This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1。手術装置を構成注：操作中と後の魚が生きたまま確保するために、魚の口から1滴/秒の速度で半分濃度（0.015％）の点滴麻酔液の3 - アミノ安息香酸エチルメタンスルホン酸塩（MS-222、またはトリカイン） 図1に示される手製のドリップシステムを用いて。 <ol><li> 図1（a）に示すようにボックスを作る（長28センチ、幅は10cmで、高さは5cmである）、途中で（長さが6cmで、幅9センチ、高さ4センチ）のスポンジを入れて開くその上にギャップ。 </li><li>その後、魚の口の中に挿入される光硬化性結合（ 図1B）と針の端部に、滑らかな球状の面を作成します。針の表面が損傷される魚の口を防ぐために、滑らかであることを確認してください。 </li><li> MS-222の半分の濃度を追加します（左室、長さが6cmで、幅は10℃ですM、高さは8cmで）と生理食塩水（右室は、長さは、図1（c）に示すように高さが貯水池に）8cmで、幅は10cmで、4センチメートルです。注意：新鮮MS-222をお勧めします。 </li><li> 2分間の0.03％のMS-222で魚を麻酔。 </li><li>スポンジの隙間に魚を置き、胸びれの後ろに、それぞれ総排泄毛穴、11の横に挿入されたピンの2組、魚を修正。 </li><li>魚の口に接続し、MS-222、及び図1Cに示すように、T型チューブで水を飼育。 </li><li> MS-222に接続されたチューブをオンにします。 </li></ol>右蓋から2。レトログラードラベルのRGC 注：以前は頭にありませんが、エタノールが残っていないことを確実に70〜75％エタノールで全ての機器を消毒する。 <ol><li> Rossing：ややsclerectomeで全体の頭蓋骨を覆う皮膚を取り除く（頭蓋骨の背ビューは、 図2Aおよび2Bに示されている</stron得&gt;）。 </li><li>クリア：生理食塩水で頭蓋骨を洗ってから耳の洗濯電球でそれを乾燥させます。 </li><li>初期の腐食：10秒間エッチング剤で頭蓋骨を腐食。 </li><li>クリア：完全に綿棒でエッチング液を乾いた布で拭き取ってくださいして、生理食塩水で領域を洗って乾燥させます。 </li><li>保護：過剰な腐食から右蓋を除く他の領域の頭蓋骨を保護するために、光硬化結合を有​​するこれらの領域をカバーし、次に携帯型光源と、青色光への曝露によってそれを硬化させる。 </li><li>完全な腐食：2分のための完全なエッチングに再び右蓋の中央部にエッチング液を入れる。注意：古い魚の頭蓋骨が石灰化しているため、腐食は時間がかかります。 </li><li>クリア：完全に綿棒でエッチング液を乾いた布で拭き取ってくださいして、生理食塩水で領域を洗って、再度乾燥させます。 </li><li>頭蓋骨の開口部：慎重に右の蓋（ 図2Dを公開する鉗子でmalacic頭蓋骨（ 図2C）を削除</strong&gt;）。 </li><li>クリア：ゲルフォームの破片で穴から粘液や血液放散を拭いて、出血が停止するまでの生理食塩水で洗って。 </li><li>色素の配置：全体右蓋に色素（組織標識ペースト）入れて、ゲルフォームでそれをカバーしています。 </li><li>分離：同じ動物から無菌規模で穴をカバーしています。 </li><li>スケール上の場所光硬​​化結合をし、次いで40秒間再度、青色光でそれを硬化：シール。 </li><li>復活：債券の表面を洗浄し、生理食塩水で動物を復活させる。 </li><li>看護や繁殖：5日間、28.5℃の胚培地（ZFIN）で魚を保持し、2回/日を養う。その色素の実験で5日間、蓋に滞在しなかった魚が含まれていません。注：胚培地（EM）ソリューションは、魚の生存に不可欠であり、温度がDiIを標識するための重要な要素です。 </li></ol> 3。Wholemounting網膜とイメージング<ol><li>誰が網膜の前に2時間、暗視野に魚を置きますlemount。 </li><li>氷水で魚を麻酔。 </li><li>角膜の余白に穴を穿刺し、金星はさみで角膜を除去することにより、アイカップを作る。 </li><li>レンズを外した後、氷冷した1×PBSで網膜の中心をフラッシュします。 </li><li>何顔料がなくなるまで、氷冷1X PBSで網膜と強膜との間のギャップをフラッシュします。 </li><li>ディスクで視神経を切った。 </li><li>上向きのRGC層を維持し、自作のガラススプーン（ 図3Aと図3B）で網膜を開催しています。ドロッパーはまた、網膜を転送するために使用することができる。 </li><li>上向きのRGC層とスライド上の氷の30％のグリセリン（ 図3C）の低下に網膜を置く。 </li><li>グリセリンを外し、4象限（ 図3D）に網膜をカットする前に、スライドガラス上に網膜を平らに。上向きのRGC層で繰り広げ網膜に保つことは、網膜を平坦化するための参考になります。 </li><li> T ONドリップ氷の50％のグリセリン彼は、顔料の断片を洗い流し網膜。 </li><li>グリセリンを削除して網膜上に氷のような75％のグリセリン20μlのドロップします。 </li><li> 1.8センチメートルX 1.8センチメートル四角いカバーガラスでサンプルをカバーしています。 </li><li>マニキュアでカバーガラスをシール。網膜の即時画像化することをお勧めします。 </li><li>ノンインタレース画像DP72 CCD（各画像は1360×1024ピクセルです）で20倍の対物レンズ（NA = 0.70）の下で全体の網膜。各フィールドは、3つの異なる焦点を持っています。 </li><li>ソフトウェアの「拡張被写界深度」のツールでそれぞれの被写界深度を拡張します。 </li><li> Photomergeの機能を備えた仮想網膜を組み立てる。 </li><li> RGCの平均密度（ 図4A）を分析するために、網膜の各方位の680×512ピクセルの3つのフィールド（実際の面積は0.092ミリメートル2、264ミクロン350ミクロンである）を選択します。 </li><li>測定値の関数で領域を得る - 画像内のポリゴンフィーチャを作成するソフトウェア。 </li><li>式でRGCの総数を計算する」のRGC数=運命のX領域」9。 </li></ol>

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We have nothing to disclose.

RGCの逆行性標識は、哺乳類動物でのRGCの生存を研究することは重要であるが、それはゼブラフィッシュで使用されていなかった。代替の方法は、彼が7と抗体染色8染色 、ラベルされたすべてのRGCでのRGC番号、およびトランスジェニック系統をカウントするための金規定するものではありません、まだ12、13を構築されていません。このビデオでは、大人のゼブラフィ?...

大人のゼブラフィッシュは、視神経損傷1の後に軸索を再生する強力な能力を持っているように、全体のRGCをカウントするための適切な方法は、RGCを生存と再生2を評価することが不可欠である。哺乳類や金魚3の逆行性標識のRGCの方法に基づいて- 5、私たちは、大人のゼブラフィッシュにおける視蓋からのRGCを標識する方法を構築した。大人のゼブラフィッシュの場合は、2の重要な技術的な問題に注意する必要があります。大人のゼブラフィッシュの頭蓋骨は、6非常に小さい。彼らは水環境に住んでいます。ここでは、5掘削に伴う危険を最小限に抑えることがエッチング液で頭蓋骨を扱う。その後、我々は手術後、動物の生存率を改善する光硬化性ボンドで穴を密閉する。 以前は、いくつかの他の技術は、間接的な方法でのRGCの数をカウントするために採用された。網膜切片における染色HEはRGCL 7内のセルのすべてのタイプのラベルを付けます。全体Rでの抗体標識このような膵島-1のようなetinaも、アマクリン細胞8にラベルを付けることができます。視神経の切り株から逆行標識は、網膜内のすべてのRGCにラベルを付けることができますが、それは視神経への追加として傷害の原因になりますので、それはクラッシュモデルで採用することができない。蓋から逆行ラベルを利用して、我々は視神経挫滅でのRGCの生存と再生を研究している。結果は、ほとんどすべてのRGCが生存し、RGCの90％以上がクラッシュモデル9の最初の週で蓋を再生成することを示している。 正常にすべてのRGCを標識するために、DiIをペーストは、他のいくつかの市販の染料10との比較後に選択されました。第一に、これは特にin vivoでの組織標識のために設計されている。第二に、それは、水に拡散することができない、親油性染料である。さらに、この蛍光はそれのRGC生存研究のための優れた候補になり、長時間持続することができます。

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

逆行性標識網膜神経節細胞（RGC）がサイトを死んでからのRGC細胞体を分離することができるように、それはのRGCの生存と再生の実験でのRGCをカウントするためのゴールドスタンダードとなっている。多くの研究は、視神経損傷後のRGC生存を検索するには哺乳類動物において行われている。いくつかの代替の方法は、網膜神経節細胞層（RGCL）で細胞数をカウントすることができますがしかし、成人のゼブラフィッシュにおけるRGCの逆行性標識はまだ報告されていない。成体ゼブラフィッシュ頭蓋骨のサイ​​ズが小さく、頭蓋骨にドリル後の死亡の高いリスクを考慮すると、我々は、酸エッチングを利用して頭蓋骨を開いて、有意に生存率を改善することができる光硬化性ボンドで穴を密封する。 5日間の染料を吸収した後、ほぼすべてのRGCをラベル付けされている。このメソッドは、視神経を横断する必要がないとして、それは大人のゼブラフィッシュにおける視神経挫滅した後のRGC生存の研究にかけがえのないです。ここではご紹介この方法ステップバイステップとは、代表的な結果を提供します。

図4B-Dが示すように 、のDiI +細胞の数は、RGCLにおけるDAPI +細胞の三分の二である。通常の網膜では、全体の網膜（ 図4E）のモンタージュ画像は中央エリアでのRGCがで彼らのターゲットに再生成されていないようにDiI +のRGCは、網膜全体にわたってますが、再生された網膜（ 図4F）に分布していることを示してい最初の...

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

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

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 ...

retrograde-labeling-retinal-ganglion-cells-adult-zebrafish-with

Research

JoVE Journal

Neuroscience

9.4K ビュー.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

ゼブラフィッシュ後脳における標識およびイメージング細胞

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

神経科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

電位感受性色素を持つカニ口と胃の神経節における神経細胞の光学的イメージング

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

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 ...

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. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

マウスの網膜神経節細胞におけるシナプスと人工コンダクタンスによるダイナミッククランプの実装

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

大人のゼブラフィッシュにおける網膜再生研究で使用するための新規な光ダメージパラダイム

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...