資金調達：この作品は、財団Bertarelli補助金（DJL）、MED-ELの助成金（DJL）、国立衛生研究所補助金のDC01089（MCB）によってサポートされていました。

注：すべての実験手順は、実験動物の愛護管理と使用に関する公衆衛生サービスポリシーを含む全国の動物保護ガイドラインに従うマサチューセッツアイ、耳診療所とハーバード大学医学部の動物実験委員会、に従って行われる、 ILARガイド、および動物福祉法。左DCNの詳細暴露下に表示実験手順。生存手術を行いながら、無菌の楽器を使用してください。 1.プライマリ開頭と背側蝸牛神経核露出<ol><li>麻酔<ol><li>キシラジンの腹腔内投与による20mg / kgのケタミンを100mg / kgの18〜24グラム体重、高齢者8-12週間、マウスを麻酔。心拍数、呼吸数、ならびに足指ピンチ引っ込め反射を監視することによって、適切な麻酔を確認する。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を置きます。一度適切に麻酔下で、髪の上に重なるTを剃る彼は、手術部位への遮るもののないアクセスを提供するために頭皮。 </li></ol></li><li>外科ポジショニング<ol><li>鼻クランプにより所定位置に保持小動物定位ホルダー、に安全にマウスを置きます。 </li><li>鼻クランプが十分な呼吸を可能にするのに十分な緩いが、完全にマウスの頭部を固定するのに十分タイトであることを確認してください。マウスヘッドが緩んでいる場合、動物は処置の開頭部分の間にヘッドホルダから脱落する可能性がある。 </li><li>心拍数のモニタリングを可能にし、標準的な方法、で聴性脳幹インプラント電極を配置します。呼吸数は、視覚化することによって監視されるべきである。通常の心拍数や呼吸数は、マウスの年齢に応じて変化するであろうことに注意してください。 </li><li>恒温加熱ブランケット上に配置介して温度計を配置し、euthermiaが確保される。 </li></ol></li><li>皮膚切開と頭蓋骨のinterparietalと後頭部の骨の露出<ol><li> microscの下でOPE、直接耳介の間で、正中線から始まる、と後頭部の尾部に至る皮膚を介して垂直切開する。正中皮膚切開の後、横方向に皮膚を移動させる。頭蓋骨の左側の後部側面をカ​​バーする筋肉を視覚化する。 </li><li>メスまたはアイリスはさみと、それぞれの骨に筋肉の添付ファイルの離断によって筋肉左頭頂、interparietalの上にある、と頭蓋骨の後頭部の骨を削除します。カット筋縁に沿って、出血の小さな度合いを観察します。 10〜15秒間綿棒で穏やかな圧力によって、これを最小化します。 </li></ol></li><li>開頭その上の蝸牛神経核<ol><li>矢状及びラムダ縫合ライン（ 図1、左パネル）を含む関連縫合線を、特定します。 </li><li>骨鉗子を使用して、interparietal骨の上に開頭手術をし、正中線のラムダ縫合線へ〜2mmの尾を残した。この領域は、DCNの上に重なる。 </li><li> FollowinGの開頭、小脳の上に重なる硬膜の薄い層を観察する。手術用メスの刃を使用して、硬膜を削除します。 （ 図1、中央のパネル）。 注：硬膜の除去は、出血の小さな度合いを引き起こす可能性があります。硬膜を除去するために外科用メスの刃を使用したこのプロセスは、脳幹の表面とDCNの曖昧な同定にプールう小脳吸引の間の血液損失の量を低減することを目的とする。マウスの全血液量を15％以上&gt;〜1.5ミリリットルを超えてはならないです。出血がこの量を超えた場合、マウスは、流体補充を用いて提供されるべきである。 </li><li>綿棒アプリケーターで小脳を重ね凝固した血液を削除します。必要に応じて、優しく血液を血液を離れてクリアする小脳の上に歯科ポイントを使用して生理食塩水を滴下する。 </li></ol></li><li>小脳吸引<ol><li> DCNのVIになるまで5フランス語吸引を使用して、DCNの上に重なる左小脳の横ほとんどの部分を吸引UAL（ 図1、右パネル）。左小脳の約1/4 1/3を削除します。 DCNに隣接した主なランドマークは、優れた半規管の膨大部である。 注：小脳の吸引がプロトコルの重要なステップである。それが出血原因になります。このような吸引の複数のパスを持つ単一の試行で発生していない場合は、小脳の監督吸引が最も成功している。吸引を補助するために、小脳の表面にわずかに遠位されるであろう、CNの予想される深さに顕微鏡の焦点面を設定する。 CNに焦点面を設定すると、可視化を改善し、鮮明な画像が保証されます。 </li><li> DCNの以下の吸引、さらに出血、脳脊髄液（CSF）、ビルドアップ、及び小脳変位が期待されている。血液凝固を防ぐためにすぐに開頭術に滅菌生理食塩水0.5ccのを植え付ける。歯科ポイントと吸引優しい軽くたたくの組み合わせは、その後かもしれDCNの直接可視化するためのパスを離れてクリアするために使用。直接DCNの表面に接触しないでください。 </li></ol></li></ol>ウイルス媒介遺伝子導入および外科回復2.圧力マイクロインジェクション<ol><li> DCNは、その上の血液およびCSFの自由であり、はっきりと見えるした後、2分間にわたって10μlのハミルトンシリンジを使用して、DCNに圧力マイクロインジェクションを作る。 </li><li>先端がDCNの表面の下に見えなくなるまで、マイクロマニピュレーターで針を導入しない。 </li><li>最適なパフォーマンスを得るには、33または34ゲージの針を使用する薄いである、鈍的外傷を最小限にし、DCN内の注入量をローカライズするために、そのような45°と、浅いベベルと（34ゲージ針で気密注射器を使用します）と、浅い脳幹構造体（&lt;厚さ300μm）。 注：他のマイクロインジェクション器具ベースを利用することができる。理想的には、注射器は若干ベンを可能にするために柔軟であるべきであるD、直接DCNをターゲットにする必要がある場合。マウスは呼吸による手続きを通じて少し移動するとさらに、楽器、運動の少量に耐えるために十分に堅牢でなければなりません。 </li></ol> 3.外科回復<ol><li>すぐに次の注射は、皮膚を再近似するとマウスが標準の回復手順ごとに回復することができます。残りの小脳および瘢痕組織が小脳の吸引に起因する空洞内に記入します。十分な意識が胸骨横臥位を維持するために回復するまで、常に動物を監視します。心拍数、呼吸数、ならびに供給する能力をモニターする。 注：完全に回復するまでは、動物の手術を受けた他の動物とのケージに戻している。 </li><li>マウスは、円術後（症例の&lt;5％）の中を歩くが開始された場合、それはおそらく困難な時期送りを持っていると同じように、通常は2〜4時間皮膚切開の閉鎖後、すぐに、マウスを生け贄に捧げる。 T彼の副作用に起因小脳の吸引に起因する可能性が高い。動物は、術後の痛みのために監視し、適切な麻薬は、制度の基準に基づいて、動物の快適性を確保するために与えられるべきである。感染の証拠があれば抗生物質は、必要であり得る。 </li></ol> 4.セカンダリ開頭と蝸牛神経核露出<ol><li>癒しとウイルス媒介遺伝子導入のインキュベーションの2-4週間後、再麻酔前に注射したマウスを繰り返しステップ1.1〜1.5の最適なインキュベーション時間は、導入遺伝子の種類によって異なります。 </li><li>ピンセット、外科用メス、および骨鉗子の組み合わせによって開頭サイト上の瘢痕組織を削除します。 </li><li>開頭術を可視化した後、残りの小脳及びDCNを覆う瘢痕組織の組み合わせを特定する。その上小脳/（前の小脳吸引と同様）瘢痕組織を吸引する5フランスの吸引を使用してください。 </li><li>吸引後、さらに出血、CSF bを期待するuildアップ、および小脳の残りのビット。すぐに血液凝固を防ぐために開頭術に生理食塩水を導入。 DCNの直接可視化するためのパスをクリアするために穏やかな歯科ポイント軽くたたくと吸引を組み合わせて使用​​してください。 </li><li> DCNの表面を観察します。 DCNがアクセスできるようになりましたように、光遺伝学ベースの生理学実験を行う。光遺伝学ベースの実験を駆動するために、光ファイバ（ 図2）と、例えば、光刺激を導入する。 </li></ol> 5.組織学<ol><li>実験の終了後、ケタミンの過剰摂取でマウスを安楽死させる。 4％パラホルムアルデヒド、続いて生理食塩水でマウスを灌流する。 </li><li> 2時間頭蓋骨とポストフィックスから脳幹を抽出します。 24〜48時間、30％スクロース中で脳幹をCryoprotect。第60ミクロン切片を用いて、標準的なクライオスタットを用いて、脳幹。 </li></ol>

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著者らは、開示することは何もない。

本稿では、中央聴覚系を操作するためのマウスモデルにおけるDCNの直接可視化の技術が記載されている。直接可視化の概略アプローチは、定位アプローチである主要な代替、上の大きな利点を提供する。定位のアプローチが直接可視化を与えないのに対し、主に、DCNの直接可視化は、脳幹の部位の即時確認することができます。注入は、ターゲットの場所を「ミス」した場合場合、ウイルス?...

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

逮捕または難聴逆転するウイルス媒介遺伝子導入の使用の調査は、主に末梢聴覚系に追いやられています。いくつかの研究は、中枢聴覚系への遺伝子導入を検討した。聴覚路の二次ニューロンが含まれている脳幹の背側蝸牛神経核（DCN）は、遺伝子導入のための潜在的部位である。このプロトコルでは、後頭蓋窩アプローチを経由してネズミDCNの直接的かつ最大の露出のための技術が実証されている。このアプローチは、急性または生存のどちらかの手術が可能になります。 DCNの直接可視化に続いて、実験のホストは、青色光レーザに結合された光ファイバによる蝸牛神経核およびその後の刺激へのオプシンの注射を含む、可能である。そのような電気刺激と神経インジェクタトレーシングのような他の神経生理学実験は、また可能である。visualizaのレベルると、刺激の持続時間は、達成可能な実験の広範囲にこのアプローチを適用する。

部分的な小脳吸引は蝸牛神経核へのアクセスをデモ 頭蓋骨を覆う皮膚と筋肉がそのような冠状及びラムダ縫合線として、頭蓋骨の表面ランドマークを、除去された後、開頭のおおよその局在を実証する。鉗子との開頭術の後、小脳が可視化される。小脳の小部分を注意深く吸引し、次に注入することができ、CN、（ 図1）の可視化を実証する。 <p cl...

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.

聴覚路の光遺伝学的刺激のためのマウス背側蝸牛神経核の直接可視化

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

Research

JoVE Journal

Neuroscience

9.2K ビュー.  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. 

ビデオ: 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.

成人および新生児マウス内耳への薬剤の局所投与への外科的アプローチとしての Canalostomy

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

遺伝学

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.

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

神経科学

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.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

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.

齧歯類の中枢神経系の生体光遺伝学的刺激

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.

へのガイド<em&gt;インビボ</emOptogenetically同定された皮質抑制性介在から&gt;シングル単位記録

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.

聴覚神経の光遺伝学的刺激

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.

新生児マウス蝸牛移植法<em&gt;インビトロ</em&gt;聴覚研究のスクリーニングツール

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.

生体内ニューロン回路接続を模倣するためのインビトロウェッジスライスの準備

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

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

同時光遺伝学的変調と電気的神経記録のためのオプトロードアレイ

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...

Preparing Wedge Brain Slices to Visualize Auditory Neurons

Source: Fischl, M. J. et al., <a href="https://app.jove.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.

聴覚ニューロンを可視化するためのくさび脳スライスの準備

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

聴覚路の光遺伝学的刺激のためのマウス背側蝸牛神経核の直接可視化

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