作者要感谢坎迪斯谢医生的建议和援助，对鸡内耳解剖成像技术和多丽丝吴医生的专业知识，在早期胚胎发育。这项工作是由美国国家科学基金会，美国国立卫生研究院IOS-0642346 T32-DC010775，NIH，NIH R01-DC010796，T32-GM008620和美国能源部GAANN P200A120165的支持。

1。准备好以下的解剖工具和试剂<ul><li>人工脑脊液（人工脑脊液; 130 mM氯化钠，3 mM KCl中，1.2毫摩尔KH 2 PO 4，20毫摩尔的NaHCO 3，3 mM的HEPES，10mM的葡萄糖，2毫摩尔的CaCl 2，1.3 mM的用MgSO 4）用95％O连续输注2/5％CO 2中 ，在室温下。输液，补到2/3的500毫升广口的Nalgene瓶的盖子钻了一个洞。坦克通过管道将被附加的玻璃干起泡，贯通通过的jar盖中的孔的人工脑脊液。调整压力以获得恒定的气泡流，达到约1/3的液体，但力度不够导致溅在轮辋。淹没的Nalgene罐分开个别样品从对方的气体湍流的5毫升玻璃小瓶（最多6个）。学联应含氧为任何组织孵育之前至少20分钟。 </li><li>小硅夹层盘（35毫米×10权衡船毫米的Petri涂层的使用Sylgard的人造橡胶包）用解剖针放入小六角聚苯乙烯（47毫米底部）。 </li><li>两个细尖镊子，一个弯曲尖端钳，一个50毫升烧杯中，和用于组织废物的容器。 </li><li>被拉玻璃微（1.2 OD / 0.9 ID）破碎至约20-50微米的前端开放，并填充与罗丹明葡聚糖胺（RDA，分子量= 3000，Invitrogen公司）中的6.25％的溶液与0.4％的Triton X-100的PBS 。安装细管，以picospritzer和稳定微操作机器人。 </li></ul> 2。微解剖，揭示基底乳头E8 4,5 <ol><li>用弯钳，切割E8胚胎的正下方脑干，直接将头权衡船到有机硅涂层的解剖盘内。 </li><li>用解剖针，头的位置，一个腹面观，所以在耳朵上侧的利益是稍微明显，头部稍微倾斜的背部和指向10-30度，距离CE印表机（ 图2A）。立即用别针固定头。如果双边跟踪需要，在0度，中心的头，或一个完整的腹面观，使双方可以访问。类似地，对于一个左双面喷射，在-10℃至-30度的头部固定从中心使左侧是可访问的。 </li><li>使用50毫升的烧杯中，30毫升的含氧脑脊液转移入菜，完全浸没在组织。 </li><li>轻轻抓住下颌细尖镊子剥离下来，然后取出下眼睑上侧的兴趣。 </li><li>上面的血管组织，包括任何剩余的腭和咽喉。这些软组织剥离露出光滑的表面，在其表面上具有鲜明的椎动脉显影chondrocranium。由这点，特征的白色otoconial质量应该是可见的。 </li><li>仔细解剖周围皮肤的外鼻道，下颌关节软骨及中耳，而离开内耳完整。使用镊子轻轻地切掉软骨结构。 </li><li>删除椎动脉和集中使用斯文扫地运动的血液用细镊子提示的。可以掩盖血和凝血可能会堵塞注射针口。 </li><li>蜗管与相邻的感觉上皮（基底乳头）和声学神经节直接位于下方chondrocranium 6的，并可以被形象化为一个细长的手指状突起从内耳腹侧延伸，与最远侧尖端（顶点）附近的前中线5。有可能是一个小的解剖和/或耳石钙化的（白色），蜗管（ 图2B），可以帮助可视化的顶点池的血（红色）。在的顶点是lagenar的黄斑，有感觉的补丁被认为是前庭功能7,8。 </li></ol> 3。 CN第八听觉纤维的选择性标记<ol><li将注射针蜗管和基底乳头区附近的胚胎表面。 </li><li>慢慢地降低的微量移液器第一穿刺头盖骨。这颅地区是厚度小于周边地区和需要减穿透力。微管现在应该蜗管内。可选:一个小的注射追踪染料，可在这里，以帮助可视化的基底乳头。 </li><li>不重新定位微管的情况下，继续降低，直到它只是违反了深边境的蜗管（ 图1）。在这里执行注射剂，并重复沿基底乳头的长度（ 图2C，2D），在多个区域内不含前庭lagena位于最近端的小费。 10-30磅之间，picospritzer，多次注射。注入染料的流体的体积的变化，并取决于所需的程度上的标签。每次注射应导致的荧光染料 - 拉贝莱德点约为200 - 400微米直径的（ 图2D）。 </li><li>一旦注射完成后，用钳子来横切脑干上面的示例中，使用移液管，放置到一个小罐子的尾部的人工脑脊液的容器内，用95％O 2/5％CO 2的不断灌注。 </li><li>对于每个胚胎，允许20-30分钟的染料转移，这取决于所需的距离上的中心跟踪。 </li><li>删除组织和准备​​组织学切片。在这里所示的例子中，在4％多聚甲醛（在1X PBS）过夜，30％蔗糖（1X PBS）过夜组织浸渍。 </li></ol> 4。 Counterstaining与分析<ol><li> Counterstaining是非常有用的帮助观察者的解剖位置，以及确定特定地区的利益。在此制备一种有效的染液是神经丝免疫标记（ 图1，图3B，3E），这强烈升abels轴突和可以划分在中枢神经系统的轴突大片。 </li><li>分析应该执行整个标记的区域中，，突起可以延长几百微米或更大沿rostrocaudal轴。基本分析的一些例子是:目标错误率，时序和/或发散的突起图形的变化。 RDA-标记的轴突与高分辨率（ 图3A，3D）的外周和中枢神经系统中的可视化。 </li></ol>

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没有利益冲突的声明。

第八次神经的早期发展研究已部分限制，因为很难确定胚胎从多个不同的神经轴突产生。一些研究探讨了指导听觉和前庭感觉细胞和神经节细胞的命运在早期发育过程中的分子信号，5,11,12，但还没有得到确定的调节中央神经支配的进程。声神经节细胞预测报告通常描述外围过程到感觉上皮13日至15日 ，而被称为中央工艺主要核突出。中央突出第八细分在胚胎发育过程中的研究也常...

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>聚苯乙烯称盘</td><td> Fisher Scientific则</td><td> 02-202-101 </td><td></td></tr><tr><td>培养皿中，35 X 10毫米</td><td> Fisher Scientific则</td><td> 50820644 </td><td>使用硅解剖菜</td></tr><tr><td> Sylgard的有机硅弹性体套件</td><td>世界精密仪器</td><td> SYLG184 </td><td>外套Petri网进行解剖盘</td></tr><tr><td>解剖销</td><td>各个</td><td></td><td>举行胚胎发生在解剖</td></tr><tr><td>氯化钠</td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>氯化钾</td><td>各个</td><td></td><td>脑脊液配方的一部分</TD&gt; </tr><tr><td> KH 2 PO 4 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 碳酸氢钠 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>葡萄糖</td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 氯化钙 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td> 硫酸镁 </td><td>各个</td><td></td><td>脑脊液配方的一部分</td></tr><tr><td>为脑脊液的容器。建议半透明的广口罐，500毫升（16盎司）的Nalgene盖。 </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td>钻孔开口顶部的盖子为玻璃bubling干到渗透液</td></tr><tr><td>空5毫升玻璃小瓶或类似的透明瓶</td><td>美国制药合作伙伴，公司</td><td> 6332300105 </td><tD&gt;使用在脑脊液孵化到样本保持独立，相​​互借鉴，从鼓泡流</td></tr><tr><td>坦克Carbogen增（95％O 2/5％CO 2）的通过管道连接到鼓泡</td><td>各个</td><td></td><td>连接通过管道输液进入脑脊液玻璃干起泡</td></tr><tr><td>玻璃干起泡</td><td>各个</td><td></td><td>进入脑脊液注入Carbogen增</td></tr><tr><td>弯尖镊子</td><td>世界精密仪器</td><td> 501008 </td><td>从鸡蛋中取出胚胎头</td></tr><tr><td>细尖镊子</td><td>世界精密仪器</td><td> 501985 </td><td>对于显微解剖</td></tr><tr><td> 50 mL烧杯</td><td>各个</td><td></td><td></td></tr><tr><td>罗丹明葡聚糖胺（RDA） </td><td> Invitrogen公司</td><td>各个</td><td>荧光轴突示踪</td></tr><tr><td>的Triton X-100的</td><td> ICN Biomedicals </td><td></td><td></td></tr><tr><td>磷酸盐缓冲盐水（1X PBS） </td><td>各个</td><td></td><td>标准的实验室试剂</td></tr><tr><td>薄壁玻璃毛细管，0.9，1.2 OD ID 4英寸（100毫米）长</td><td>世界精密仪器</td><td> TW120F-4 </td><td>加载RDA。每个毛细管两个玻璃微</td></tr><tr><td>针/移液管拉马</td><td>大卫KOPF工具</td><td>型号720 </td><td>设置:热16.4，电磁阀2.2 </td></tr><tr><td> Picospritzer </td><td>帕克仪表</td><td>各个</td><td>附加细管玻璃微</td></tr><tr><td>微操作机器人</td><td>成重</td><td>各个</td><td></td></tr><tr><td>解剖显微镜与荧光</td><td>各个</td><td></td><td></td&gt; </tr><tr><td> 4％多聚甲醛</td><td>各个</td><td></td><td>标准的实验室试剂</td></tr><tr><td>抗神经丝抗体，可选</td><td>密理博</td><td> AB1991 </td><td>按照组织的协议，制造商推荐的</td></tr><tr><td>低温恒温器和相关材料的切片</td><td>徕卡</td><td>各个</td><td></td></tr><tr><td>外荧光显微镜成像</td><td>蔡司，各种</td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

鸡胚是一种广泛使用的外周和中枢的神经节细胞预测模型的研究。在听觉系统，听觉选择性标记的第八次颅神经轴突内将提高研究听觉中枢电路的发展。这种方法是具有挑战性的，因为多种感官的内耳器官的第八次神经。此外，可靠区分的标记，听觉与前庭禽流第八次神经轴突内还没有得到确定。听觉和前庭通路不能区分功能在早期胚胎中，感觉诱发反应不存在之前形成的电路。中心突出的第八次神经轴突已经在一些研究中追踪，伴随着但听觉神经轴突标签的标签从其他的第八次神经组件2,3。在这里，我们描述的方法，顺行追踪声节有选择地拉贝升听觉的第八次神经轴突内。首先，在为期8天的鸡胚沉浸在氧合人工脑脊液的前头部的局部解剖，解剖标志确定的蜗管。接着，罚款拉玻璃微被定位成注入少量罗丹明葡聚糖胺进入管和相邻的位于较深的区域中的声学的神经节细胞。在注射30分钟后，听觉神经轴突集中到后脑被跟踪，以后可以可视化的组织学准备。这种方法提供了一个有用的工具，周边听觉中枢电路形成发育研究。

第八次神经和神经本身的解剖结构的组成部分是复杂的，错综复杂的（ 图1，图3）。通过选择性地跟踪从声学的神经节细胞所产生的纤维，可以干净地跟踪段的第八次神经，以及初级听觉传入脑干内区别于他们的前庭同行（ 图2，图3）。同样地，这种技术可用于研究声学的神经节细胞的周缘凸部（ 图3G），或修改，以研究从个别前庭器官所产生的凸起。 ，因...

Watch this Scientific Journal Video about Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve at JoVE.com

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

在这里，我们描述了一个显微切割技术，用荧光染料注射到声早期鸡胚神经节选择性地跟踪听觉神经轴突的神经纤维和后脑。

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

selective-tracing-auditory-fibers-avian-embryonic-vestibulocochlear

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K 次观看.  University of California, Irvine. 在这里，我们描述了一个显微切割技术，用荧光染料注射到声早期鸡胚神经节选择性地跟踪听觉神经轴突的神经纤维和后脑。

视频: Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

与耳蜗内毛细胞传入树突:多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

神经科学

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 ...

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. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that the chicken embryo is an effective research model for studying basic biological functions of auditory system development. More recently, the chicken embryo has become particularly valuable in studying gene expression, regulation and function associated with hearing. In ovo electroporation can be used to target auditory brainstem regions responsible for highly specialized auditory functions. These regions include the chicken nucleus magnocellularis (NM) and nucleus laminaris (NL). NM and NL neurons arise from distinct precursors of rhombomeres 5 and 6 (R5/R6). Here, we present in ovo electroporation of plasmid-encoded genes to study gene-related properties in these regions. We show a method for spatial and temporal control of gene expression that promote either gain or loss of functional phenotypes. By targeting auditory neural progenitor regions associated with R5/R6, we show plasmid transfection in NM and NL. Temporal regulation of gene expression can be achieved by adopting a tet-on vector system. This is a drug inducible procedure that expresses the genes of interest in the presence of doxycycline (Dox). The in ovo electroporation technique &#8211; together with either biochemical, pharmacological, and or in vivo functional assays &#8211; provides an innovative approach to study auditory neuron development and associated pathophysiological phenomena.

In Ovo Electroporation in the Chicken Auditory Brainstem

Auditory brainstem neurons of avians and mammals are specialized for fast neural encoding, a fundamental process for normal hearing functions. These neurons arise from distinct precursors of embryonic hindbrain. We present techniques utilizing electroporation to express genes in the hindbrain of chicken embryos to study gene function during auditory development.

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 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.

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

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

禽流感胚胎听神经听觉神经纤维的选择性跟踪

摘要

探索更多视频

此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

与耳蜗内毛细胞传入树突:多泡发布监测功能区突触的突触后录音

鸡脑干切片的制备及文化

基因转移到鸡听觉器官通过

在奥运电击鸡听觉脑干中

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

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

禽流感胚胎听神经听觉神经纤维的选择性跟踪

摘要

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此视频中的章节

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

与耳蜗内毛细胞传入树突:多泡发布监测功能区突触的突触后录音

鸡脑干切片的制备及文化

基因转移到鸡听觉器官通过

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