失明とEY02687（ワシントン大学眼科視覚科学部）を防ぐため失明を防ぐための研究からキャリア開発賞、NIHの助成金EY19312とEY19543（VJK）によって、同様の研究から無制限の助成金によってサポートされています。

1。電極を作る<ol><li>ガラス電極を準備します。 120mgの寒天を量り、10 mLの蒸留水（最終寒天濃度1.2％）でそれを混ぜる。湯浴中で寒天溶液を溶かす。プラスチックシリンジを用いて寒天溶液を用いて：ガラスキャピラリー（長さ= 100ミリメートル、外径/内径= 1/0.75 mmとし、内部容積= 44μL我々は以下の寸法を使用してWordのPresisionインスツルメンツTW100-4キャピラリーを使用します）を入力します。室温で10分間寒天を固める。したがって、最大約200毛細血管が（寒天混合物の残りの部分は-4℃と、再使用されるいくつかの時点で保存することができます）入力することができます。 </li><li>ダイヤモンドナイフを使用して半分に毛細血管を切った。 </li><li> 4℃で少なくとも24時間のマウス電極溶液中で得られたガラス電極を浸す4℃で電極を保存し、3〜4ヶ月の期間にわたってそれらを使用します。 </li></ol> 2。実験のセットアップ<ol><li>一晩マウスを暗適応します。 （例えば、C野生型を使用します。57Bl / 6）ロッド駆動型transretinal録音とTのα - / -マウスコーンtransretinal録音8シグナリングロッドを欠損したマウス。 </li><li> 1リットルの潅流液を、100mLの電極液、20mLのインキュベーション溶液（下記参照）を準備し、灌流液の谷のミリポア0.45μmまたは0.22μmのフィルター（オプション）をフィルタリングします。 </li><li>網膜の平面に配置された光度計を用いた光源（505 nmのLEDやハロゲン電球の光刺激）を校正します。 LED光源の場合には、光の均一性を達成するために、光拡散板を使用しています。 </li><li>実験（チャンバー構造は異なる場合があります）の灌流チャンバーを準備します。私たちの部屋は、小さなプラスチック製のペトリ皿の蓋から作られた底に、3 mmのプレキシグラス製で、〜400μL（L = 25ミリメートル、W = 8ミリメートル、H≈2ミリメートル）の活性灌流量を持っています。 2mMのL-グルタミン酸及び10mM BaCl 2を含む電極溶液をチャンバーの下部電極の空間を埋める。プラスチックのチューブやガラスキャピラリーを用い、下部電極空間内の溶液（チャンバーの底部に取り付けられたプラスチック製のチューブコネクタ製）と電極ホルダーの間の接続を行います。電極には気泡、接続チューブ、または電極ホルダー（必要に応じてプラスチックシリンジを使用してソリューションをパージ）が存在しないことを確認してください。 </li><li>顕微鏡のステージ（：顕微鏡は、防振テーブル上に取り付けられ、機械的、電磁ノイズを最小限に抑えるためにファラデーケージ内に遮蔽することができるオプション）に録音室をマウントします。差動増幅器の正のステージのポール（例えばワーナー·インスツルメンツDP-311アンプ）に下部電極ホルダーを接続します。センターと刺激光スポットで開いてチャンバー下部電極の位置を合わせます。録音室に流線を接続し、灌流をオンにします。 </li><li> 95％O 2/5％CO 2のバブル灌流液40にそれをインキュベートしながら°CワットERのお風呂。 CO 2は 7.2に溶液のpHを調整します。解決策は、36から37にそれを再加熱セラミック抵抗体を通過する重力℃にして瓶から録音室に流れる熱損失を低減するために、ヒーターは、理想的には顕微鏡のステージ上で、可能な限り記録室の近くに配置する必要があります。 </li><li>フローレギュレータで約1 mL / minの流量を調整します。 </li><li>第二の電極ホルダーを使用して、ステージの第二の（マイナス）極に上部電極を接続します。 O-リングを使用して、上部電極に熱電対プローブをバインドします。電極と熱電対の先端が温度の読み取りが可能な限り、網膜の中心部に近いと解釈されるように、接近していることを確認します。灌流液中の上部電極の先端を浸し、室の中央に配置します。ベースライン安定化のための10-15分を許可します。 </li><li>溶液の温度は36になるようにヒータのDC電圧を調整する -37℃ </li></ol> 3。マウスの網膜を分離<ol><li>薄暗い赤い光の下で、CO 2および頸椎脱臼で暗順応マウスを安楽死させる。オプション：焼灼ペンまたは加熱された解剖ピンと強膜の中で最も背点を焼灼することにより、各マウスの眼球上の基準マークを付けます。後続のすべての手順は、解剖顕微鏡に合わせる赤外線画像コンバータを使用して赤外光の下で実行されています。 </li><li>穏やかな圧力を適用し、各眼の両側に皮を延伸することによって湾曲したハサミで目を解剖する。 microscissorsまたはカミソリの刃を使用して、赤外線照明の下で両方の眼球を二等分する。 </li><li>角膜とレンズを外します。できるだけ硝子の限りを削除します。 </li><li>色素上皮層からの最初の網膜剥離及び、必要に応じて、色素上皮の残りの顆粒を削除するには、ピンセットでそれを徹底的に清掃してください。我々は一般的にロッドとコーンtransretの両方に全体の網膜を使用しているinal録音。ただし、特に網膜は、参考として焼灼マークを使用してかみそりの刃またはmicroscissorsとアイカップの腹の部分を削除する剥離する前に、より多くのM / L錐体9を有しているマウスの網膜の背側から録音したい場合。 </li><li>インキュベーション溶液小さなペトリ皿の中で第二hemisected眼球を配置し、使用時まで純酸素で飽和した遮光ボックス内の準備をインキュベートします。通常、これらの条件下で二個目のマウスのアイカップは、室温で数時間保存することができ、録音に使用されます。 </li><li>ガラスやプラスチック製のピペットを使用してペトリ皿のふたの上灌流溶液の液滴（約300μL）に分離された網膜を転送します。 BaCl 2でプレインキュベート網膜にBaCl 2を含む電極液の5-6小滴を追加します。コー​​ナー塗っ裏面の四角い部分（約5×5 mm）を置きます（私たちはダウコーニング111バルブ潤滑剤とシーラントを使用します）ミリポアろ紙（0.45μmのは、同じソリューションのドロップに、その中心に既​​製の穴（直径2〜2.5ミリメートル）で）型をHARG。ろ紙の上（最大受光側）に鉗子の位置網膜を使用して、それをフラットマウントするために周囲にその端を押してください。網膜は、ろ紙の開口部を介して中心にする必要があります。 </li><li>鉗子を使用すると、灌流チャンバーに網膜にろ紙を転送し、刺激スポット揃え下部電極空間上に配置します。若干チャンバーと結合、それにろ紙の油を塗ったエッジを押してください。 </li><li>網膜の中心部（少し感光層に触れて）の上に上部電極を配置します。網膜上に上部電極を中心とする信号の振幅（最大〜網膜の端にそれに近い配置に比べて20〜40％）増加します。このような電極の位置がわずかに網膜の小セクタ（その総面積の〜12から15パーセント）に達する効果的な光刺激強度を歪めるが、それはまだ電気を配置する最適な方法です。trode。準備は今transretinal録音の準備ができています。 </li></ol> 4。 Transretinal録音<ol><li>マウスのライン、LED光源または光刺激から提供されたキャリブレーションさ505nmの光の明るい強度に薄暗いからレコードロッドやM / L-錐体駆動型テストのフラッシュ応答に応じて。当社のLED光源の場合には、フラッシュの強さは、LED入力電圧、切替器、および中性密度フィルタのセット（ここ​​ではE-カラーロスコ研究所から＃211 0.9 ND膜フィルタを使用してのコンピュータ制御の組み合わせによって制御されます。 ）LEDと網膜の間に配置されます。 LEDテストフラッシュ（20 ms）の期間は、コンピュータによって制御されます。ハロゲン電球の光刺激、テスト発光強度と波長を使用している場合は、それぞれ、中立的な密度フィルタおよび狭帯域干渉フィルタのセットによって制御することができます。テスト発光持続時間は、コンピュータ主導のシャッターで制御することができます。 </li><li>オプション：効率的な維持のため終日BaCl 2の光応答のグリア細胞成分の抑制は、新鮮な溶液を充填したプラスチックシリンジを使用してそれをパージすることによって（例えば、前のそれぞれの網膜をテストする）時折下部電極のソリューションを変更します。 </li></ol> 5。代表的な結果ソリューション<ol><li>マウス網膜灌流液：112.5 mMの塩化ナトリウム、3.6 mMの塩化カリウム、2.4 mMのMgCl 2、1.2mMのCaCl 2を 、10mMのHEPES（pH7.2）を、20mMのNaHCO 3を 、3 mMのコハク酸ナトリウム、0.5mMのナトリウム、グルタミン酸、0.02mMのEDTA、 10 mMグルコース。さらに、このソリューションは、2 mMのL-グルタミン酸と光応答10,11の高次成分をブロックするために、10μMのDL-AP-4を添加され、0.1％MEMビタミンやMEMアミノ酸溶液（Sigma）で網膜を向上させるために可能性。 </li><li>マウスの電極液：140mMのNaCl、3.6 mMの塩化カリウム、2.4 mMのMgCl 2、1.2mMのCaCl 2を 、3 mMのHEPES液（pH7.4、NaOH）を。下ELEのソリューションctrodeスペースも光応答3,12のグリア細胞成分を抑制するシナプス伝達10と、さらに、10mMのBaCl 2をブロックするように2mMのL-グルタミン酸を含んでいます。 </li><li>網膜インキュベーション溶液：272 mgのL-15培地（Sigma）および20 mLの脱イオン水に20mgのBSAを溶解する。最後のL-15とBSAの濃度は、それぞれ13.6 mg / mLとし、1 mg / mLのである。 1 M HClでpHを7.4に調整します。 </li></ol><img alt="図1。" src="/files/ftp_upload/3424/3424fig1.jpg" /> 図1。transretinal ERG記録用のマウス網膜の分離を示す模式図。 <img alt="図2。" src="/files/ftp_upload/3424/3424fig2.jpg" /> 図2：灌流チャンバーとその所有者と記録電極の写真。赤い矢印は血流の流れ方向を示しています。 <img alt="図3。" src="/files/ftp_upload/3424/3424fig3.jpg"/&gt; 図3。transretinal ERGの記録のための実験セットアップの模式図。赤い矢印は血流の流れ方向を示しています。 <img alt="図4。" src="/files/ftp_upload/3424/3424fig4.jpg" /> 図4代表的な結果：マウスロッド駆動型transretinal応答の記録家族。光応答は、ローパス1 kHzでデジタル化し、さらなる分析のためのコンピュータに保存され、30 Hzの（8極ベッセル）でろ過した。に示すトレースは飽和光強度で薄暗い光強度および2-3応答で5月6日応答の平均を表しています。 <img alt="図5。" src="/files/ftp_upload/3424/3424fig5.jpg" /> 図5代表的な結果：マウスコーン主導transretinal応答の記録家族。光応答は、ローパス1 kHzでデジタル化し、さらなる分析のためのコンピュータに保存され、30 Hzの（8極ベッセル）でろ過した。に示すトレースはアベレージれ飽和光強度で薄暗い光強度および3-5応答で5〜10応答のES。

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著者らは、開示することは何もありません。

ロッドとコーン主導transretinal ERG記録法は、野生型および遺伝子改変動物の両方で、マウスの光受容体の機能を調べるための強力なツールになっている上記の。基本的な光応答特性の簡単な特性に加えて、この単純な手法に近いから無傷の網膜の準備で実行長期的な実験中に大きな応答の安定性を提供します。野生型マウスの暗順応ロッドの最大応答振幅と感光性の両方が録音の開始から少な...

<table border="1"><tbody><tr><td> 材料名 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> DL-AP-4 </td><td></td><td>バイオサイエンスTOCRIS </td><td> 0101 </td><td></td></tr><tr><td>すべての他の試薬</td><td></td><td> Sigma-Aldrich社</td><td></td><td></td></tr><tr><td>ガラスキャピラリー</td><td></td><td>世界の精密機器</td><td> TW100-4 </td><td>電極を作るため</td></tr><tr><td>ろ紙</td><td> HARG </td><td>ミリポア</td><td> HABG01300 </td><td></td></tr><tr><td>光度計</td><td></td><td> UDTインスツルメンツ</td><td> S350 </td><td>光キャリブレーションのために</td></tr><tr><td>放射シリコンセンサ</td><td></td&gt; <td> UDTインスツルメンツ</td><td> 221 </td><td>光キャリブレーションのために</td></tr><tr><td>防振テーブル</td><td></td><td>テクニカル·マニュファクチャリング株式会社</td><td> TMC 78から239-02R、TMC63-26012から01 </td><td>メカニカルノイズを最小限に抑えるために</td></tr><tr><td>エアコンプレッサーパンサーP 15TC </td><td></td><td>ウェルテルインターナショナル</td><td> P 15TC </td><td>防振テーブルに接続</td></tr><tr><td>実体顕微鏡</td><td></td><td>ライカ</td><td> MZ9.5 </td><td>マウスの眼の解剖のために</td></tr><tr><td>赤外線イメージコンバータ</td><td></td><td> BEMeyers </td><td> ProwlerTM </td><td>実体顕微鏡にバインドされている</td></tr><tr><td>差動増幅器 DP-311 </td><td></td><td>ワーナー·インスツルメンツ</td><td> DP-311 </td><td></td></tr><tr><td>軸索Digidata 1440Aデジタイザ</td><td>; </td><td>モレキュラーデバイス</td><td> 1440A </td><td></td></tr><tr><td>デュアルチャネル8極フィルタ</td><td></td><td>クルーン-HITE株式会社</td><td> 3382 </td><td></td></tr><tr><td>セラミック抵抗</td><td></td><td> TEの接続</td><td> CGS SBCHE618RJ </td><td>灌流液を加熱するための</td></tr><tr><td>熱電対</td><td> T </td><td> Physitempインスツルメンツ</td><td> IT-18 </td><td></td></tr><tr><td>温度モニター</td><td></td><td>オメガ</td><td> DPi32 </td><td>熱電対に接続</td></tr><tr><td> LEDは505nmの</td><td></td><td> TTエレクトロニクス/ Optek Technology種類</td><td> Digi-KeyのP / N 365-1185-ND </td><td>テストが点滅/漂白ライトを適用するには</td></tr><tr><td>焼灼ペン</td><td></td><td> Bovie </td><td> AA25 </td><td>背の部分をマーキングするためのマウスの眼球の</td></tr><tr><td> 10電気生理学的データ収集と解析ソフトウェアをpCLAMP </td><td></td><td>モレキュラーデバイス</td><td></td><td></td></tr></tbody></table>

transretinal erg recordings from mouse retina: rod and cone photoresponses

ロッドとコーン：脊椎動物の網膜に結像光受容体の2つの別個のクラスがあります。ロッドは、コーンが急速に明るい光条件の変化の下で継続的に動作するのに対し、光の単一光子を検出することができます。光受容体の外側のセグメントのロッドとコーン特有の視覚的な顔料による光の吸収は、最終的に細胞膜と細胞の過分極における環状ヌクレオチド依存性チャネルの閉鎖につながる光情報伝達カスケードをトリガします。現在および潜在的な膜で、この光誘起変化はどちらか古典的な吸引電極記録手法1,2、または薬理学的にブロックされたシナプス応答コンポーネントを3月5日孤立網膜からtransretinal電図記録（ERG）によって、光応答として登録することができます。後者の方法は、マウスの光受容体からの薬物がアクセス可能な長期的な録音を可能にし、安定した光応答frを得るために、特に便利です。OM乏しいと壊れやすいマウスコーン。円錐の場合には、そのような実験は暗順応6,7の間にコーン感光性回復のプロセスを監視するために、両方の本質的にすべての視覚色素を漂白暗順応条件と、以下の強烈な照明で行うことができます。このビデオでは、ロッドと暗順応マウス網膜からM / L-錐体主導transretinal録音を実行する方法を示します。ロッドの録音は、野生型（C57BL / 6）マウスの網膜を用いて実施される。 8シグナル伝達ロッドを欠くマウスを簡単にするために、コーンの録音は、遺伝子組換えロッドトランスデューシンαサブユニットのノックアウト（ - / - Tα）から取得されます。

Watch this Scientific Journal Video about Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses at JoVE.com

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

我々は、無傷のマウス網膜から桿体と錐体の光応答を取得するためのtransretinal電図（ERG）の録音の比較的簡単な方法について説明します。このアプローチは、それらの光反応を分離し、分離されたフラットマウントされている網膜の間に配置フィールド電極を使用してそれらを記録する光受容体からシナプス伝達のブロックを活用しています。

マウス網膜からERG Transretinal·レコーディング：ロッドとコーンの光応答

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

transretinal-erg-recordings-from-mouse-retina-rod-cone

Research

JoVE Journal

Neuroscience

17.6K ビュー.  Washington University School of Medicine. 我々は、無傷のマウス網膜から桿体と錐体の光応答を取得するためのtransretinal電図（ERG）の録音の比較的簡単な方法について説明します。このアプローチは、それらの光反応を分離し、分離されたフラットマウントされている網膜の間に配置フィールド電極を使用してそれらを記録する光受容体からシナプス伝達のブロックを活用しています。

ビデオ: Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

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

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

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

暗順応スライス標本におけるマウス網膜神経細胞からパッチクランプ記録

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

神経科学

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

の巨大線維経路からの電気生理学的記録 D.キイロ</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

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

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

胚ゼブラフィッシュマウスナー細胞からパッチクランプ記録

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

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

ビブラトームセクショニングマウス網膜は、感光体文化を準備する

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

イメージングカルシウム<sup&gt; 2+</supマウス網膜の光受容コーン軸索末端で&gt;ダイナミクス

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

事項をカットする場所: 郭清と眼のランドマークからマウス網膜の空間的なオリエンテーションの解析ガイド

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

子豚を自由に移動するから非侵襲的脳波

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

軸索の成長円錐の破壊とマウスの培養神経細胞の初期のアミロイド β 効果の可視化

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...