资金来源:这项工作是由基金会资助贝尔塔雷利（DJL），一个MED-EL补助（DJL），以及健康教育资助DC01089国家研究院​​（MCB）的支持。

注:所有的实验程序按照马萨诸塞州眼的动物护理和使用委员会及耳科医院和哈佛医学院，这遵​​循国家动物保健指南，包括公共卫生服务政策上的人文关怀和使用实验动物，在执行ILAR指南，以及动物福利法。上市实验步骤如下左DCN的细节曝光。使用无菌器械，同时执行的生存术。 1.主开颅手术和背耳蜗核曝光<ol><li>麻醉<ol><li>通过腹腔内给药麻醉小鼠，年龄8-12周，体重18-24克，用甲苯噻嗪20mg / kg的氯胺酮和100毫克/公斤。确定合适的麻醉监测心脏，呼吸率，以及脚趾捏撤回反射。对眼睛的地方兽医软膏，以防止干燥，而在麻醉下。一旦充分麻醉下，头发剃覆吨他头皮以提供无障碍地访问手术部位。 </li></ol></li><li>手术定位<ol><li>将鼠标牢固在小动物立体定位架，由一吻夹具保持在适当位置。 </li><li>确保吻钳位足够松，以便有足够的呼吸，但不够紧完全固定在鼠标的头部。如果鼠标头是松散的，动物是可能的过程的开颅部分期间掉出的头支架。 </li><li>放置在标准的方式，这允许心脏速率监测听觉脑干植入电极。呼吸频率应该由可视化监控。需要注意的是正常的心脏速率和呼吸速率会根据鼠标的年龄而变化。 </li><li>温度计放置和euthermia确保通过放置在恒温加热毯。 </li></ol></li><li>切开皮肤及颅骨interparietal和枕骨骨曝光<ol><li>下一个microscOPE，使透过皮肤的垂直切口处开始的中线，耳廓直接之间，并延伸到后头部的尾部。下面正中切开皮肤，横向位移皮肤。可视覆盖颅骨的左后侧方面的肌肉。 </li><li>肌肉附件离断取下的肌肉覆左顶叶，interparietal和颅骨枕骨骨各自的骨头用手术刀或虹膜剪刀。观察一个小的程度以及切割边缘的肌肉出血。通过用棉签10-15秒温和的压力减少了。 </li></ol></li><li>开颅覆耳蜗核<ol><li>确定相关缝合线，包括矢状面和lambda缝合线（ 图1，左图）。 </li><li>用咬骨钳，作开颅手术在interparietal骨，中线左侧，〜2毫米尾鳍在lambda缝合线。这个区域覆盖了DCN。 </li><li>跟随着克开颅手术，观察薄层硬膜覆盖在小脑。使用手术刀，删除硬脑膜。 （ 图1，中图）。 注:去除硬脑膜可能导致小程度的出血。使用手术刀刀片去除硬脑膜的这个过程是为了减少小脑抽吸，这将集中在脑干表面和模糊的识别与DCN期间血液损失量。小鼠总血量为〜1.5毫升不应超过&gt;低于15％。如果出血超过这个数额，鼠标应提供与补液。 </li><li>取出凝固的血液覆盖的棉签涂抹小脑。或者，用牙科点到小脑清除血液离开血淌轻轻生理盐水。 </li></ol></li><li>小脑吸<ol><li>使用5法语吸，吸左侧小脑覆DCN的最外侧部分，直到与DCN是相UAL（ 图1，右图 ）。卸下左侧小脑大约1/4到1/3。毗邻DCN的主要地标是上半规管的壶腹。 注:小脑心愿是协议的关键一步。如果它发生在一个单一的企图，而不是与吸多遍，因为这会导致出血是最成功的进行了小脑导演的愿望。以协助抽吸，设置显微镜的焦平面处，CN，这将是略微远端小脑的表面的期望的深度。焦平面设置到CN将改进的可视化，并确保一个鲜明的图​​像。 </li><li>下面愿望，再出血，脑脊液（CSF）集结起来，小脑排量的DCN预期。灌输0.5毫升的无菌盐水的迅速进入开颅以防止血液凝固。然后轻柔涂抹的用牙科点和吸入的组合可以是使用扫清了DCN直接可视化的路径。不要直接接触DCN的表面。 </li></ol></li></ol> 2.压力显微注射的病毒介导的基因转移和手术恢复<ol><li>与DCN是免费的覆盖血液和脑脊液后和清晰可见，用10微升Hamilton注射器在2分钟内使压力显微注射入DCN。 </li><li>用显微操作引入针，直到针尖不再与DCN的表面下可见。 </li><li>为了获得最佳性能，使用33或34号针（使用气密注射器用34号针头）与浅斜面，如45°，以减少钝伤和本地化DCN内注射量，这是一种薄和浅脑干结构（&lt;300微米厚）。 注意:其他注射手段可以基于利用。理想情况下，注射仪器应具有灵活性，允许一些轻微的奔D，如果需要直接针对DCN。另外，作为小鼠轻微移动整个手术过程中，由于呼吸，仪器应是坚固足以承受运动的少量。 </li></ol> 3.手术恢复<ol><li>紧接着注射，重新接近皮肤，让鼠标按标准恢复过程来恢复。其余小脑和疤痕组织将填补引起小脑愿望腔。不断地监测动物，直到足够的意识恢复，保持胸骨斜卧。监测心脏速率，呼吸速率，以及饲料的能力。 注:没有动物已经动过手术返回到与其他动物的笼子，直到完全康复。 </li><li>如果鼠标开始走在圈子手术后（&lt;5％的病例），皮肤切口关闭后通常2-4小时，立即牺牲鼠标，因为它很可能有困难的时候喂食。牛逼他的副作用，可能是由于小脑的愿望。动物应为疼痛，术后进行监测，并适当麻醉品应给予保证根据机构的标准动物的舒适性。抗生素可能是必要的，如果感染的证据。 </li></ol> 4.二次开颅手术和人工耳蜗核曝光<ol><li> 2-4周的愈合和病毒介导的基因转移的潜伏期后，重新麻醉与基因转移的类型预先注射小鼠，并重复步骤1.1-1.5孵育时间而变化。 </li><li>由镊子，解剖刀，和咬骨钳的组合去除瘢痕组织在开颅站点。 </li><li>可视化开颅后，确定剩余的小脑和疤痕组织覆盖在DCN的组合。使用5弗伦奇吸入到吸覆小脑/疤痕组织（类似于前述小脑抽吸）。 </li><li>下面的心愿，期待再出血，脑脊液Build镜头，和小脑的其余位。快速引入盐水进开颅以防止血液凝固。用温和的牙齿涂抹点和吸力的组合来清除的DCN直接可视化的路径。 </li><li>观察到DCN的表面上。执行光遗传学基础生理学实验为DCN现在访问。引入光刺激，例如用光纤（ 图2），以驱动一个光遗传学系的实验。 </li></ol> 5.组织学<ol><li>下面的实验结论，安乐死鼠标氯胺酮的过量。灌注小鼠生理盐水后跟4％多聚甲醛。 </li><li>提取的头骨和后固定2小时脑干。 Cryoprotect在30％蔗糖的脑干中24-48小时。使用一节使用60微米的部分标准的低温​​恒温器脑干。 </li></ol>

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作者什么都没有透露。

本文介绍了DCN在操纵中枢听觉系统的小鼠模型直接可视化的技术。直接可视化的概括方法提供了主要的替代显著的优势，这是立体的方法。为主，DCN的直接可视化允许对脑干部位的立即确认，而立体定向的方法不能提供直接可视化。在于必要延长潜伏期，如在病毒介导的基因转移的情况下的实验中，存在用于低感染效率的潜力，如果注射液"射门"时，目标位置。另外，直接可视化的方法允许用于?...

Virus-mediated gene transfer to reverse hearing loss has largely been focused on the peripheral auditory system.1 Targeting the cochlea, investigators have examined a host of delivery routes, including osmotic minipump infusion2, vector-transgene complex-soaked Gelfoam&#174;2 or gelatin sponge3, direct microinjection4; numerous gene transfer vectors, including adeno-associated viral vectors5,6, lentiviral vectors7, and cationic liposomes2; and the dissemination of gene transfer vectors beyond the target tissue2. Most recently, adeno-associated virus (AAV)-1 has been introduced in the cochlea in order to treat deafness in mice due to loss of vesicular glutamate transporter-3.8 Further, the application of optogenetics in peripheral auditory system has recently been described.9
Few studies, however, have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem contain second order neurons of the auditory pathway. While gene transfer techniques in the cochlear nucleus (CN) may be utilized for a host of investigations, gene transfer of opsins, light-sensitive proteins, to the DCN may also be utilized to enable optogenetics-based experimental techniques. Following virus-mediated gene transfer delivery of an opsin, such as channelrhodopsin-2 (ChR2), the neurons of the DCN becomes sensitive to light stimuli. Optogenetic gene transfer has been previously attempted in several brainstem regions, including the rat retrotrapezoid nucleus, mouse locus coeruleus, monkey superior colliculus, and mouse ventral tegmental area.10-14
Recently, investigators have examined the use of optogenetics in the DCN.15,16 The DCN is the location of placement of auditory brainstem implants in humans, making it an attractive part of the auditory system to study for translational studies on auditory neuroprostheses. However, given the location of the DCN, surgical exposure is challenging. The technique described herein provides a protocol for maximal exposure of the DCN via posterior fossa approach to enable viral vector gene transfer and optogenetics-based experiments in a murine model. Previous studies used stereotactic microinjection into the DCN with channelrhodopsin-2.16 Stereotaxic injections, however, are potentially less accurate than injections made by direct visualization, especially in a nucleus as small and deep along the brainstem as the DCN. Transgenic mice expressing tissue specific proteins in the CN are also an attractive option and would obviate the need for gene transfer. Our protocol for exposure of the DCN would also work in transgenic mice as the DCN would need to be directly exposed for optical stimulation. This technique for surgical exposure of the DCN is adapted from previous protocols involving recordings from the auditory nerve and cochlear nucleus in mice and rat models.15,17-20
The overall goal of the protocol is to provide direct exposure to the CN to allow for gene transfer techniques. More specifically, the approach is compatible with both acute and survival surgery and the preparation can be repeated in the same animal for subsequent neurophysiological testing. The direct exposure of the DCN protocol has implications for optogenetics- and virus-mediated gene transfer-based experimentation in other nuclei of the brainstem.

<table><tbody><tr><td>Stereotaxic holder</td><td>Stoelting</td><td>51500</td><td></td></tr><tr><td>Homeothermic blanket</td><td>Harvard</td><td>507214</td><td></td></tr><tr><td>Scalpel blade #11</td><td>Fine Surgical Tools</td><td>10011-00</td><td></td></tr><tr><td>Iris scissor</td><td>Fine Surgical Tools</td><td>14084-08</td><td></td></tr><tr><td>5 French suction</td><td>Symmetry Surgical</td><td>2777914</td><td></td></tr><tr><td>Dental Points</td><td>Henry Schein</td><td>100-8170</td><td></td></tr><tr><td>Bone rongeur</td><td>Fine Surgical Tools</td><td>16020-14</td><td></td></tr><tr><td>10 &amp;micro;l Hamilton syringe</td><td>Hamilton&amp;nbsp;</td><td>7633-01</td><td></td></tr><tr><td>34 gauge, needle</td><td>Hamilton&amp;nbsp;</td><td>207434</td><td></td></tr><tr><td>Rongeurs</td><td>Fine Surgical Tools</td><td>16021-14</td><td></td></tr></tbody></table>

direct visualization of the murine dorsal cochlear nucleus for optogenetic stimulation of the auditory pathway

调查使用病毒介导的基因转移到阻止或逆转听力丧失在很大程度上被转移到外周听觉系统。很少有研究探讨基因转移到中央听觉系统。脑干的背侧耳蜗核（DCN），它包含了听觉通路的二阶神经元，是用于基因转移的潜在位点。在这个协议中，对于鼠DCN经由后颅窝的方法直接和最大曝光技术被证实。这种方法允许对急性或存活手术。继DCN的直接可视化，一台主机的实验是可能的，包括注射视蛋白进入耳蜗核和随后刺激的由光纤耦合到蓝色光的激光。其他神经生理学实验，如电刺激和神经注射器描也是可行的。visualiza的水平化和刺激的持续时间可达到使这种方法适用于广泛的实验。

部分小脑吸入演示进入耳蜗核 后的皮肤和肌肉覆盖在颅骨被除去，颅骨表面的地标，例如冠状和LAMDA缝合线，表明开颅的大致定位。下面开颅咬骨钳，小脑可视化。小脑的小部分仔细吸出表明该CN，然后可将其注入（ 图1）的可视化。 二级耳蜗核暴露的刺激与蓝光激光器 继DCN，与视蛋白的基因转移，和潜伏...

Watch this Scientific Journal Video about Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway at JoVE.com

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

direct-visualization-murine-dorsal-cochlear-nucleus-for-optogenetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.2K 次观看.  Harvard Medical School. The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model. 

视频: Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

科研

遗传学

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

神经科学

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

Optogenetically有针对性的前脑电路的激光扫描光刺激

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

生物学

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

指南<em&gt;在体内</em&gt;单单元从Optogenetically鉴定皮质抑制性录像

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

新生儿鼠耳蜗外科手术技术<em&gt;体外</em&gt;听力研究筛选工具

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

医学

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/61664">In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity.</a>&#160;J. Vis. Exp.&#160;(2020)
This video demonstrates the process of preparing wedge-shaped brain slices in a laboratory setting. Using a vibratome, a wedge-shaped slice from a mouse&#39;s brain is crafted with varying thickness. This slice contains the auditory nerve root positioned on the thicker end, while the hearing-related neurons are located on the thinner end.

准备楔形脑切片以可视化听觉神经元

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