由美国国家科学基金会（0854551）资助。

1。实验前的准备工作<ol><li>准备6的解决方案（见表1的组合物）：（一）100毫升NZR（一般斑马鱼林格氏的），（二）50毫升NZR +三卡因（MS222），（三）10毫升NZR + BSA;（四）100毫升的LOCA （低Ca 2 +的溶液）和（e）10毫升LoCaS +木瓜蛋白酶，（六）100毫升ķ+内部 ​​溶液。解决方案（一），（二），（d）和（f）可以被存储在4℃下长达一个月（三）及（e）应准备好一天的实验。在开始实验前，所有的解决方案应该是在室温下。 </li><li>标签和填补四张35毫米的培养皿中约一半的解决方案（a）中，（c）中，（d）和（五）。 </li><li>解剖盘填补了60毫米的培养皿中，用Sylgard（道康宁密歇根州米德兰市），然后把它切割腔，让鱼坐直没有翻倒。 </li><li>准备至少两个细胞的分离工具，通过胶合卵泡端的狗毛玻璃结束巴斯德吸管与强力胶，使头发过去的移液管的端部延伸，由约0.5厘米。软毛的狗，拉布拉多猎犬威玛猎犬一样的毛发正常工作。 </li></ol> 2。分离，听觉和前庭迷路<ol><li>牺牲我一个成年斑马鱼浸泡在一个烧杯中NZR +三卡因​​。可视化监控的鳃，鳃盖运动停止，直到所有的。再等待10分钟，然后取出鱼和新鲜NZR冲洗。这些程序已被批准的实验动物护理和使用委员会（IACUC），佩珀代因大学（Pepperdine University），但应该从自己的机构批准。 </li><li>引脚的鱼解剖菜背侧通过插入一个标准缝纫直针（约2.5厘米长）通过每个眼窝和三分之一引脚通过背腹轴约三分之一到二分之一的距离，从头部所示的尾图1A中。 </li><li>在一个变焦（0.62-5X）配有10倍的目镜的立体解剖显微镜下，用春风似剪刀（精细的科学工具目录号15000-02），暴露大脑和脊髓一小段取出的头骨屋顶从约0.5厘米尾鳍着鼻子的鱼鳃的水平。切脊髓，用细镊子（精细的科学工具目录号11251-30）的同时，减少脊髓神经和其他附件，解除大脑，消除大脑和丢弃（ 图1B）。 </li><li>腹一半的头骨胶囊，仍然形成一个“碗”，其中包含的内耳器官。遵守突出的，白色的，不透明的耳石在lagenas和sacculi对称地位于尾端的每个内耳和横向放置的椭圆囊耳石吻侧（ 图1C和4A）。 </li><li>取出一碗骨头切割腹部和侧依恋TS，同时轻轻抬起它的头用细镊子。将它放置到培养皿中的LOCA（ 图1D和4B）。 </li><li>使用2双细钳子，裂纹在中线通过敛骨的横向边缘和拉动它们分开，或者，分离插入钳子的各点在中心和撬半部左，右两半分开，碗分开。从骨头中取出两个迷宫用细镊子（ 图2）。 </li><li>检查的迷宫，，识别听觉和前庭终器官：半规管，椭圆囊，sacculi和lagenas（ 图4C）。根据每个耳石的粉红色的斑疹，含毛细胞。同样地，带粉红色的里面的半圆形运河壶腹标识位于毛细胞的吸盘。 </li><li>小心地取出用细镊子从lagena和囊耳石（ <strong&gt;的图2和图4C）。这是没有必要的球囊取出耳石，因为这样做可能会损坏头发的囊状细胞。 </li></ol> 3。毛细胞分离<ol><li>使用的塑料移液管（Fisher科学产品目录号13-711-上午09点），移动结束器官的培养皿中LoCaS +木瓜蛋白酶转移的流体的体积最小化。 </li><li>孵育30分钟，在室温下在此溶液中的这些结构。 </li><li>在年底的潜伏期，NZR + BSA培养皿中移动的结构，并在此溶液中孵育至少30分钟。在此解决方案中，毛细胞保持健康，长达四个小时，而且会损坏隔离后1-2小​​时后（请参阅下面的步骤）。 </li><li>在筹备细胞的分离，移动的最终器官的培养皿中NZR。 </li><li>对于的lagenas和椭圆囊，狗的头发轻轻抬离斑疹张粉红色的上皮TIssue包含毛细胞（图3A）。斑疹可以舀出（用狗毛）从在耳石的sacculi，从他们的位置的半规管壶腹内的cupulae。 </li><li>使用两个准备步骤1.4中的细胞分离工具，捣碎，而斑疹或吸盘产生的碎片字段中包含的毛细胞（图3B）。允许细胞定居的菜的底部上移动它之前的至少五分钟。 </li><li>移居他的菜，配有相衬光学倒置显微镜的阶段。 40倍的放大倍率下观察细胞，并注意形态的多样性。许多细胞，而另一些牛油果形长而薄的。顶束发的存在将其标明为毛细胞相亮度的保证他们的健康。孤立的毛细胞的显微照片示于图5。 </li></ol> 4。膜片钳斑马鱼的毛细胞<ol><li>在准备射孔录音5,6额定补丁，准备两性霉素含内部解决方案如下：将5毫克两性霉素B（适马A4888）至1.5 ml离心管中，并填写用100μlDMSO。立即涡此溶液中10秒至20秒，直到所有的两性霉素溶解。然后，移液管625微升K +内部 ​​解决方案，成为第二个离心管中。加入10μl的两性霉素溶液的K +-内部溶液和涡如上。股票两性霉素溶液会持续数个小时，但一小时后丢弃的最终解决方案。 </li><li>制作补丁移液器从BF 150-86-10硼硅酸盐玻璃（萨特仪器），使用多级的牵拉（萨特P-1000）。吸量管应该尖端约2微米的直径，并且将有1-3的电阻MΩ与表1中的解决方案。拉拔器上的设置工作如下：热量=斜坡minus10，拉= 0时，速度= 18，延迟= 1，压力= 600。的形状的良好的吸液管的尖端是显而易见的，从图5中的插图c的照片。钝的“子弹头形”的吸液管是首选，因为他们提供的电生理记录至少访问时的阻力。 </li><li>使用的28号的Microfil注射器针（MF28G-5，世界精密仪器），以填补记录电极与两性霉素含K +内部 ​​解决方案的一半。 </li><li>贴上电极膜片钳放大器的探头。应用-10毫伏，10毫秒的电压的步骤在10赫兹，并保持正的压力，因为它是通过气/液界面操纵和向下的记录盘的底部附近的细胞在电极中。 </li><li>垂直的电极放置一个健康的细胞。当电极是足够近的小区，这样它开始移动远离满分流动的溶液，快速反向压力，轻微的真空施加到所述电极，直到细胞在其上的“跳跃”。立即停止对日的吸E电极，并应用到所述电极的内侧的保持电位为-70 mV。在几秒钟内将形成一个gigaohm密封。 </li><li>观察后不久出现密封形成的电压的步骤的起始和终止的容性电流瞬变。继续监视的膜穿孔电极两性霉素看的瞬态增长幅度最大尺寸（约十分钟）。 </li><li>要确认建立在一个健康的细胞的全细胞（穿孔）的配置，通过施加超极化和去极化电压的步骤引起外向K +电流。大多数毛细胞具有显着的的向外电流（ 见图6）。 </li></ol> 5。代表性的成果 图4示出了在细胞分离步骤期间拍摄的图像。在面板的lagenas，saccluli和椭圆囊耳石可以看到THROUGH一层薄薄的骨之上。这些不透明的结构提供了方便地标有助于剥离在随后的步骤中的安全移除的端部从该动物器官。图B显示的“碗”的骨骼包含完整的椭圆囊，lagenas和sacculi的在迷宫和突出的耳石。面板C示出了已经从骨骼上移除的迷宫。注意大的扇形在lagena边缘耳石，的冰柱形耳石，在球囊和椭圆囊豆状耳石。某些可见在从lagena分离的细胞的大小和形态的多样性是图5中所示。一个健康的细胞很容易识别相亮用锋利的周边。细胞形态大致可以分为两大类：鳄梨形（避免）和长和薄（THN）虽然在每一组内的单元格的大小可以显着不同（例如，避免1和避免3比较）。插图示出的群集3细胞，并没有完全被彼此隔离。 插图 B中的单元格的黑色和颗粒状的外观容易标识此细胞为死亡。插图c显示了示出用于记录从这些细胞的形状和尺寸适当的一个补丁吸管尖端。 响应于超极化和去极化上lagena细胞类似于图5中所示的避免2所施加的电位的步骤中得到的在图6中所示的电流的痕迹。电流反应在不同大小和形状的细胞，可以很宽的范围内变化暗示在补体的电压门控通道的多样性。 图1-3：卡通毛细胞的隔离步骤说明。 <img alt="图1" src="/files/ftp_upload/4281/4281fig1.jpg" /> 图1去除头骨capsu的。乐从斑马鱼内耳的迷宫。针牺牲斑马鱼背解剖盘。B。开放性颅骨，删除和丢弃的大脑。C。观察耳石在椭圆囊，lagenas和sacculi。D。删除腹侧部分头骨胶囊含有的迷宫。 <img alt="图2" src="/files/ftp_upload/4281/4281fig2.jpg" /> 图2。去除头骨胶囊的迷宫。A。除了 ​​头骨胶囊在中线破解。B。删除迷宫骨C。取出使用产钳的lagenas和椭圆囊耳石。 <img alt="图3" src="/files/ftp_upload/4281/4281fig3.jpg" /> 图3。迷宫毛细胞的分离。轻轻抬离马与狗的头发culae。B。捣碎斑疹与两个狗的毛发。 C.使用隔离膜片钳毛细胞。 <img alt="图4" src="/files/ftp_upload/4281/4281fig4.jpg" /> 图4。拍摄的图像的单个细胞中的隔离在步骤A.除去后的大脑，两个lagenas和sacculi（箭头）和小囊（箭头）与耳石可以可视化。B.除去的腹侧后部：颅骨胶囊，它包含从动物的内耳机关。卡通中的右半B 的标识左边的的囊，lagena和球囊的位置。虚线表示的近似位置的左侧的迷宫。C。右迷宫（中间图）D：卡通说明在 C中的关键部分迷宫。答：前半B：圆形管，水平管，C：后路椎管，D：E：椭圆囊耳石，囊状耳石，F：瓶状囊耳石。在D中的比例尺为A和B表示1毫米，0.5毫米，C和D。 <img alt="图5" src="/files/ftp_upload/4281/4281fig5.jpg" /> 图5。lagena说明形态的多样性; AVO：牛油果形细胞; THN：长，薄的细胞隔离，健康的细胞。插入一个三不完全分离出细胞群; 插图 B：死细胞;插图ç：在斑马鱼的毛细胞的电生理记录使用的贴片电极尖端。比例尺= 20微米的主图和所有插图。 <img alt="图6" src="/files/ftp_upload/4281/4281fig6.jpg" /> 图6。修补程序中的电流钳位毛细胞。平均反应的细胞（类似TØ避免在图5），适用于从-140 mV的10 mV为增量从保持电位为-70 mV至+70 mV的电压步骤介绍。请注意，出现在更多的去极化电位的失活电流。阶跃电压幅值下的痕迹。

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+the+action+of+acetylcholine+on+isolated+outer+hair+cells+of+the+guinea+pig+cochlea.">Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea.</a> Proc. R. Soc. Lond. B. 244, 161-167 (1991).</li><li>Nicolson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+hearing+and+balance+in+zebrafish.">The genetics of hearing and balance in zebrafish.</a> Ann. Rev. Genetics. 39, 9-22 (2005).</li></ol>

没有利益冲突的声明。

小心剥离结束的器官和温和处理的细胞是健康的，完整的和生理活性的毛细胞的成功的隔离至关重要。遵循以下提示，确保成功： <ol><li>这是比较容易得到健康的细胞从较小的鱼（2-2.5厘米长）。更大的鱼有更多的脂肪滴在剥离过程中，晦涩的可视化。 </li><li>必须小心除去的的耳石，其中叠置毛细胞时。避免接触细胞时，用钳子抓住了耳石。 </li><li>时，得到最好的结果的斑疹和cupulae的被剥离纸片和细胞从上皮研制。 </li><li>我们找到狗的头发，捣碎细胞优于其他方法，包括使用人的睫毛是锥形和灵活的使用。 </li><li>在膜片钳，应用正压吨他电极，因为它通过的气/液界面是重要的是保持清洁的前端。的正压力的释放，使细胞在电极上的“跳”也是很重要的。此方法清除小体积的含两性霉素-溶液远离尖端，从而增加与细胞形成gigaohm密封的可能性。在我们的实验中，我们使用的更传统的全细胞记录技术，而不是因为脆弱的细胞穿孔修补程序。 “全细胞”，往往造成损失的gigaohm密封。 </li></ol>这里描述的方法，将产生数百个健康的头发细胞，每只动物。该技术可以在室温下进行，并超出在解剖显微镜不需要专门的设备。朱庇特先前的一份报告中说明使用的腹侧的方法8斑马鱼内耳的解剖。这些表明，狄氏ction完整的，多聚甲醛固定的内耳器官。我们鼓励读者观看此视频相比，我们的方法。这里提出的方法的优点之一是获得有用的活细胞的生理调查。除了 ​​它们的使用在膜片钳实验中研究的电压门控离子通道（如这里所示）可以扩展到使用这些细胞研究细胞共振9,10神经递质的释放，显示器通过使电容测量11,12，调查神经调节诱导以及挖掘到丰富的信息，我们可以从使用影响听力和平衡14的突变体，配体门控离子通道的激活13。

<table border="1"><tbody><tr><td> 解决方案名称 </td><td> 食谱 </td></tr><tr><td> （一）NZR（普通斑马鱼林格氏）（以mM） </td><td> 116 2氯化钠，氯化钾，2 3的CaCl 2，葡萄糖，5的Na +-HEPES，pH值7.35。 </td></tr><tr><td> （二）NZR +三卡因</td><td> NZR + 0.02％3 - 氨基苯甲酸乙酯甲磺酸（Sigma公司A5040）。 </td></tr><tr><td> （三）NZR + BSA </td><td> NZR + 0.4％牛血清白蛋白（适马A2153）。 </td></tr><tr><td> （四）LoCaS（低Ca 2 +的溶液）（以mM） </td><td> 100 2氯化钠，氯化钾，0.05的CaCl 2的MgCl 2，3葡萄糖，30的Na +-HEPES，pH值7.35，0.05。 </td></tr><tr><td> （五）LoCaS +木瓜蛋白酶</td><td>的LOCA + 0.05％L-半胱氨酸（Sigma公司C1276）+ 0.2％的木瓜蛋白酶（Sigma公司P3375）。 </td></tr><tr><td> （六）K +-内部溶液（以mM） </td><td> 52 K 2 SO 4，38氯化钾，1 K +-EGTA，5 K +-HEPES，pH值7.3。 </td></tr></tbody></table> 表1。解决方案。

patch clamp recordings in inner ear hair cells isolated from zebrafish

从不同的物种中分离的感觉毛细胞的电压门控通道的膜片钳分析前面描述的1-4，但这个视频的第一个应用程序，这些技术从斑马鱼的毛细胞。在这里，我们表现出的内耳器官，lagena：球囊，椭圆囊和半规管的方法分离健康，完整的毛细胞。此外，我们证明了毛细胞的大小和形态的多样性，举一个例子，可以得到的膜片钳记录种。这种斑马鱼模型系统的使用比其他的优点源于斑马鱼突变体的可用性，同时影响听觉和平衡。结合使用转基因株系和其他技术，利用遗传分析和处理，细胞分离和电生理的方法，这里介绍的角色毛细胞p应促进更深入地了解躺在调解这些感觉。

Watch this Scientific Journal Video about Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish at JoVE.com

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

这个视频的目的是为了证明程序获得的成年斑马鱼的内耳器官的健康，完整的毛细胞，然后利用他们为特征的生物物理特性的电压门控离子通道的膜片钳研究的目的。

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

patch-clamp-recordings-in-inner-ear-hair-cells-isolated-from-zebrafish

Research

JoVE Journal

Neuroscience

16.6K 次观看.  Pepperdine University. 这个视频的目的是为了证明程序获得的成年斑马鱼的内耳器官的健康，完整的毛细胞，然后利用他们为特征的生物物理特性的电压门控离子通道的膜片钳研究的目的。

视频: Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...