Name: auditory brainstem response and outer hair cell whole-cell patch clamp recording in postnatal rats
Uploaded: 2018-05-24T12:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats at JoVE.com

这项工作得到了973计划 (2014CB943002) 和中国国家自然科学基金 (11534013, 31500841) 赠款的支持。

所有涉及动物学科的实验性协议均由南方医科大学动物伦理委员会批准。
1. 准备实验解决方案
<ol><li>准备麻醉剂 (参见材料表): 1.5% 戊巴比妥钠溶解在 ddH2O。</li>
	<li>准备解剖解决方案 (参见材料表): 在 ddH2的1升中溶解一袋 Leiboviz 的 L-15 粉, o 将 pH 值调整为 7.3, 用1米氢氧化钠。使用前使用 osmometer 和 10 mM HEPES 将渗透调整为 300-330 mOsm。</li>
	<li>溶出2毫克/毫升胶原酶 IV (参见材料表) 在步骤1.2 中制备的解剖溶液中。</li>
	<li>准备染色解决方案 (参见材料表): 将4% 多聚甲醛溶解在磷酸盐缓冲盐水 (PBS) 中。 注意: 多聚甲醛有毒;穿戴适当的保护。</li>
	<li>稀释0.3% 渗透剂在 PBS。将 PBS 中的10% 正常山羊血清稀释为阻塞缓冲液。在阻塞缓冲器中稀释 prestin 抗体1:200。在阻塞缓冲器中稀释 Alexa488-conjugated 抗体1:600。在 PBS 中稀释罗丹明-四甲基罗丹明 B 异硫氰酸酯1:200。</li>
	<li>准备胞外溶液 (参见材料表): 准备 Leiboviz 的 L-15 介质, 如上文所述 (步骤 1.2)。</li>
	<li>准备细胞内溶液 (参见材料表)。</li>
</ol>2. ABR 记录
注意: 以前的研究9中已经详细描述了 ABR 记录。
<ol><li>麻醉大 (SD) 大鼠 (1-14 天的老幼崽和2月大的成年人, 两性) 与单一注射1.5% 戊巴比妥钠 (22 毫克/千克为幼崽和30毫克/公斤成人, 腹腔 (ip))。</li>
	<li>将麻醉动物放入聚乙烯泡沫霉菌中, 使动物的身体固定。将动物放在隔音室的防振台上。用暖气垫把动物的体温保持在37.5 摄氏度。</li>
	<li>用 70% (卷/卷) 乙醇擦拭头部区域。使用细剪刀, 使1-2 毫米切口延髓到外部耳廓放置参考电极或地面。真皮下针电极 (记录电极) 位于头骨顶点 (图 1A)。</li>
	<li>使用函数发生器, 产生校准的音调爆发 (1 毫秒上升/下降, 3 ms 高原) 的各种频率 (2, 4, 8, 16, 24, 和32赫) 和强度 (从85分贝到10分贝在 5 db 步骤)。手动或使用计算机改变频率和振幅。通过一个位于10厘米处的静电扬声器向动物头部传递声音刺激。</li>
	<li>过滤器 (100-1, 000 Hz), 放大和平均 (256 倍) 的声音产生的潜力使用多功能处理器获得 ABRs。ABR 跟踪被在线监视并存储以进行离线分析。从一个动物那里完成 ABR 记录需要大约40分钟。 注: 通常, 三到七峰 (波 I 到波 VII) 的潜伏期小于15毫秒, 可以识别 (图 1B)。ABR 阈值定义为可以检测到波 I 和 II 的最小声级。</li>
	<li>弄死在动物醒之前从麻醉 (与腹腔注射0.3 毫升1.5% 戊巴比妥钠)。</li>
</ol>3. Corti (OC) 解剖机构
<ol><li>麻醉鼠崽。对于不到6天的老鼠 (< P6), 将动物放在100毫米的培养皿中, 用冰覆盖盘子10分钟。对于老鼠 > P6, 麻醉的动物与单一注射1.5% 戊巴比妥钠 (22 毫克/千克, ip)。麻醉后斩首鼠幼崽。</li>
	<li>用 70% (卷/卷) 乙醇喷雾消毒头部。</li>
	<li>用剪刀将头骨沿矢状中线打开;去掉大脑, 露出内耳。</li>
	<li>把内耳转成一个35毫米的培养皿, 里面装满3毫升冰冷的 L-15 (图 2A)。</li>
	<li>在解剖显微镜下, 使用细钳去除耳蜗骨胶囊 (图 2B)。</li>
	<li>从蜗轴中打开 OC 和相关的纹耳蜗 (SV)。</li>
	<li>通过将 sv 的基底部分与镊子保持在一起, 完全通过从基到顶点 (图 2C) 慢慢展开来将 OC 与 SV 分开。</li>
	<li>使用细剪刀将 OC 均匀地切成三块 (图 2D)。</li>
</ol>4. 免疫荧光染色
<ol><li>通过使用200µL 吸管尖端, 将 OC (步骤 3.8) 的段转移到玻璃滑块上, 然后用100µL 4% 多聚甲醛, 在4摄氏度上修复至少4小时。 注意: 多聚甲醛有毒;穿戴适当的保护。</li>
	<li>用100µL 的新鲜 PBS 取代多聚甲醛来冲洗组织。把纸巾洗三次, 10 分钟。</li>
	<li>室温下在 PBS 中用0.3% 通透剂孵化组织, 室温30分钟。</li>
	<li>在 pbs 中丢弃渗透剂, 用 pbs 清洗组织三次。</li>
	<li>在室温下, 在 PBS 中用10% 正常山羊血清阻断1小时。</li>
	<li>在室温或4摄氏度时, 用抗 prestin-C 终点抗体 (1:200) 在2小时的阻塞溶液中孵化。</li>
	<li>用 PBS 洗三次, 5 分钟。</li>
	<li>用 Alexa488-conjugated 次生抗体 (1:600) 在室温下的1小时的阻塞溶液中孵化。</li>
	<li>在黑暗中用 PBS 洗三次, 5 分钟。</li>
	<li>用罗丹明-罗丹明 (1:200) 在室温下在黑暗中孵育10分钟。</li>
	<li>在黑暗中用 PBS 洗三次, 5 分钟。</li>
	<li>安装在带有安装介质的玻璃滑梯上 (含 DAPI)。使用 405 nm、488 nm 和 594 nm 激光的共焦显微图像。将激光功率设置为 0.1-0.5 兆瓦, 扫描速度为0.5 帧/秒。</li>
</ol>5. 隔离 OHCs 的膜片钳记录
<ol><li>使用吸管拉拔和微伪造器, 使修补吸管与尖端直径2-3 µm. 背部填充吸管细胞内的溶液。通常, 在浴缸溶液中, 贴片吸管的初始电阻为 2.5-3.5 MΩ。</li>
	<li>通过使用200µL 吸管尖端, 将 OC 的一块 (从步骤 3.8) 转移到35毫米培养皿中。用100µL 酶消化培养基 (胶原酶 IV, 参见材料表) 在室温下5分钟消化组织。</li>
	<li>用100µL 的 L-15 取代酶消化培养基。用微型手术刀将组织切成小块, 以隔离毛发细胞。</li>
	<li>经过温和的吹打, 将细胞转移到自制的小塑料室 (直径 ~ 1.5 厘米), 填充无酶浴溶液 (~ 1.5 毫升, 见材料表)。</li>
	<li>把房间放在倒置显微镜的舞台上。找到健康出现的孤独 OHCs。</li>
	<li>将贴片吸管加载到700B 放大器的头部阶段。小心地移动机器人, 并将其定位在外发单元格的底部 (图 3A)。</li>
	<li>全细胞贴片通过封闭细胞体的外侧壁来夹紧 OHC。应用轻吸, 直到细胞膜破裂。设置保持电位在-70 mV。具有接触阻力的细胞从10到 17 MΩ, 膜阻力范围从100到 500 MΩ, 被认为是一个成功的全细胞构型。</li>
	<li>使用计算机控制, 应用极化和去极化型电压 (250 毫秒的持续时间和范围从−140到 +94 mv 在 13 mv 步骤) 到细胞, 以引出全细胞电流。</li>
	<li>放大整个细胞电流, 过滤器 (5 赫的角频率) 由700B 放大器。将数据转换为1440A 转换器并存储为离线分析。</li>
	<li>用膜片钳软件控制的两个正弦波电压刺激协议测量 OHCs 的膜电容。将刺激电压范围设置为-140 到 110 mV。存储数据以进行离线分析。</li>
</ol>

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作者没有什么可透露的。

在11岁以下的老鼠中, 听觉皮层可能没有反应出对声音刺激的动作电位6,7。因此, 后天11被描述为 "听觉发作"10。听力发病前听力功能的发展尚未得到很好的研究。使用类似的方法, 成人 ABR 记录, 我们证明, ABRs 可以引起的纯音调爆裂的鼠幼崽比 P11 (图 1)。然而, 从大鼠幼崽获得的 ABR 波形显示出与成年动物不同的特?...

耳蜗感觉毛细胞的两个不同功能是哺乳动物听觉的关键: mechanoelectric 转导 (见) 和 electromotility1,2。通过在发束中遇到的通道, IHCs (IHCs) 和 OHCs 将声音振动转换成膜电位的变化, 以及支配螺旋神经节神经元的电信号。OHCs 改变其细胞长度与膜电位, 放大低电平声音的振动。这种活动称为 electromotility 是由位于 OHCs3侧壁的运动蛋白 prestin 所衍生。
在许多物种中, 包括啮齿类动物, 听觉功能是不成熟的早期出生的时代4,5。在听觉皮层中, 在听觉皮质上没有任何动作电位可以检测到声音信号6,7。耳蜗的形态学和功能的发展已被广泛研究的老鼠, 沙鼠, 鼠4,5,8。毛发细胞的 mechanotransduction 和 electromotility 也在早期生命世纪的5中发展起来。
为评价大鼠不同出生年龄的听觉敏感性, 研制了一种听觉脑干反应 (ABR) 记录方法。全细胞贴片钳是研究 OHCs electrophysiologically 的理想技术。然而, 与神经元和其他上皮细胞中的膜片钳相比, 全细胞封闭率低, 限制了孤立 OHCs electromotility 的调查。
在这里, 我们描述了一个程序, 以调查 OHCs 的形态和 electrophysiologically 在急性分离的耳蜗从产后大鼠。该方法可对调节内毛细胞发育和功能的分子机制进行改进。

<table><tbody><tr><td>&lt;strong&gt;Anesthetic&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pentobarbital sodium</td><td>Sigma</td><td>P3761</td><td>1.5% in water</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Dissection solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leiboviz&#039;s L-15 Medium</td><td>Life Technologies</td><td>41300-039</td><td>1 pack in 1 L water</td></tr><tr><td>Collagenase IV</td><td>Sigma</td><td>C5138</td><td>2 mg/mL in L-15</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunostaining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td>PH 7.3</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>4% in PBS</td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>ZS-0694</td><td>0.3% in PBS</td></tr><tr><td>Normal goat serum</td><td>Thermo Fisher Scientific</td><td>10000C</td><td>10% in PBS</td></tr><tr><td>prestin antibody</td><td>Santa Cruz</td><td>SC-22694</td><td>dil 1:200</td></tr><tr><td>Alexa Fluor 488-conjugated antibody</td><td>Thermo Fisher Scientific</td><td>A-11055</td><td>dil 1:600</td></tr><tr><td>Phalloidin-Tetramethylrhodamine B isothiocyanate</td><td>Sigma</td><td>P1951</td><td>dil 1:200</td></tr><tr><td>DAPI</td><td>Solarbio</td><td>C0060</td><td>dil 1:20</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Extracellular solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Leiboviz&#039;s L-15 Medium</td><td>Life Technologies</td><td>41300-039</td><td>1 pack in 1 L water</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Intracellular solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CsCl</td><td>Sigma</td><td>7647-17-8</td><td>140 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>7791-18-6</td><td>2 mM</td></tr><tr><td>EGTA</td><td>Sigma</td><td>67-42-5</td><td>10 mM</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>10 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Osmometer</td><td>Gonotec</td><td>OSMOMAT 3000 basic</td><td></td></tr><tr><td>Forcep</td><td>WPI</td><td>14095</td><td>Tweezers dumont</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>MODLE-P97</td><td></td></tr><tr><td>Micro Forge</td><td>Narishigen</td><td>MF-830</td><td></td></tr><tr><td>Mini Operating System</td><td>Sutter Instrument</td><td>MP-285</td><td></td></tr><tr><td>MultiClamp</td><td>Axon</td><td>700B</td><td></td></tr><tr><td>Low-Noise Data Acquisition System</td><td>Axon</td><td>1440A</td><td></td></tr><tr><td>ES1 speaker</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>TDT system 3</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SigGenRP software</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>BioSigRP software</td><td>Tucker-Davis Technologies</td><td></td><td></td></tr><tr><td>jClamp</td><td>Scientific Solutions</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Animal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>SD rat</td><td>Experimental Animal Center of Southern Medical University</td><td></td><td></td></tr></tbody></table>

auditory brainstem response and outer hair cell whole-cell patch clamp recording in postnatal rats

外毛细胞是哺乳动物耳蜗内两种感觉毛细胞之一。它们通过受体电位改变细胞长度, 放大低电平声信号的弱振动。OHCs 外毛细胞的形态和电生理特性在产后早期发育。外毛细胞的成熟可能有助于听觉系统的发展。然而, OHCs 的发展过程还没有得到很好的研究。这部分是由于难以用电生理方法来测量它们的功能。为了开发一种简单的方法来解决上述问题, 我们在这里描述了一步一步的协议, 以研究 OHCs 在急性离型耳蜗从产后大鼠的功能。通过这种方法, 我们可以对纯色调刺激的耳蜗反应进行评价, 并对 OHCs 中运动蛋白 prestin 的表达水平和功能进行研究。该方法也可用于研究内毛细胞 (IHCs)。

ABR 可以从7岁以上的麻醉鼠幼崽 (P7) 使用纯音调爆发 (图 1A) 中诱发。如图 1B所示, 从鼠幼崽获得的 ABR 波形仅显示三到四个小振幅的不同波。通常在成年动物的 ABR 波形中观察到七峰值 (图 1B)。
对于以下染色和电生理测量, 大鼠内耳从颞骨进行了新的解剖。我们...

Watch this Scientific Journal Video about Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats at JoVE.com

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

本协议描述的方法, 记录的听觉脑干反应的产后鼠幼崽。为研究外发细胞的功能发育情况, 本文分步介绍了全细胞膜片钳在分离外毛细胞中的实验过程。

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

auditory-brainstem-response-outer-hair-cell-whole-cell-patch-clamp

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Southern Medical University. 本协议描述的方法, 记录的听觉脑干反应的产后鼠幼崽。为研究外发细胞的功能发育情况, 本文分步介绍了全细胞膜片钳在分离外毛细胞中的实验过程。

视频: Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

膜片钳记录的成年大鼠完整背根神经节

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

科研

生物化学

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

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

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 video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

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  ...

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

经鼓膜药物治疗耳毒性的临床研究

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could ...

医学

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

脑干中的数据采集与分析 在小鼠中引发响应音频测量

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

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 ...

Measuring the Auditory Brainstem Response in Mice

<a href='https://app.jove.com/v/64286/modified-experimental-conditions-for-noise-induced-hearing-loss-mice'>Source: Lee, D., et. al. Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell Damage. J. Vis. Exp. (2023)</a>This video demonstrates the measurement of auditory brainstem response (ABR) in a mouse. Electrodes are placed on the head, behind the ear, and near the tail of an anesthetized mouse. Acoustic stimuli at various intensities are delivered, and electric signals generated are recorded as waveforms.

Source: Lee, D., et. al. Modified Experimental Conditions for Noise-Induced Hearing Loss in Mice and Assessment of Hearing Function and Outer Hair Cell ...

JoVE Encyclopedia of Experiments

Neuropathology

Neuromuscular Disorders

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Source: Iyer, M. R., et al. <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

内耳感觉神经元细胞体的穿孔膜片钳记录

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

Neurophysiology

Imaging and Optical Techniques

Recording Auditory Brainstem Responses in a Rat Pup

Source: Chang, A. et al., <a href="https://app.jove.com/v/56678">Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats.</a> J. Vis. Exp. (2018).
This video demonstrates a method for recording auditory brainstem responses (ABRs) from rat pups. It outlines the steps for electrode placement, sound stimulus application, and signal processing analysis in the brainstem to assess neural responses.

记录大鼠幼崽的听觉脑干反应

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Electrophysiology Techniques

Whole-Cell Patch Clamp Recordings on Intact Rat Dorsal Root Ganglia

Source: Gong, K.,et al. <a href="https://app.jove.com/v/54287">Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats.</a> J. Vis. Exp. (2016)
This video demonstrates the examination of sensory neuron excitability in the dorsal root ganglia using the patch clamp technique to measure the rheobase and membrane potential, analyzing the neurons&#39; responses to incremental current injections.

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

摘要

探索更多视频

此视频中的章节

膜片钳记录的成年大鼠完整背根神经节

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

膜片钳记录在内耳毛细胞的分离斑马鱼

经鼓膜药物治疗耳毒性的临床研究

脑干中的数据采集与分析在小鼠中引发响应音频测量

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

Measuring the Auditory Brainstem Response in Mice

内耳感觉神经元细胞体的穿孔膜片钳记录

记录大鼠幼崽的听觉脑干反应

完整大鼠背根神经节上的全细胞膜片钳记录

产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录

摘要

探索更多视频

此视频中的章节

膜片钳记录的成年大鼠完整背根神经节

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

膜片钳记录在内耳毛细胞的分离斑马鱼

经鼓膜药物治疗耳毒性的临床研究

脑干中的数据采集与分析 在小鼠中引发响应音频测量

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

Measuring the Auditory Brainstem Response in Mice

内耳感觉神经元细胞体的穿孔膜片钳记录

记录大鼠幼崽的听觉脑干反应

完整大鼠背根神经节上的全细胞膜片钳记录

脑干中的数据采集与分析在小鼠中引发响应音频测量