著者らは、初期胚発生時にひよこ内耳の解剖学上の専門知識のためのイメージング技術と博士ドリス·ウーとの提案と支援博士キャンディHsieh氏に感謝したいと思います。この作品は、NSF、IOS-0642346、NIH T32-DC010775、NIH T32-GM008620、NIHのR01-DC010796、とDOE GAANN P200A120165によってサポートされていました。

1。以下解剖ツールおよび試薬を準備<ul><li>連続して95％Oを流し込んで、人工脳脊髄液（130mMのNaCl、3mMのKClを、1.2mMのKH 2 PO 4、20mMのNaHCO 3を 、3 mMのHEPES、10mMグルコース、2mMのCaCl 2、1.3mMのMgSO 4を ACSF）室温で2/5％CO 2で 。点滴は、2月3日ふたに穴を開けた500ml広口ナルゲン瓶に記入してください。タンクは瓶のふたの穴に通しACSFを貫通ガラスステムバブラーにチューブで接続されます。液体の約1/3に達するが、リムの上に飛散引き起こすのに十分な説得力がありません泡の一定のストリームを取得するために圧力を調整します。互いから、ガス流の乱れから、個々のサンプルを分離するナルゲン瓶に沈める5ミリリットルガラスバイアル（最大6）。 ACSFは、どの組織のインキュベーションの前に少なくとも20分間酸素化されるべきである。 </li><li>小さなシリコーン解剖皿（35ミリメートル×10小さな六角形のポリスチレンに配置された2つの解剖ピン付きシルガード弾性体キットを使用してmmペトリコーティング）（47ミリメートルの下）ボートの重量を量る。 </li><li>二つ精密チップ·鉗子、1湾曲先端鉗子、50mlビーカー、および組織廃棄用のコンテナ。 </li><li>ガラスマイクロピペット（1.2外径/ 0.9 ID）の先端の開口は約20から50程度に分割して0.4パーセントと6.25パーセント·ソリューションのトリトンX-100、PBS中におけるローダミンデキストランアミン（RDA、MW = 3000、Invitrogen社製）を充填した引っ張ら。マイクロマニピュレーターにpicospritzerして安定化する細かいチューブで取り付けます。 </li></ul> 2。顕微解剖E8 4,5で乳頭基部を明らかにする<ol><li>湾曲したピンセットで、ボートの重量を量る内にシリコーン被覆解剖皿に直接頭を置いて、ちょうど脳幹下E8胚を切った。 </li><li>興味のある側の耳が少し見えるように位置腹望むヘッドは、解剖のピンを使って、頭がわずかに背側に傾けて、CEから10から30度を指しているNTER（ 図2A）。すぐにピンと頭を固定します。二国間のトレースは、中心0度ヘッド、または両側がアクセスできるように、完全な腹ビューを希望する場合は。左側がアクセスしやすいように同様に、左サイド注入のために、中心から-30度〜-10で頭部を固定します。 </li><li> 50ミリリットルのビーカーを使用して、完全に組織を浸漬、皿に酸素化ACSFの30ミリリットルを転送します。 </li><li>そっとつかんで、精密チップ·鉗子を用いて下顎ダウンはがし、次に興味のある側に下瞼を削除します。 </li><li>残りの口蓋と食道を含め、上層の血管組織を削除します。これらの軟部組織が剥がれ、その表面に独特の椎骨動脈と軟骨頭蓋の開発の滑らかな表面を明らかにする必要があります。この時点では、特徴的な白いotoconial質量が見えるはずです。 </li><li>に残したまま、慎重に外尿道口、顎関節の軟骨、中耳の周りの​​皮膚を切開NERの耳無傷。そっとこれらの軟骨構造を切り取るために鉗子を使用しています。 </li><li>椎骨動脈を外し、細かい鉗子のヒントと穏やかな弧を描く動きを用いて血液をプール。血がビューと凝固がインジェクションピペットの開口部を塞ぐことが見えにくくなる場合があります。 </li><li>隣接感覚上皮（乳頭基部）と聴神経節と蝸牛管は軟骨頭蓋6の直下に位置し、前方の近くの最も遠位の先端（頂点）で、腹側内耳から延びる細長い指状の突起として可視化することができる正中線5。視覚化するのを助けることができる蝸牛管（ 図2B）の頂点に解離および/ ​​またはotolithic石灰（白）から少量の血液プーリング（赤）があるかもしれません。頂点にlagenar黄斑ですが、感覚パッチは前庭機能7,8であると思った。 </li></ol> 3。 CN VIII聴覚繊維の選択的標識<ol><li&gt;蝸牛管と乳頭基部領域における胚の表面付近インジェクションピペットを置きます。 </li><li>最初はゆっくり穿刺頭蓋にマイクロピペットを下げます。この頭蓋領域は、周辺地域よりも薄く、小さい力が浸透している必要があります。マイクロピペットは現在、蝸牛管の内側になければなりません。オプション：トレース染料の小さな注射は乳頭基部を視覚化するのを助けるためにここで行うことができます。 </li><li>マイクロピペットの位置を変更せずに、単に侵害それまで蝸牛管（ 図1）の深い境界線を下げ続けている。ここに注射を行うと前庭つぼが置かれている最も近位チップを除く、乳頭基部（ 図2C、2D）の長さ方向に沿って複数の地域で繰り返す。 10から30 psiの間に設定picospritzerで、いくつかの注射が行われます。注入された色素の流体の体積は変化し、所望の標識の程度に依存する。各注射は蛍光色素ラをもたらすべきである約200のbeledスポット- 400μmの直径（ 図2D）。 </li><li>注入が完了すると、脳幹上記サンプルを横断すると、継続的に95％O 2/5％CO 2下で灌流さACSFのコンテナ内に小さな瓶に尾部を配置するためにホールピペットを使用する鉗子を使用しています。 </li><li>各胚については、目的の中央トレースの距離に応じて、移染のために20-30分を許可します。 </li><li>組織を除去し、組織学的切片の準備をします。ここに示す例では、組織は4％パラホルムアルデヒド（1X PBS中）を一晩一晩、30％スクロース（1X PBS中）に浸漬されている。 </li></ol> 4。対比と分析<ol><li>対比は、解剖学的位置に向きをオブザーバを助けるだけでなく、関心のある特定の領域を識別するのに便利です。この製剤における有効対比を強くリットルニューロフィラメント免疫標識（ 図1、図3B、3E）であり、abelsは、軸索と中枢神経の軸索路の区分を可能にします。 </li><li>予測はrostrocaudal軸に沿って数百ミクロン以上を拡張することができますように分析は、標識された地域全体で行うべきである。基本的な分析のいくつかの例は以下のとおりです。ターゲットエラーレート、タイミングおよび/または投影パターンの発散の変化。 RDA-標識軸索は、高解像度（ 図3A、3D）と末梢および中枢神経系において可視化される。 </li></ol>

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利害の衝突は宣言されていません。

VIIIth神経の早期開発の研究があるため、複数の別個の神経節から生じる胚軸索を識別するのに難しさの一部に限られていた。いくつかの研究は、初期の開発中に、聴覚と前庭感覚細胞と神経節細胞の運命を導く分子シグナル、5,11,12を検討されているが、中枢神経支配を規制するプロセスがまだ決定されています。レスがプライマリ核に投射する中央のプロセスについてはほ?...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>ポリスチレンは皿を計量</td><td>フィッシャー·サイエンティフィック</td><td> 02-202-101 </td><td></td></tr><tr><td>シャーレ、35 X 10ミリメートル</td><td>フィッシャー·サイエンティフィック</td><td> 50820644 </td><td>シリコーン解剖皿を作るために使用</td></tr><tr><td>シルガードシリコーンエラストマーキット</td><td>ワールド·精密機器</td><td> SYLG184 </td><td>コー​​トペトリは、解剖皿を作る</td></tr><tr><td>解剖ピン</td><td>さまざまな</td><td></td><td>解剖時に所定の位置に胚を保持</td></tr><tr><td> NACL </td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td> KClを</td><td>さまざまな</td><td></td><td> ACSFレシピの一部</TD&gt; </tr><tr><td> KH 2 PO 4 </td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td> NaHCO 3を </td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td>グルコース</td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td> CaCl 2を </td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td> MgSO 4を </td><td>さまざまな</td><td></td><td> ACSFレシピの一部</td></tr><tr><td> ACSF用のコンテナ。蓋付き透明広口ナルゲン瓶、500ミリリットル（16オンス）を示唆している。 </td><td> CPLabSafety </td><td> QP-PLC-03717 </td><td>液体浸透するガラスbublingステム用蓋の上部の穴の開口部をドリル</td></tr><tr><td>空の5ミリリットルのガラスバイアルまたは同等の透明バイアル</td><td>アメリカの製薬パートナーズ株式会社</td><td> 6332300105 </td><tD&gt;互いにおよびバブリングストリームからサンプルを別々に保管するACSFのインキュベーション中に使用</td></tr><tr><td>バブラーにチューブで接続されたcarbogenのタンク（95％O 2/5％CO 2）で</td><td>さまざまな</td><td></td><td> ACSFへの注入用ガラス幹バブラーに管によってアタッチ</td></tr><tr><td>ガラス幹バブラー</td><td>さまざまな</td><td></td><td> ACSFにcarbogenを注入する</td></tr><tr><td>先の曲がった鉗子</td><td>ワールド·精密機器</td><td> 501008 </td><td>卵から胚の頭部を削除するには</td></tr><tr><td>二つ精密チップ·鉗子</td><td>ワールド·精密機器</td><td> 501985 </td><td>顕微解剖用</td></tr><tr><td> 50mlビーカー</td><td>さまざまな</td><td></td><td></td></tr><tr><td>ローダミンデキストランアミン（RDA） </td><td>インビトロジェン</td><td>さまざまな</td><td>蛍光軸索トレーサー</td></tr><tr><td>トリトンX-100 </td><td> ICNバイオメディカル</td><td></td><td></td></tr><tr><td>リン酸塩は、（1×PBS）、緩衝生理食塩水</td><td>さまざまな</td><td></td><td>標準的な実験用試薬</td></tr><tr><td>薄壁ガラスキャピラリー、外径1.2、0.9番号4 &quot;（100mm）の長さ</td><td>ワールド·精密機器</td><td> TW120F-4 </td><td> RDAを使用したロード。各キャピラリーは、2枚のガラスマイクロピペットを作る</td></tr><tr><td>ニードル/ピペットプラー</td><td>デビッドKopfはインスツルメンツ</td><td>モデル720 </td><td>使用される設定：熱16.4、ソレノイド2.2 </td></tr><tr><td> Picospritzer </td><td>パーカー計装用</td><td>さまざまな</td><td>ガラスマイクロピペットを微管によって取り付け</td></tr><tr><td>極微操作装置</td><td>ナリシゲ</td><td>さまざまな</td><td></td></tr><tr><td>蛍光と解剖顕微鏡</td><td>さまざまな</td><td></td><td></td&gt; </tr><tr><td> 4％パラホルムアルデヒド</td><td>さまざまな</td><td></td><td>標準的な実験用試薬</td></tr><tr><td>抗ニューロフィラメント抗体、オプション</td><td>ミリポア</td><td> AB1991 </td><td>メーカーが推奨する組織学的なプロトコルに従ってください</td></tr><tr><td>クライオスタットとセクショニングの関連材料</td><td>ライカ</td><td>さまざまな</td><td></td></tr><tr><td>イメージングのための落射蛍光顕微鏡</td><td>ツァイスは、様々な</td><td></td><td></td></tr></tbody></table>

selective tracing of auditory fibers in the avian embryonic vestibulocochlear nerve

ニワトリ胚は、末梢および中枢神経節細胞の突起の研究のために広く使用されているモデルです。聴覚系では、VIIIth脳神経内聴覚軸索の選択的標識は、中枢聴覚回路発達の研究を強化するであろう。内耳の複数の感覚器官がVIIIth神経1に貢献するため、この方法は困難である。また、確実に鳥VIIIth神経軸索内の前庭グループに対して聴覚を区別するマーカーが同定されていない。回路が形成される前に、感覚誘発反応が存在していないとして、聴覚と前庭の経路は、初期胚において機能的に区別することはできません。一元的に突出したVIIIth神経軸索は、いくつかの研究でトレースされているが、聴覚軸索標識は他のVIIIth神経コンポーネント2,3からラベリングを伴っていた。ここでは、音響神経節から選択ラベへのトレースを順行するための方法を説明します開発VIIIth神経内のL聴覚軸索。まず、酸素人工脳脊髄液中に浸漬し、8日、ニワトリ胚の前頭部の部分切開した後、蝸牛管は、解剖学的なランドマークによって識別されます。次に、罰金引かガラスマイクロピペットは、音響神経節細胞が配置されているダクトと隣接深い領域にローダミンデキストランアミンを少量注入するように配置されている。注射後30分以内に、聴覚軸索は後脳に集中的に追跡され、後に、次の組織学的準備を可視化することができる。この方法では、中枢聴覚回路形成に周辺の発達研究のための便利なツールを提供します。

VIIIth神経と神経自体の解剖学的構造のコンポーネント（ 図1、図3）、複雑で入り組んである。選択的に音響神経節細胞から生じる繊維をトレースすることにより、脳幹内VIIIth神経だけでなく、一次聴覚求心性神経のセグメントがきれいにトレースすることができるとその前庭の対応（ 図2、図3）とは区別される。同様に、この技術は、音響神経節細胞の周辺の突起?...

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

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

ここでは、神経と後脳における聴覚軸索線維の選択的なトレースのための初期のニワトリ胚の音響神経節への蛍光色素を注入し顕微解剖法を説明します。

鳥類胚性内耳神経における聴覚繊維の選択的なトレース

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

9.1K ビュー.  University of California, Irvine. ここでは、神経と後脳における聴覚軸索線維の選択的なトレースのための初期のニワトリ胚の音響神経節への蛍光色素を注入し顕微解剖法を説明します。

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

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

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

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

発生生物学

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

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

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

蝸牛内有毛細胞に接触求心性樹状突起でのシナプス後録音：リボンシナプスでのモニタリングの多胞体のリリース

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

神経科学

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

チキンの聴性脳幹スライスの準備と文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

チキン聴覚器官への遺伝子導入によって、<em&gt;卵内</em&gt;マイクロエレクト​​ロポレーション

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

In Ovo Electroporation in the Chicken Auditory Brainstem

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

In Ovo Electroporation in the Chicken Auditory Brainstem

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

鶏の聴覚脳幹における卵の電気穿孔

Electroporation is a method that introduces genes of interest into biologically relevant organisms like the chicken embryo. It is long established that ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

胚性ニワトリ聴覚脳幹をスライスしてトノトピック勾配とマイクロ回路を評価する

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

ニワトリ孵化子の聴覚脳幹応答の評価

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

聴覚回路における脆弱X精神遅滞タンパク質の細胞自律機能をIn Ovo エレクトロポレーション法で解剖

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

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

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

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

パッチクランプ記録のための新生児げっ歯類からの前庭およびらせん状神経節体細胞の分離と培養

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