原稿にジョシュア歌手、ジョナサン ・ シェンク、コメントに対して思いやりのある校閲者に感謝したいと思います。我々 も感謝したいベン ホワイトとハロルド法テクニックについて議論します。ジョナサン ・ シェンクは、図 3 aのデータを提供します。この作品は、ホワイト ホールの基盤助成金、QG を NIH R21 によって支えられました。

1. ハエや識別 GAL4 プロモーター ライン
<ol><li>GAL4 プロモーターのラインを選択し、UA NaChBac 遺伝子と活性化のための光遺伝学的遺伝子を運ぶハエとそれを交差します。 注: その高い電気伝導度と NaChBac 媒介活動電位がアクティブ化、使いやすさのためには、UA CsChrimson11が選択されます。ブルーミントン株式リポジトリからは UA NaChBac と UA CsChrimson の両方があります。</li>
	<li>エタノール (35 mM) の原液としてすべて trans 網膜を準備し、-20 ° C での冷凍庫保管ソリューション</li>
	<li>約 5 mL の水分を補給されたジャガイモにこの株式のミックス 28 μ L フレーク フードと従来のショウジョウバエ媒体のバイアルの先頭にこの混合物を追加します。</li>
	<li>0.2 mM すべて-トランス-レチナールの 1-3 日11を含む食品に csChrimson を表現する大人のハエを発生させます。</li>
</ol>2. TTX 在庫を準備します。
<ol><li>蒸留水または脱イオン水で原液 1 mm TTX を作成します。数ヶ月の研究室の冷蔵庫にこのソリューションを保存します。多くの機関が有害または制御物質として TTX を分類、TTX のストレージに関する機関の方針を確認してください。</li>
</ol>3. 電気生理学のハエを準備します。
<ol><li>すべて trans 網膜に食品に発生する古い大人のハエ 1-3 日を収集します。 注: は、パッチクショウジョウバエのニューロン12,13の詳細については 2013 年にマーシーとターナーを参照してください。</li>
	<li>ハエをシンチレーション バイアルに入れてください。ハエを固定する 1 分の氷にバイアルを配置します。</li>
	<li>粗鉗子を使用すると、カスタム記録室にハエを挿入します。商工会議所は、3 ½"サークル レーザー カット 1/16" アクリルからから成っています。カット ¾"穴は 2 液性エポキシでカスタム ホイルがアタッチされているセンターです。ハエが挿入希望の形をした三角形の箔が含まれます。</li>
	<li>ワックスまたは記録室の中でしっかりとそれを動物に UV 硬化型エポキシ樹脂を適用します。</li>
	<li>グースネック光ファイバー顕微鏡下で可視化のための準備を照らします。彼らは側から直接その場を照らすので、それらを調整することにより斜照明用グースネック繊維を設定します。</li>
	<li>まだハエの明確に可視化を可能にする最低の設定に光のレベルを調整します。この光の強度は、個々 の実験の間で異なります。 注: このプロトコルは、適応することができますでその場の脳を使って移植法14。</li>
	<li>外部録音の生理食塩水を取得します。MM で生理食塩水を含む: 3 103 塩化ナトリウム、KCl、5 N-トリス (ヒドロキシメチル) メチル-2-— ジアルキルアミノエタン-スルホン酸、8 トレハロース、ブドウ糖 10、26 NaHCO3、1 改良近距離音響ホログラフィ2PO4、1.5 CaCl2、および 4 MgCl2 (270-275 mΩ に調整)。95% O25% CO2 (カーボゲン ・) 生理食塩水をバブルし、7.3 の15に pH 調整をしています。</li>
	<li>フライの準備上の細胞外記録生理食塩水 4-6 滴を配置します。この時点で、優れた視認性解剖顕微鏡で光のレベルを上げます。</li>
	<li>電解を用いたタングステン細線をシャープに。ワイヤー自演3の飽和溶液を調製し、先端を削ってまで 20 回 100 ms のためのソリューションにワイヤの先端を浸漬しながら約 20 V の AC 電流を適用します。場で、脳がむき出しの頭にキューティクルを削除するのに尖ったタングステン ワイヤを使用します。</li>
	<li>さらに気管とそれを囲む脂肪体を削除することによって、脳を公開します。触角の丸い突出部にアクセスする、記録室に可視性を高める箔下にアンテナを優しく挟むに曲がったタングステン線または同様のツールを使用します。レコーディングが作成される興味の領域をカバー グリアの被覆を離れて引き裂く軽くシャープな鉗子を使用します。</li>
	<li>電気生理学のリグに録音室を転送します。すぐに開始外部録音生理食塩水で灌カーボゲン ・脳を維持するために、バブル。食塩貯留層は、顕微鏡ステージ上に配置し、準備に重力供給します。廃棄物の生理食塩水を収集するために、真空ラインと 2 L の三角フラスコを使用します。</li>
</ol>4. 全細胞記録を取得します。
<ol><li>市販のピペット引き手を引いて、パッチ ピペット。7 mΩ の近くの先端抵抗と適切なピペットを達成するための設定については、マニュアルを参照してください。</li>
	<li>パッチ ピペットに内部録音ソリューションの 4 μ L を追加します。アスパラギン酸カリウム 140、10 HEPES、4 MgATP、0.5 Na3GTP、1 グリコールエーテルジアミン四酢酸、および 1 から成っている内部の生理食塩水で mM KCl です。</li>
	<li>全細胞記録用パッチ クランプ アンプを設定します。アンプのオフセットをゼロし、テスト パルスを提供するアンプを設定します。ここでは、午前のシステム パッチ クランプ アンプ 2,400 が使用されます。</li>
	<li>ピペットを肯定的な圧力を適用し、セルのアプローチします。ピペットを直接セルに連絡してマイクロマニピュレーターを使用します。肯定的な圧力をリリースします。セット-60 に膜の保持可能性 mV。ピペットは、膜低サブ ピコアンペアの保持電流によって立証されるように gigaohm シールを形成する必要があります。 注: GFP を用いた細胞標的を導かれて、いる場合 ChR2 活性化と潜在的なシナプスを防ぐために epifluorescent ライトの使用を最小限にする試み。また手順 5 を適用する前に数分を待ちます。</li>
	<li>-50 からテスト パルスを提供するアンプを設定する-60 mV mV。この設定は、標準パッチ ・ クランプ アンプです。パッチ ピペットを充電に必要な流れを表す大きな容量性過渡テスト パルスを明らかにします。午前 2400 容量補正ノブを調整することによって容量性過渡または同じようなアンプを削除します。</li>
	<li>細胞膜を破壊し、細胞構成を取得する否定的な圧力の短いパルスを適用します。 注: ニューロンの静電容量を示す向けのトランジェントの大幅な増加がある場合、セル全体構成が観察されます。保持電流 (ベースライン) のわずかな増加はまた観察する可能性があります。アンプを現在のクランプ モードに設定し、-50-60 ~ セルの可能性を調整 mV。</li>
</ol>5. シナプス接続をテストします。
<ol><li>光パルスを生成する LED ドライバーをトリガーするデータ集録 (DAQ) システムを構成します。ここでは、Matlab とナショナルインスツル メンツの DAQ システムを使用して、LED ドライバーにアナログ信号を提供します。アナログ信号をドライバーに LED の輝度は調整可能します。LED は LED 620-630 nm の波長です。</li>
	<li>準備の下に直接にハイパワー赤色の LED を配置します。0.238 mW/mm2に達する場光の強度を調整します。</li>
	<li>関心のシナプス後細胞から録音中のシナプス前ニューロンをアクティブにします。細胞活性化生じる、pharmacogentically を達成するまたは神経刺激を介して。ここでは、csChrimson と赤色光の短いパルスが適用されます。 注: Epsp として現在のクランプまたは工として電圧クランプでは、シナプス接続を監視できます。シナプス接続が 40 ms 光パルス応答の TTX を適用する前に表示されます。そうだ場合は接続を認められなかったニューロンがシナプスがある接続されているいずれかのモノラル - または polysynaptically。潜在的な保有物は、それに応じて興奮または抑制の接続を強調する調整ができます。</li>
	<li>1 の最終的な集中を達成するために灌流システムに TTX を追加通常生理食塩水中での接続が発生した場合 μ m の範囲を使って再循環ポンプ キャプチャして TTX を節約するために生理食塩水を再利用します。蠕動性ポンプはお風呂から食塩を描画し、生理食塩水の貯水池に戻る。</li>
	<li>上昇し、下落を風呂で生理食塩水レベルを許可しない強力な一定真空を提供するためにポンプの回転数を調整します。 注: TTX のアプリケーションは全部携帯で監視中のニューロンの活動をスパイクの停止になります。これは、生理食塩水に十分な TTX が追加されていることを確認します。</li>
	<li>赤色光 (パルスごとの 40 ミリ秒) の短いパルスを再度適用します。この時点で、残っている任意のシナプス接続は可能性がシナプス。TTX のままシナプス結合の化学的性質をテストするのには循環の生理食塩水に薬理学的拮抗薬を追加します。 メモ: 光遺伝学とニューロンの蛍光ターゲットを結合する場合があります永続的記録を取得しようとしたときのニューロンのシナプス前の人口をアクティブにすることが重要。これはシナプスうつ病につながることができるし、の接続を参照してくださいすることは困難になります。ニューロンと channel2rhodopsin を視覚化する赤の蛍光体を使用ことができます csChrimson は緑の蛍光蛋白質の範囲に広く拡張する長い励起帯があるため (Ch2R) は細胞を刺激する使用ことができます。そう、特定の遺伝学およびカスタム フィルター キューブが必要です。適切なハエを生成、LexA または Q システム バイナリ表現システムの下で赤の蛍光体 (RFP または mCherry) を表現するハエを行います。これらは、Gal4 プロモーターと UAS Ch2R と UAS NaChBac のエフェクター遺伝子を表現するハエと交差する必要があります。落射蛍光の最適フィルター キューブ赤の蛍光体を励起が Ch2R。 さらに刺激しない狭い加振範囲、フィルター セットは、同様に多くの放射光を収集するために長いパス排出フィルターを含める必要があります。パーツ番号 et580/25 x と t600lpxr (49306 セット) からクロマから設定カスタム フィルターを使いましたが et610lp バリア/排出フィルターを使用。</li>
</ol>

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著者が明らかに何もありません。

テレピン油分析は褒め割れ特定ニューロン間のシナプスの検出を有効にする回路に使用される現在の技術です。具体的には、アプローチは広く TTX を区別しないナトリウム チャネル NaChBac ニューロンの興奮性を復元中 TTX と活動電位の沈黙によってシナプス接続を明らかにします。シナプス後イベントの監視全体セルの録音をしながら、シナプスのリリースは光刺激によって誘発されます。?...

神経科学の主な目標は、回路を介して情報の流れを理解するニューロン間の接続をマップすることです。多数のアプローチおよびリソース モデル システム1,2の範囲で神経細胞間の機能的結合をテストする利用可能となっています。電気生理学を使用して最も正確な配線図を生成するには、2 つのセル間の観察された接続が直接と間接とシナプス シナプスであるかどうかを解決することが重要です。哺乳類細胞におけるこの区別を作るためのゴールド スタンダードは、シナプス前の細胞の 1 つの action potentials と 2 番目のニューロンに興奮性シナプス電流 (工) の発症との間の待機時間を計測することです。短い待機時間は数ミリ秒と変動の少ない3シナプスの接続が必要です。長い電位シナプス コンダクタンスと記録間隔による検出の遅れを引き起こしている体細胞記録サイトどころか、樹状突起でシナプスがありますのでこのアプローチは無脊椎動物の神経細胞に複雑なすることができます。電極。このような遅延は、ポリ - シナプス貢献対に関するあいまいさを導入できます。さらに、小さなシナプス イベントは体性録音のサイトに到達する前に腐るかもしれないし、強いシナプス前の活動を運転はヒヨコ イベントを採用する可能性が高い。
様々 な技術は、無脊椎動物のシナプス接続をテストするために開発されています。1 つのアプローチは、過剰の Mg+ + , Ca+ +を含む高二価陽イオン溶液 (やあ Di) を使用します。このソリューションでは、削減の放出確率とシナプス入力4、5の検出を支持する活動電位のしきい値を増加シナプス接続をブロックします。必要な Ca ++ ++ Mg の正確な比率を決定するただし、簡単ではありません、ヒヨコの貢献は均一で適度な刺激6持続可能性があります。シナプス シナプス接続7を推論するは、前及びシナプス後膜間の近接の GFP 再構成間シナプス パートナー (把握) と呼ばれる別のアプローチを活用します。ここでは、緑色蛍光タンパク質 (GFP) のコンポーネントは、1 つのニューロンで表され、分子の相補的なフラグメントは推定シナプス パートナーで表されます。蛍光の存在は、2 つのニューロン GFP 分子の再構成を許可するように十分に近い近接は、シナプスの存在を意味することを示します。把握報告できるシナプス虚偽、しかし、2 つのセルが密接に神経や束のように膜をあてる場合。把握の亜種はシナプトブレビン、アクティブなシナプス8時のみ再構成がように直接に GFP のシナプス前のフラグメントをテザリングでこのような偽陽性を排除します。把握とその亜種は、ショウジョウバエ中枢神経系の機能的結合を決定するのに尽力されている、いくつかの接続が見えるようにすることによって把握前及びシナプス後のパートナーとの間の距離が比較的大きい場合。これは特にニューロモデュレーション9または gaba 作動性抑制10に関連付けられているボリューム伝送の評価に関連します。
ここでは、補完・新規技術を示すショウジョウバエ中枢神経系に直接シナプス接続をテストするため。このメソッドと呼ばれるテトロドトキシンに設計された抵抗のプロービング シナプス (テレピン油)、フグ毒 (TTX) の共発現で機能-耐性ナトリウム チャネル、NaChBac、および推定から録音中のシナプス前細胞の光遺伝学的活性化シナプス パートナー9。、TTX 存在下で NaChBac を表現する以外の細胞で活動電位を介したすべての活動は抑制されます。NaChBac チャネル選択的に対象となるシナプス前セル光誘発のシナプス伝達を許可の興奮性を復元します。このメソッドは、接続はシナプス回路を採用の確率を削減しながら体の記録によって postsynaptically 解決できるようにシナプス前ニューロンの強力な活性化を許可します。重要なは、この手法はボリューム伝送の検討を許可し、回路を通して単一ニューロンからリリースされた送信機の広がりを明らかにします。また、テレピン油は、従来の薬理学を通じた接続の化学的性質を明らかにできます。テレピン油は TTX を区別しないナトリウム チャンネルの遺伝子組換え発現を可能にするモデルにおける使用に適しています。

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UAS-csChrimson</td>
			<td style="width:219px;">Bloomington Drosophila Stock Center</td>
			<td style="width:344px;">55135</td>
			<td style="width:167px;">Used as a neural activator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UAS-NaChBac</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9466</td>
			<td style="width:167px;">Resotores excitibility in cells in TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetrodotoxin</td>
			<td>Tochris</td>
			<td>1078</td>
			<td style="width:167px;">Special permission may be needed to purchase TTX as it is a controlled substance</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">all trans-Retinal</td>
			<td>Sigma-Aldrichall trans-Retinal</td>
			<td>R2500</td>
			<td style="width:167px;">Require co-factor for channelrhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Weldable 321 Stainless Steel&#160;Sheet,&#160;0.002&#34;&#160;Thick,&#160;10&#34;&#160;Wide</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td style="width:167px;">Used for dissection of preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Waxer</td>
			<td>Almore</td>
			<td>Eectra Waxer 66000</td>
			<td style="width:167px;">Used during dissection to secure fly in foil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraffin Wax</td>
			<td>Joann</td>
			<td>4917217</td>
			<td style="width:167px;">Used with waxer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Number 5 forceps</td>
			<td>Fine Science Tools</td>
			<td>#5CO</td>
			<td style="width:167px;">Used for dissection and desheathing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td style="width:167px;">Used to remove parts of the cuticle during dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tungsten wire</td>
			<td>A-M Systems</td>
			<td>797500</td>
			<td style="width:167px;">Use with electrolysis to make sharpened needles for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reciculating Peristaltic Pump</td>
			<td>Simply Pumps (Amazon)</td>
			<td>PM200S</td>
			<td style="width:167px;">for recirculating TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speed Controller for peristaltic pump</td>
			<td>Zitrades (Amazon)</td>
			<td style="width:344px;">N/A</td>
			<td style="width:167px;">PWM Dimming Controller For LED Lights or Ribbon, 12 Volt 8 Amp,Adjustable Brightness Light Switch Dimmer Controller DC12V 8A 96W for Led Strip Light B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Versa-Mount&#160;Precision Compressed Air Regulator</td>
			<td>McMaster Carr</td>
			<td style="width:344px;">1804T1</td>
			<td style="width:167px;">For applying positive pressure during patching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass capilaries</td>
			<td>World Precision Instruments</td>
			<td style="width:344px;">TW150F-3</td>
			<td style="width:167px;">For patch pipettes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multipurpose Gauge</td>
			<td>McMaster Carr</td>
			<td>3846K431</td>
			<td style="width:167px;">Gauge for pressure regulator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrophysiology Camera</td>
			<td>Dage MTI&#160;</td>
			<td>IR-1000</td>
			<td style="width:167px;">Any camera that works in the IR range (850 nm) will work. You do not want to use red illumination as this can activate csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IR LED</td>
			<td>Thorlabs&#160;</td>
			<td>M850F2</td>
			<td style="width:167px;">For oblique illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">fiber optic for IR LED</td>
			<td>Thorlabs</td>
			<td>M89L01</td>
			<td style="width:167px;">Couples to IR LED</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective lens&#160;</td>
			<td>Olympus 40X</td>
			<td>LUMPlanFLN</td>
			<td style="width:167px;">This can be used on most microscipes and works well for visualizing fly neurons.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amber LED</td>
			<td>Thorlabs</td>
			<td>M590L3d</td>
			<td style="width:167px;">For visualizing RFP and mCherry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue LED</td>
			<td>Thorlabs</td>
			<td>M470L3d</td>
			<td style="width:167px;">For visualizing GFP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GFP filter set</td>
			<td>Chroma</td>
			<td>49011</td>
			<td style="width:167px;">For visualizing GFP or stimulating channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Custom mCherry Filter Set</td>
			<td>Chroma</td>
			<td style="width:344px;">&#160;et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic</td>
			<td style="width:167px;">Use only if you wish to patch identified neurons with channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dichroic to combine Amber and blue LED</td>
			<td>Thorlabs</td>
			<td style="width:344px;">DMLP550R</td>
			<td style="width:167px;">Use only patch under mCherry and excite channel2rhodopsin with blue light.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Red LED</td>
			<td>LEDSupply</td>
			<td>Cree XPE 620 - 630 nm</td>
			<td style="width:167px;">Used to drive csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LED Driver</td>
			<td>LEDSupply</td>
			<td>3021-D-E-1000</td>
			<td style="width:167px;">Used to drive LEDs for optogenetic stimulation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Manipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td style="width:167px;">Used to position pipette during recordings</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Patchclamp Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2400</td>
			<td style="width:167px;">An equivalent amplifier is suitable</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bessel Filter</td>
			<td>Warner Instruments&#160;</td>
			<td>LPF 202A</td>
			<td style="width:167px;">Auxillary filter used to filter current trace to oscilloscope during patching.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Acquisition System&#160;</td>
			<td>National Instruments</td>
			<td>NI PCIe-6321&#160;&#160;&#160;&#160;&#160;&#160; 781044-01</td>
			<td style="width:167px;">Used to record data from amplifier to computer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Connector Block - BNC Terminal BNC-2090A</td>
			<td>National Instruments</td>
			<td>779556-01</td>
			<td style="width:167px;">Used to connect amplifier to DAQ card.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Foil&#160;</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Steel foil for custom recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnets</td>
			<td>K&#38;J Magnetics</td>
			<td>D42</td>
			<td style="width:167px;">To secure recording chamber to ring stand</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">1/16&#34; Cell Cast Acrylic Clear</td>
			<td>Pololu</td>
			<td></td>
			<td style="width:167px;">Used to make custom recording chamber. Acrylic can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
	</tbody>
</table>

examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels

ここでは、新しい技術と呼ばれる Tetrotoxin (TTX) 設計された抵抗のプロービング シナプス (テレピン油) は、ターゲット ニューロン間のシナプス接続のテストに適用されます。メソッドは、特定のシナプス前ニューロンのテトロドトキシン耐性ナトリウム チャネル、NaChBac、遺伝子活性化因子の共発現に依存します。推定されるシナプス パートナーとの接続は、NaChBac を表現しないニューロンの電気的活動をブロックする TTX 存在下で全体セルの録音によって決定されます。このアプローチは、活性化または接続の記者としてカルシウム イメージングと動作するように変更できます。テレピン油は、ネットワーク内で接続を識別するために使用できるツールの成長のセットに追加します。しかし、テレピン油は、一括またはボリューム伝送と波及伝達も確実に報告という点でユニークです。

テレピン油は、モノとニューロン間のシナプス結合でヒヨコの貢献を区別するために使用されます。セルの弱い刺激は、直接接続のテストに使用できるよりシナプス前の活動を多くの場合運転シナプス接続 (図 1 a) を募集しています。テレピン油は、TTX を区別しないナトリウム チャネル NaChBac と光活性剤の共発現し、シナプス接続 (<strong class="x...

Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

この記事は、フグ毒、テトロドトキシン耐性ナトリウム チャネル、NaChBac を用いてニューロン間のシナプス接続をテストするためのメソッドを備えています。

テトロドトキシン耐性ナトリウム チャネルを用いたショウジョウバエのシナプス接続を調べる

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

examining-monosynaptic-connections-drosophila-using-tetrodotoxin

Research

JoVE Journal

Neuroscience

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

9.9K ビュー.  University of Maryland. この記事は、フグ毒、テトロドトキシン耐性ナトリウム チャネル、NaChBac を用いてニューロン間のシナプス接続をテストするためのメソッドを備えています。

ビデオ: Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

神経損傷を調べるための器官型海馬スライス培養モデル

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

神経科学

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

マルチ連絡ラミナーの電極の録音を使用してローカルネットワークの処理を検討

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

人間の前頭前野皮質からfMRI信号への行動応答の相関：タスク分析を用いた認知プロセスを調べる

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

CA1錐体ニューロンにおけるグルタミン酸のUV媒介焦点アンケージングと組み合わせることで、多光子細胞内ナトリウムイメージング

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

臭気誘導挙動の高分解能定量<em&gt;キイロショウジョウバエ</em&gt;使用<em&gt; Flywalk</em&gt;パラダイム

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

勉強するリニアアガロースチャネルを使用します<em&gt;ショウジョウバエ</em&gt;幼虫クロールの動作

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

使用して報酬を求める行動の変化を測定するための簡単な方法<em&gt;キイロショウジョウバエ</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

ImageJ を用いた蛍光タグ付きタンパク質の公平な分析によるショウジョウバエの神経変性の定量細胞生物学

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

ショウジョウバエ幼虫を用いた熱遺伝学的神経細胞スクリーンにおけるマクロ栄養素摂取量の定量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

テトロドトキシン耐性ナトリウム チャネルを用いたショウジョウバエのシナプス接続を調べる

テトロドトキシン耐性ナトリウムチャネルを用いたショウジョウバエのシナプス接続を調べる