TKにG2205（学術振興のために国立科学財団（BACにIOS-1050701）、国立衛生研究所（SMにNS54174とAML-WへF30DC0111907。）、上原記念財団と日本協会が提供する資金）。我々は細胞外の軸索の録音のトピックに関する彼の支援とご指導をユリウスミークスに感謝。我々はプロトタイプ記録室を提供するためにカールホプキンスに感謝します。

<ol>
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利害の衝突は宣言されていない。

一度習得し、この手法は1つが多くのモデル系におけるin vivoでの録音のために個々の軸索を含む識別ニューロンを、ターゲットにできるようになります。さらに、この技術は1つが確実に挑戦インビボ記録方式で行う従来のユニークな解剖学的特徴を有するニューロンのスパイク出力を記録することができる。私たちは、モルミルス科弱電気魚でELA &quot;小さなセル&quot;から録音するために、この技術を利用してきた。 &quot;小さなセル&quot;のチューニング特性を研究するための以前の試みは、挑戦的な記録条件11、14のために失敗した。類似の解剖学的特徴は、多くの異なる脊椎動物聴覚とelectrosensoryニューロン8-10から単一のユニットの録音を得るための障壁を作成します。我々のシステムでこれらの課題を克服するために、我々は &quot;小さな細胞が&quot; ELPのプロジェクトそのELAで唯一の細胞であるという事実を利用しました。したがって、ELPに置か色素の逆行輸送は &quot;小さなにELAでラベリング制限セル&quot;細胞体と軸索。軸索の蛍光標識にはアクセスできなくSOMAのにもかかわらず、可能性識別細胞から単一ユニット記録を作り、ラベルの付いた軸索の隣に正確な電極配置を可能にした。私たちは、周囲を巻き込むのシナプス11のため、おそらく、体の録音を試みたが、成功しなかった、17があるが、体ラベリングは、この技術は、他の細胞型と他の回路の不定記録を標的化するために使用され得ることを示唆してはっきりと見えた。in vivoでの逆行輸送を介してニューロンの蛍光標識は、 インビトロ 19-21 に標的記録を案内するために使用されている。類似の技術はゼブラフィッシュ脊髄22で運動ニューロンからin vivoでの録音の対象に使用された。私たちの仕事はラベリングと記録の両方が脳内で生体内で行われているこのアプローチの新規拡大を表し、私たちの方法は、生体内であることを示してい逆行tと共に中枢神経系領域のラベリングレーサーは、同様に選択的な投影ニューロンを持つ他の無傷の回路の研究に拡張することができます。例えば、哺乳類の聴覚処理で、下丘（IC）は、複数の菱脳構造23からの入力のための重要な中継センターとして機能しています。 ICへの色素の注入が選択的にこれらの核のそれぞれから投影細胞を標識だろう。上丘（SC）は、ビジョン24と同様の機能を提供しています。脊髄調製物が特に良く、この技術に適している、脊髄が容易にアクセスされるように、色素注入は、記録部位から遠くに発生することができ、それは、より詳細な解剖学的情報25を取得する選択ニューロンの細胞内記録と充填と組み合わせることができる。最後に、管·トレースは複雑な回路26をマッピングするために、中枢神経系全体で使用されるよく確立された技術である。我々の方法は、wを行われているように、これらの研究に機能情報を追加するために利用することができるネコ視覚野27のi番目のカルシウム感受性の指標。 十分に確立されている手術は、信頼性が高く、定期的にin vivoでの録音16 でブラインドに使用、最小限の出血や動物と生き残るために組織を可能にするため、脳の表面にダメージなしで完了しなければならない。練習すれば、手術と染料アプリケーションが30-45分で完了することができます。我々は成功した準備の67％で、 &quot;小細胞&quot;軸索のラベルが付いた。ほとんどの準備はわずか1または2目に見えるラベル軸索を持っているが、一部は8できるだけ多く持っている。試み119ラベル単位の、私たちは12製剤（ 表2）上に分散26台から単一ユニット記録を得た。従って、データは、25％の全体的な成功率のために標識された軸索を有する製剤の41％から採取した。 染料アプリケーションの重要な側面は、深さをラベリングされています。タングステン線の浅い挿入を洗浄AWという染料になりますAY。浸透が深すぎる場合は、ラベルの付いた軸索は、ターゲティングのために表示されません。さらに、細胞にはいくつかの機械的な損傷が十分に25、28に取り込まれるように染料のために発生する必要があります。しかし、あまりにも多くの被害は、細胞を殺すでしょう。我々は、他の染料やELP中の標識軸索（ 表3）からの録音とペアELAに染料注入を通じて順行性ラベルなどの他の方法（ 表2）で標識試み。ラベルはそれらが応答しなくなり、体細胞の損傷を持つ細胞に限られていたので、私たちは順行性ラベルが成功しなかったと仮定。さらに、シナプス前端末が邪魔されている可能性があり。これとは対照的に、逆行性標識は、これらの懸念の両方を最小限に抑えます。染料塗布量と位置が検討されて特定の回路に応じて変更することができる。最大のラベルは、我々がコーティングされたタングステン線を使用して達成する最大の色素濃度、で発生します。しかし、デと軸索を標識するそれが進んでいるようにEP予測は、染料は、タングステン針をオフに来るかもしれない。これらのケースでは、圧力注入がより適切であろう。染料の取り込みと輸送は2時間後に注射し、6時間後に注射限り遅く現れる追加ラベル軸索には早くも目に見えるある私達の準備中の標識軸索で、迅速です。従って、標識および記録が生存手術に伴う技術的な問題を排除し、一日で達成することができる。タイミングは、染料の輸送のために必要な距離に応じて、アプリケーションごとに異なります。 もう一つの重要な側面は、電極の配置である。これは、先端の目詰まりを防ぐために、ラベルの付いた軸索のサイトへの組織の近くに入ることが重要です。などELAのよう高密度組織の場合は、記録電極上に長い、細いシャンクは、周囲の組織の過剰な動きを最小限に抑えることができます。成功した記録が最初の試みは、記録が得られるまで、新たな電極を有する繰り返し又はTISS上で達成されていない場合UEは、軸索は、もはや表示された時点に中断されます。しかし、すぐに漂白光毒性を引き起こす可能性蛍光露光、の量を最小限にし、セル29-31の生理学的特性に影響を及ぼす可能性軸索の横に電極を配置することも重要である。 軸索のセグメントを正常に記録電極に吸引されると、記録が数時間にわたって得ることができる。ユニットは常に1時間未満で失われている場合、抜け軸索を防ぐために、より小さい電極チップを作って検討してください。一方、先端の小さすぎには軸索に詰まり、低い信号対雑音比、または損傷につながる可能性があります。追加の吸引ユニットのスパイク振幅の着実な減少と &#39;リターン&#39;先端が大きすぎることを示しています。多すぎると吸引軸索に取り返しのつかないダメージを与えることがあります。一つの解決策は、圧力が徐々に0に戻るように空気ラインに小さな漏れを可能にすることである。急速にイコライジング番目電子圧力が軸索を追放することが一時的な相対外側 &#39;プッシュ&#39;になります。 この技術は識別された投射ニューロンからターゲットの記録を得るための主要な利点であるが、色素を注射部位でのすべての種類の細胞によって取り込まれるように、それは、ローカル介在を区別するために有用ではありません。理論的には、別個の注入部位を持つ複数の蛍光体の使用は、この方法を拡張することを可能にすることができる。例えば、単一の対二重標識の比較回路32の2つの点で二重注射後に投射ニューロンからニューロンを区別するために使用することができる。同様に、逆行性トレーサーはキンカチョウハイボーカルセンター（HVC） は32で、最近行われたような二光子イメージングなど他の高度なイメージング技術、と組み合わせることができます。落射蛍光顕微鏡を使用しているとき、我々は唯一の1＆を解決することができたとしても、記録領域は、表面付近のものに限定されているムー、組織の最初の30ミクロンm以内の構造。しかし、この深さは、このような二光子顕微鏡33又は対物結合面状照明顕微鏡34のような他の顕微鏡技術の使用を介して拡張することができる。相対的にアクセスできないものも含め、 -それはモデル系の様々な多くの異なる回路における単一ニューロンから記録するために使用することができるので、全体として、この技術は、in vivoで神経回路の研究において重要な進歩を表している。

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

この方法の全体的な目標は、ニューロンの識別集団から単一ユニット応答を記録することです。 生体内では個々のニューロンから電気生理学的記録は、神経回路が自然条件下でどのように機能するか理解するために重要である。伝統的に、これらの記録は記録セルのアイデンティティは記録の開始時は不明である意味、 &#39;ブラインド&#39;行われてきた。細胞のアイデンティティはその後細胞内juxtacellular 1,2、または緩いパッチ染料の3イオン導入を介して決定することができるが、これらの記録は、機能的に不均一な細胞型と地域の特定のニューロンにあらかじめ対象とすることができません。蛍光タンパク質を視覚的に誘導単一細胞電気生理学4-6を可能にする細胞型特異的に発現させることができる。しかし、これらの遺伝のツールが利用できない者である多くのモデル系があります。でも、遺伝的にアクセス可能なモデル系で、目的のプロMOTERが不明であってもよいし、遺伝的に均一なニューロンは、投影パターンを変化させている可能性があります。同様に、ウイルスベクターは、投影ニューロンの特定の亜群7を標識するために使用されているが、この方法の使用は、毒性およびトランス-シナプス特異性の欠如によって制限される。このように、 生体内で識別される単一のニューロンから記録するために特定のプレビジュアライゼーションを提供する付加的な技術が必要とされている。ターゲットニューロンのプリビジュアライゼーションは、古典的な単細胞レコーディングはしばしば8-11極めて困難であるため困難な記録条件、特に便利です。迅速かつ選択的に標的と電気生理学的記録を取得するための視覚的な手がかりを提供し、独自の軸索投射に基づいて、特定の脳領域内の細胞の特定のサブセットを標識するためにタングステン針を使用して適用本稿で述べた新しい技術は、蛍光色素の逆行性輸送を使用しています無傷回路withiがで識別ニューロンからNAはCNS脊椎動物。 本手法の最も重要な新規な進歩は非遺伝子アクセス可能なモデル系における特定の細胞型を標的とする蛍光標識の使用である。弱電気魚は目を覚まし、行動動物12に神経回路を研究するための優れたモデルシステムである。私たちは弱い電気モルミルス科魚類の前方exterolateral核における &quot;小さなセル&quot;（ELA）によって感覚処理を研究するために、この手法を利用した。 &quot;小細胞は&quot;シナプス前スパイク13の到着時間のミリ秒以下の違いを検出するための重要な時間比較器ニューロンであることが仮定されている。しかし、このような緻密なミエリン、巻き込むシナプス、小細胞体としての解剖学的特徴は、それが極めて困難従来の方法11,14を使用して、これらの細胞から記録するために行った。ここでは、信頼性の高い、堅牢な録音ます特性を考慮し、我々の新しい方法は、選択的に準備の28％にこれらの細胞を標識することを実証electrosensory刺激に対する応答のrization。

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

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

蛍光色素ラベル解剖投影に基づくニューロンの亜集団の逆行性輸送。標識された軸索は、視覚的に対象とすることができます<em生体内で&gt;</em&gt;、識別された軸索から細胞外記録を可能にする。この手法は、ニューロンが遺伝子操作を通してラベルまたは &#39;ブラインド&#39;を使用して分離することが困難であることができない場合に記録が容易<em生体内で&gt;</em&gt;アプローチ。

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 ...

retrograde-fluorescent-labeling-allows-for-targeted-extracellular

Research

JoVE Journal

Neuroscience

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

19.8K ビュー.  Washington University in St. Louis. 蛍光色素ラベル解剖投影に基づくニューロンの亜集団の逆行性輸送。標識された軸索は、視覚的に対象とすることができます、識別された軸索から細胞外記録を可能にする。この手法は、ニューロンが遺伝子操作を通してラベルまたは 'ブラインド'を使用して分離することが困難であることができない場合に記録が容易

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

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

視床下部スライスに緑色蛍光タンパク質（GFP）によって識別されるゴナドトロピン放出ホルモン（GnRH）ニューロンから二重の体細胞の録音

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

神経科学

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 ...

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 Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

神経、地帯、および蛍光色素で脊髄根の逆行ローディング

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

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 ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

蛍光色素で大人のゼブラフィッシュの網膜神経節細胞の逆行性標識

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

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

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

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

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

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

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

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

無傷の神経上皮におけるマウス嗅細胞の穿孔パッチクランプ記録：同定された嗅覚受容体を発現するニューロンの機能解析

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...