This work was funded by the Whitehall Foundation and the NIH. We thank Clifford Dax (University of Oregon Technical Support Administration) for his help and expertise in designing a circuit for light delivery.

注：オレゴン大学の動物実験委員会によって承認され、次のプロトコルが健康ガイドラインの国立研究所によるものである。 1.急性手術<ol><li>腹腔内（ip）注射した（ 表1）を介して、ケタミン-メデトミジンカクテルで動物を麻酔。 注：これらの実験で使用したマウスは、ドライバ·ライン（; SST-iCre12、SOMの+; CrをiCre12、CR + Pvalb-iCre11、PV +）を介在するCRE依存ChR2を-のeYFPトランスジェニックline10を交配することによって生成されます。 ChR2をまたは関連オプシンのウイルス送達は、同様の発現レベルが得られると仮定して、同様にうまく動作するはずです。 </li><li>手術を開始する前に、確実に、動物の展示穏やかつま先のピンチに応答がないという。麻酔のこの深さを維持するために必要に応じて、実験を通して麻酔薬を再投与する。注射用麻酔薬を使用する場合は、必要に応じてメンテナンス注射用のIPカテーテルを埋め込む。 </li><li>メンテナンス注射（ 表1）のために適切に希釈した麻酔薬のカクテルを使用することにより、たとえば、（約3ミリリットル/ kg /時間）、実験を通して生理食塩水または乳酸加リンゲル液で水和動物を保管してください。 </li><li>定位や他のヘッド保持装置における動物を置きます。頭蓋骨はよく固定されていることを確認してください。これにより、安定した単一細胞記録を維持するために不可欠である。 </li><li>乾燥を防ぐために、目にopthalamic軟膏を適用します。 36.5から37℃の体温を維持する。 </li><li>追記安定性のための脳槽ドレーンを実行します。手術用メスの刃を使用して、大槽マグナを露出するために頭蓋骨の後部面から組織を除去する。脳脊髄液を排出する硬膜に小さなニックを作る。刃のごく先端を使用し、小脳や脳幹に接触することは避けてください。 </li><li>メスを使用して、聴覚のために（対象の皮質領域の上に小さな開頭術（1~2 mm 2で） を行うブレグマの皮質、おおよそ-2.3 mmの後方、正中線の横4.5ミリメートル）。 </li><li>硬膜を取り出し、暖かいアガロースの層で0.5さらさ皮質をカバー - 1mm厚を（1.5％アガロース生理食塩水に、0.9％のNaCl;〜37℃で適用されます）。定期的に生理食塩水を数滴を適用することにより、実験を通して湿ったアガロースを保管してください。 </li></ol> 2.録画セットアップ<ol><li>タングステン電極（7〜14MΩ、127ミクロンの直径、12°テーパーチップ、エポキシコーティングされた）を準備します。ガラス毛細管に電極を瞬間接着剤と、グリップ（ 図1）のための熱収縮チューブを追加する。 </li><li>皮質表面に直交する移動するように設定電動（または油圧）マイクロマニピュレーター上に電極を、マウントします。頭蓋骨に対して、皮膚の下にワイヤー地面をスライドさせます。彼らは筋電図の成果物を生成することができますように頭部の側面と首の後ろに筋肉との接触を避ける。 </li><li>細胞外のアンプを用いて電気信号を増幅シングルユニット記録に適したLi FiのERが、好ましくは、インピーダンスチェックモードを装備。 2オシロスコープやパワードスピーカーのセット - 進行中のスパイク活動（5000 Hzのバンドパス300）を監視します。ここで、記録されたデータは、10kHzのサンプリングレートで連続的にデジタル化される。スパイクは、オフラインで抽出される。 </li><li>先端が皮質の表面に到達するまで、アガロースを介して電極を進める。顕微鏡でこの手順を守ってください。マイクロマニピュレータ上の &quot;ゼロ&quot;はこの位置。 </li><li>最高の記録の深さに近づけるために、視覚的に浸透の終わりに電極を引き出す際に電極先端がゼロの深さで皮質表面を出ることを確認します。 注：マニピュレータの読み取りと観測された電極位置の間に不一致は、それが組織に比べてドリフトしたことを示しています。これは、脳または動物、またはマニピュレータドリフトの不安定性によって引き起こされ得る。 <ol><li>ことを確実にするための追加のチェックとしてこのゼロ設定点は皮質の表面に対応する、組織の最初の数百メートルを進行しながら、刺激誘発電場電位を監視します。電場電位を監視する場合、10 Hzに細胞外アンプの低い帯域通過フィルタのカットオフを下げます。局所電場電位の極性は層I / IIの国境付近で13、〜100μmの深さを中心に反転させることを確認します。これは聴覚皮質のための信頼性の基準点であるが、それは他の皮質領域のために異なる場合があります。 </li></ol></li><li>手動マイクロマニピュレーター上に青色光源（ 図2）に結合された光ファイバをマウントします。顕微鏡を用いて、可能な限り、アガロースの表面に近いファイバの先端を位置決めする。電極が組織に入るビームをセンター。 </li><li>指定された幅とduratioのTTL（トランジスタ - トランジスタ論理）パルス列を送達することができる制御ユニットと、光源の出力を調整するn型、例えば、アルドゥイーノ（ 図3）、コンピュータI / Oカード（図示のように）、または市販のパルス発生器。 <ol><li>両方のオシロスコープ上の信号を監視します。 </li></ol></li><li>パワーメータを用いた光ファイバの先端に全光パワー出力（mW）を測定する。ファイバコアの断面積で割ることによって放射照度（ミリワット/ mm 2で） を計算するためにこの値を使用します。 10の範囲内の強度値で始まる- 15ミリワット/ mm 2であり、必要に応じて、アーチファクトが除去されるまで、下方向に調整する。 </li><li>組織に入るために態勢を整えて、アガロース内の電極、光のアーティファクトを確認してください。検索パルス列を起動します（ 例えば、30ミリ秒光パルス、500ミリ秒刺激間間隔（ISI））。 <ol><li>入射光の角度を変更するために、電極に対する光ファイバを再配置することにより、過渡光アーチファクト（ 図4）を除去する。アーティファクトが解決しない場合は、光パワーを減らしてみてください。目の中アーチファクトは、完全に排除することができない電子稀なケース（のみ最小化）、探索パルスの持続時間を延長する。これが本当のニューロンのスパイクを識別するのを助けることができる。 </li></ol></li></ol> 3.ストレートPINPイン」 <ol><li>約1μm/秒の速度で脳をゆっくり電極を進める。継続的な活動を監視するために、1つのオシロスコープを使用してください。他方では、レーザパルスをオフトリガー。 </li><li>電極は、ChR2を+細胞に近づいていることが多い電気信号がオシロスコープ上明らかであるだけでなく前に知覚できることを示しオーディオモニター上に光誘発スパイクのかすかな「ハッシュ」、音を聞きます。前進速度を遅くする。 注：セルは、電極の経路内に直接にある場合、光誘発活動は大きな（そして大声で）成長します。電極は、単一の介在ニューロンに近づくと、ハッシュは、均一なサイズおよび形状の小さいが明確に定義されたスパイクに解決される。 </li><li>とすぐにLiなどGHT誘発スパイクは、そうすることを開始、オシロスコープ上で個別のオフを引き起こすのに十分な大きさである。スパイク波形の正確な形状を観察するために横軸にスケーリングを調整します。 </li><li>停止し、組織が安定するのを待つ。近いセルに電極を移動する誘惑に抵抗する。このステップでは、安定した記録のために重要である。数分後に、信号対雑音比が改善されない場合 - それは、組織が定着しているが、それは任意に近い電極の先端に細胞を持っていない - 事前さらに5ミクロンおよび再び待つ。 <ol><li>ピークまたは活動電位の谷のいずれかが確実に良くノイズフロア（ 例えば、+/- 300μV以上）の上に設定された電圧閾値と捉えることができるまで、このプロセスを繰り返します。 注：抑制性介在ニューロンをPINPingとき偽陽性または曖昧さの少ないリスクがあります。ほとんどの細胞は、容易に30ミリ秒の持続時間と間PULとのパルス列に従うことができます2-5ミリ秒（ 図5）の信頼性のある最初のスパイクレイテンシを持つSE間隔500ミリ秒、またはより速く、。 PV +細胞の大部分は、特に、完全な1秒（ 図6）のための焼成に耐えることができます。これらは抑制性ニューロンであるため、disynaptic（間接）活性化は主要な関心事ではありません。 </li></ol></li><li>記録しながら、最初のオシロスコープで継続的な活動を監視する。第二オシロスコープを使用したスパイクの大きさや形状に近い目を離さない。スピーカー上の彼らのサウンドの品質に注意してください。 <ol><li>セルは（それが刺しや損傷危険性がある）は、電極に接近しすぎて描画されるか、または離れて漂流していることを示す急激な変化のために聞き入る。スパイクが大きく歪む場合は、バックアウト。彼らは小さく成長する場合には、電極を進める。 2μmのステップでゆっくりと移動します。 </li><li>ピークまたは谷に揃え、すべてのスパイク波形を、重ね合わせることにより記録事後の品質を確認してください。電圧THRESを変えるホールド。しきい値の幅広い、しか均一な高さ（ 図5a）のいずれかの一貫性のあるスパイク形状があるはずです。 </li></ol></li><li>いずれかの時点で信号が隣接ニューロンからのスパイクで汚れた場合は、上に移動します。電極は、隣接セルの範囲外に渡すが、標的ニューロンの範囲内に残ることをオフのチャンスに、ゆっくりと進める。 （これは通常、成功しません。） </li><li>各浸透皮質の深さ全体（900 +μm）を介して電極を移動します。何光応答性ニューロンは逆に、記録は日常の隣接ChR2-細胞からのスパイクで汚染されている多くの侵入や、後に遭遇されていない場合は、新しい電極を試してみてください。 注：でも、単一のバッチ内で、個別電極のインピーダンスと先端形状がかなり変化することができます。両方の要因は、WH-光誘発スパイクを検出するのに十分な大きさでなければならない電極の有効」を聞い半径」に貢献Ileの検索は、まだ単独での単一ニューロン記録を可能にするために十分な制限された。 </li><li>必要に応じて、電極のインピーダンスをテストします。組織の損傷を避けるため、よく脳の外側に、アガロース層の上部に電極の先端でこれを行う。 7-14MΩの範囲内で、正確なインピーダンスは、このアプリケーションの性能、歩留まりの予測を確認し、ない。とは言う、それは半径を聴くための合理的なプロキシです：低インピーダンス電極は、より多くのユニットを拾う。高インピーダンス、少ない。 </li><li>実験の最後に、そのような麻酔薬の過剰投与、または麻酔の深い面下に頸椎脱臼などの制度的認定の手段により動物を安楽死させる。 </li></ol>

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	<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PINP:+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording.">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4, (2009).</li><li>Moore, A. K., Wehr, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-expressing+inhibitory+interneurons+in+auditory+cortex+are+well-tuned+for+frequency.">Parvalbumin-expressing inhibitory interneurons in auditory cortex are well-tuned for frequency.</a> J Neurosci. 33, 13713-13723 (2013).</li><li>Merchant, H., de Lafuente, V., Pena-Ortega, F., Larriva-Sahd, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+impact+of+interneuronal+inhibition+in+the+cerebral+cortex+of+behaving+animals.">Functional impact of interneuronal inhibition in the cerebral cortex of behaving animals.</a> Prog Neurobiol. 99, 163-178 (2012).</li><li>Markram, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interneurons+of+the+neocortical+inhibitory+system.">Interneurons of the neocortical inhibitory system.</a> Nat Rev Neurosci. 5, 793-807 (2004).</li><li>Atallah, B. 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The authors have no competing financial interests.

PINPは、概念的には簡単であるが、それは実際に挑戦することができます。成功の主要な決定は、電極の選択である。電気リスニング半径は重要なパラメータである。それは1つがそれに応じて前進速度を調整できるように、先端が、少し離れChR2を+細胞からまだあるときに光誘発スパイクを検出するのに十分な大きさでなければならない。同時に、それは優れた単体分離を可能にするために?...

Characterizing the myriad cell types that comprise the mammalian brain has been a central, but long-elusive goal of neurophysiology. For instance, the properties and function of different inhibitory cell types in the cerebral cortex are topics of great interest but are still relatively unknown. This is in part because conventional blind in vivo recording techniques are limited in their ability to distinguish between different cell types. Extracellular spike width can be used to separate putative parvalbumin-positive inhibitory neurons from excitatory pyramidal cells, but this method is subject to both type I and type II errors2,3. Alternatively, recorded neurons can be filled, recovered, and stained to later confirm their morphological and molecular identity, but this is a pain-staking and time-consuming process. Recently, genetically identified populations of inhibitory interneurons have become accessible by means of calcium imaging or visually guided patch recordings. In these approaches, viral or transgenic expression of a calcium reporter (such as GCaMP) or fluorescent protein (such as GFP) allows identification and characterization of cell types defined by promoter expression. These approaches use 2-photon microscopy, which requires expensive equipment, and are also limited to superficial cortical layers due to the light scattering properties of brain tissue. 
Recently, Lima and colleagues1 developed a novel application of optogenetics to target electrophysiological recordings to genetically identified neuronal types in vivo, termed &#8220;PINP&#8221; &#8211; or Photostimulation-assisted Identification of Neuronal Populations. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. Unlike many other optogenetic applications, the goal is not to manipulate circuit function but simply to identify neurons belonging to a genetically-defined class, which can then be characterized during normal brain function. The technique can be implemented with standard extracellular recording equipment and can therefore serve as an accessible and inexpensive alternative to calcium imaging or visually-guided patching. Here we describe an approach to PINPing specific cell types in the anesthetized auditory cortex, with the expectation that the more general points can be usefully applied in other preparations and brain regions.
In cortex, PINP holds particular promise for investigating the in vivo response properties of inhibitory interneurons. GABAergic interneurons comprise a small, heterogeneous subset of cortical neurons4. Different subtypes, marked by the expression of particular molecular markers, have recently been shown to perform different computational roles in cortical circuits5-9. As genetic tools improve it may eventually be possible to distinguish morphologically- and physiologically-separable types that fall within these broad classes. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. This strategy was developed specifically for targeting parvalbumin-positive (PV+) cells, but we have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons. Although PINPing is conceptually straightforward, it can be surprisingly unyielding in practice. We learned a number of tips and tricks through trial-and-error that may be useful to others attempting the method.

<table border="0" cellpadding="0" cellspacing="0" width="1482">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Name of Material/Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:341px;">Product/Stock Number</td>
			<td style="width:708px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChR2-EYFP Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">12569</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pvalb-iCre (PV) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">8069</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sst-iCre (SOM) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td style="width:341px;">13044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cr-iCre (CR) Line</td>
			<td style="width:171px;">Jackson Colonies</td>
			<td>10774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:263px;">Agarose</td>
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			<td height="20" style="height:20px;width:263px;">Micro Point (dural hook)</td>
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			<td height="20" style="height:20px;width:263px;">Surgical Scissors</td>
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			<td>10003-12 (handle), 10011-00 (blades)</td>
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			<td height="20" style="height:20px;width:263px;">Puralube Ophthalmic Ointment</td>
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			<td height="20" style="height:20px;">Tungsten Microelectrodes</td>
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			<td height="20" style="height:20px;width:263px;">Capillary Glass Tubing</td>
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			<td height="20" style="height:20px;width:263px;">Heat Shrink Tubing</td>
			<td style="width:171px;">DigiKey</td>
			<td>A332B-4-ND</td>
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			<td height="20" style="height:20px;width:263px;">Zapit Accelerator</td>
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			<td>Use with standard Super Glue.&#160;</td>
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			<td height="20" style="height:20px;width:263px;">Microelectrode AC Amplifier 1800</td>
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			<td height="20" style="height:20px;width:263px;">MP-285 Motorized Micromanipulator</td>
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			<td height="20" style="height:20px;width:263px;">4-channel Digital Oscilloscopes</td>
			<td style="width:171px;">Tektronix</td>
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			<td height="20" style="height:20px;width:263px;">Powered Speakers</td>
			<td style="width:171px;">Harman</td>
			<td>Model JBL&#160;Duet</td>
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			<td height="20" style="height:20px;width:263px;">Manual Manipulator</td>
			<td style="width:171px;">Scientifica</td>
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			<td height="20" style="height:20px;width:263px;">800 &#181;m Fiber Optic Patch Cable</td>
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			<td height="20" style="height:20px;">Power Meter</td>
			<td style="width:171px;">ThorLabs</td>
			<td>PM100D (Power Meter), S121C (Standard Power Sensor)</td>
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			<td height="20" style="height:20px;width:263px;">475 nm Cree XLamp XP-E</td>
			<td>DigiKey</td>
			<td>XPEBLU-L1-R250-00Y01DKR-ND</td>
			<td>LED power and efficiency are continually increasing, so we recommend checking for the latest products (www.cree.com).</td>
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			<td height="20" style="height:20px;">Arduino UNO</td>
			<td style="width:171px;">DigiKey</td>
			<td>1050-1024-ND</td>
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	</tbody>
</table>

a guide to in vivo single-unit recording from optogenetically identified cortical inhibitory interneurons

神経生理学における主要な課題は、大脳皮質に多数の阻害性の細胞型の応答特性および機能を特徴づけることであった。 ここで私たちは、リマと同僚1によって開発された方法を使用して麻酔をかけたマウスの大脳皮質内で識別抑制性介在ニューロンから安定し、十分に単離され、単一ユニット記録を取得するための戦略を共有しています。記録は、特定のニューロンの亜集団にチャネルロドプシン-2（ChR2を）を発現するマウスで行われる。集団のメンバーは、青色光の短いフラッシュに対する反応により同定される。 「PINP」と呼ば、またはニューロン集団の光刺激アシスト識別 - - この技術は、標準的な細胞外記録装置で実現することができる。これは、遺伝的に同定された細胞に細胞外記録を標的とする目的のために、カルシウムイメージングまたは視覚誘導パッチに安価でアクセス可能な代替物として機能することができる。 HERE私たちは毎日の練習方法を最適化するための一連のガイドラインを提供する。我々は、具体的にはパルブアルブミン陽性（PV +）細胞を標的化するための戦略を洗練が、それはそのようなソマトスタチン発現（SOM +）とカルレチニン発現（CR +）介在ニューロンとして、同様に他の介在ニューロンタイプのために働くことを見出した。

ここでは、「リマら 1。 表1の詳細を示唆麻酔カクテル、ケタミン-メデトミジン-アセプロマジンを（によって開発された光遺伝学的方法を使用して、麻酔したマウス皮質で遺伝的に分類された抑制性介在からシングルユニット記録を取得するための我々の戦略を共有するKMA」）。 図1は、4図3は、Arduinoのマイクロコントローラと光出力をゲーティング?...

Watch this Scientific Journal Video about A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons at JoVE.com

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

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

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

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

a-guide-to-vivo-single-unit-recording-from-optogenetically-identified

Research

JoVE Journal

Neuroscience

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

19.0K ビュー.  University of Oregon. Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching. 

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

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

シングルユニット in vivoで</emラットの視交叉から>録音

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

神経科学

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

ザリガニのリフレクションに行動反応の記録

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

単一ユニット活動とElectrocorticographic信号を記録するための慢性神経電極の外科的移植

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

同定されたニューロンと摂食行動からカルシウム信号の同時記録キイロショウジョウバエ</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行性蛍光標識は、特定された神経細胞から標的細胞外シングルユニット記録を可能にします<em生体内で&gt;</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

トランスジェニックフィッシュの無傷の脳内で識別ニューロンからの電気的活動を記録する

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

細胞内記録、感覚野のマッピング、および培養は、リーチのニューロンを同定しヒルドmedicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮質介在ニューロンのサブタイプ選択的エレクトロポレーション

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically-と神経化学的に特定し海馬介在ニューロンからホールセルパッチクランプ記録

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同時電気生理学的記録およびげっ歯類の脳における阻害剤のマイクロ注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...