门用于快速神经编码的。其中包括鸡核细胞核（NM），核心层流（NL）及其哺乳动物类似物，前内耳蜗核（AVCN）和内侧上橄榄（MSO） 5 。然而，在听觉脑干中，调节快速神经编码的发育机制知之甚少。因此，研究负责快速神经编码的具体基因是为了更好地了解其在au中的表达，调控和功能是有利的发展。 发展中的鸡胚是研究听觉系统发展基础生物学问题的有效和成熟的研究工具。最近的分子进展已经在发展中的鸡胚中通过表达或敲低感兴趣的基因来解决这些生物学问题，以分析体内基因功能8,9 。研究特定基因的调节作用是理解与听觉缺陷相关的病理学的重大进步。在这里，我们将质子编码基因的ovo电穿孔进入鸡听觉脑干，其中声音的快速神经编码发生10 。通过靶向与菱形体5和6相关的听觉神经祖细胞区域11,12（R5 /R6），我们显示了NM和NL中质粒转染的空间控制。此外，我们通过采用tet-on向量系统显示表达的时间调节。这是在多西环素（Dox） 8存在下表达感兴趣的基因的药物诱导性程序。

<table><tbody><tr><td>Fertilized white leghorn chicken eggs</td><td>Sunnyside Inc. (Beaver Dam, WI)</td><td></td><td></td></tr><tr><td>Picospritzer</td><td>Parker Hannifin</td><td>052-0500-900</td><td>Picospritzer III, single or dual channel</td></tr><tr><td>Current/voltage stimulator</td><td>Grass Technologies</td><td>SD9</td><td>SD9</td></tr><tr><td>Microfil syringe needles</td><td>World Precision Instruments</td><td>MF28G67-5</td><td>28 Gauge, 67 mm Long, (Pack of 5)</td></tr><tr><td>Electrode holder</td><td>Warner Instruments</td><td>64-1280</td><td>MP Series: Non-Electrical Pressure Applications</td></tr><tr><td>Stimulating microelectrode</td><td>FHC</td><td>PBSA1075</td><td>PBSA1075</td></tr><tr><td>Air tank/regulator</td><td>NU Laboratory Services</td><td></td><td>Air dry 300 CF</td></tr><tr><td>Fast green</td><td>Sigma Aldrich</td><td>F7258-25G</td><td>F7258-25G</td></tr><tr><td>Clear plastic tape</td><td>Scotch</td><td>191</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma Aldrich</td><td>D9891-1G</td><td></td></tr><tr><td>Egg refrigerator</td><td>Vissani Wine Refrigerator</td><td></td><td>13.3 - 16.1 &amp;deg;C (56-61&amp;deg; F)</td></tr><tr><td>Incubator</td><td>Hova-Bator</td><td></td><td>38 &amp;deg; C (100 &amp;deg; F), ~50% humidity</td></tr><tr><td>Dissection scope</td><td>Zeiss</td><td>4.35E+15</td><td>SteREO Discovery, V8 Microscope, 50.4X</td></tr><tr><td>Cold-light source</td><td>Zeiss</td><td>4.36E+15</td><td>CL6000 LED</td></tr><tr><td>Micromanipulators</td><td>Narishige Japan</td><td>Model: MM-3</td><td>2 Micromanipulators</td></tr><tr><td>Capillary tubes</td><td>Sutter Instrument</td><td>BF150-86-10</td><td>Thick-walled borosilicate (dimensions)</td></tr><tr><td>Syringes</td><td></td><td></td><td>1 mL, 3 mL</td></tr><tr><td>Needles</td><td>BD Precision Glide&amp;nbsp;</td><td></td><td>27 G x 1 1/4, 19 G x 1 1/2</td></tr><tr><td>Forceps</td><td>Stoelting</td><td></td><td>No. 5 Super Fine Dumont</td></tr><tr><td>Egg holder</td><td>Custom Made</td><td></td><td>Clay base works as well</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td></td><td>Model P-97</td></tr><tr><td>Syringe filter</td><td>Ultra Cruz</td><td>sc-358811</td><td>PVDF 0.22 &amp;mu;m</td></tr></tbody></table>

in ovo electroporation in the chicken auditory brainstem

电穿孔是将感兴趣的基因引入生物相关生物体如鸡胚的方法。长期以来，鸡胚是研究听觉系统发育基本生物学功能的有效研究模式。最近，鸡胚在研究与听力相关的基因表达，调节和功能方面已经变得特别有价值。 在ovo电穿孔可用于瞄准听觉脑干区域负责高度专业化的听觉功能。这些区域包括鸡核细胞（NM）和核层（NL）。 NM和NL神经元来自菱形体5和6的不同前体（R5 / R6）。在这里，我们介绍质粒编码基因的ovo电穿孔研究这些区域的基因相关性质。我们显示一种促进功能表型增益或丧失的基因表达空间和时间控制的方法ES。通过靶向与R5 / R6相关的听觉神经祖细胞区域，我们显示在NM和NL中的质粒转染。通过采用tet-on载体系统可以实现基因表达的时间调控。这是在多西环素（Dox）存在下表达感兴趣的基因的药物诱导性程序。 卵内电穿孔技术 - 与生物化学，药理学和/或体内功能测定一起提供了一种创新的方法来研究听觉神经元发育和相关的病理生理现象。

我们在这里显示， 在ovo电穿孔允许在正常发展的生物系统中的基因表达。质粒编码的基因被重点注射到覆盖R5 / R6的神经管中。 图1A中示出了相对于重要的解剖学标记的电极和移液器放置的示意性示例。在电穿孔后24小时证实质粒注射的正确位置， 如图1B所示 。将质粒靶向注射到R5 / R6上的神经管中，允许在特定的听觉脑干区域（即NM和NL <sup...

Watch this Scientific Journal Video about In Ovo Electroporation in the Chicken Auditory Brainstem at JoVE.com

In Ovo Electroporation in the Chicken Auditory Brainstem

鸟类和哺乳动物的听觉脑干神经元专门用于快速神经编码，这是正常听力功能的基本过程。这些神经元来自胚胎后脑的不同前体。我们提出利用电穿孔技术在鸡胚后脑中表达基因以研究听觉发育过程中的基因功能。

在奥运电击鸡听觉脑干中

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

in-ovo-electroporation-in-the-chicken-auditory-brainstem

Research

JoVE Journal

Developmental Biology

In Ovo Electroporation in the Chicken Auditory Brainstem

8.4K 次观看.  Northwestern University. 鸟类和哺乳动物的听觉脑干神经元专门用于快速神经编码，这是正常听力功能的基本过程。这些神经元来自胚胎后脑的不同前体。我们提出利用电穿孔技术在鸡胚后脑中表达基因以研究听觉发育过程中的基因功能。 

视频: In Ovo Electroporation in the Chicken Auditory Brainstem

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

鸡脑干切片的制备及文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

科研

神经科学

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

发育生物学

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

生物学

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

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

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

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

在蛋 和 卵外 研究中研究鸟类内耳发育的方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

对胚胎鸡听觉脑干进行切片以评估异位梯度和微电路

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

鸡幼崽听觉脑干反应的评价

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

在奥运电击鸡听觉脑干中

摘要

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此视频中的章节

鸡脑干切片的制备及文化

基因转移到鸡听觉器官通过

在OVO的HH阶段10个鸡胚Electroporations

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

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

在蛋和卵外研究中研究鸟类内耳发育的方法

对胚胎鸡听觉脑干进行切片以评估异位梯度和微电路

鸡幼崽听觉脑干反应的评价

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

在奥运电击鸡听觉脑干中

摘要

探索更多视频

此视频中的章节

鸡脑干切片的制备及文化

基因转移到鸡听觉器官通过

在OVO的HH阶段10个鸡胚Electroporations

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

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

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