NEI助成金EY014850によってサポートされています。

1。 Sylgardに覆われた皿、実験チャンバー、そり及びその刃の準備<ol><li> Sylgardのエラストマーでコーティングさ35ミリメートルファルコンペトリ皿は、単一の視細胞を得るために単離網膜の適切なチョッピングのために必要とされる。エラストマーは、供給者の指示と少量の層で、その底部をカバーするためにそれぞれの皿に注がれるにしたがって調製される。皿はコーティング後にカバーを取り付け、それらを保存する。数日後にはエラストマーが硬化すると料理が準備が整いました。 </li><li>彼らはイメージング実験の過程で固定化されるように孤立した光受容体は、実験室の底に固執する必要があります。これは、ポリ- L -リジンまたはポリ- L -オルニチンでコーティングすることによりチャンバーの底を実現しています。室ごとにいずれかの0.01％溶液200μLを追加し、ほこりからそれらを保護するためにペーパータオルでチャンバーを覆う。解決策が乾燥した後、密閉ボックス内の蒸留水とストアを持つチャンバーを洗浄する。 2週間以内でご使用ください。 </li><li>実験終了時に室をクリーニングするには、油浸レンズと細胞の破片から任意の油を除去するために100％エタノールでそれらを洗ってください。細胞破片を除去するために、綿棒のアプリケーターを使用し、慎重にチャンバーの底をスクラブ。その後、蒸留水で洗浄し、再塗装の前にチャンバーを乾燥させる。 </li><li>金属カッターで小片（8つのブレードから）に両刃のカミソリの刃をカット。 </li></ol> 2。溶液の調製<ol><li>ソリューションの構成は、種に依存します。両生類のため、リンゲルは（ミリモル/ L）を持っている：110のNaCl、2.5 KClを、1.6のMgCl 2、1のCaCl 2、5 HEPES、pH値= 7.55。 pHはNaOHで最終的な値に調整する必要があります。哺乳類の場合は、リンゲルは（ミリモル/ Lで）持っています：130塩化ナトリウム、5のKCl、0.5のMgCl 2、2のCaCl 2、25 hemisodium - HEPES、pH値= 7.40を。リンゲル溶液は数ヶ月、室温で十分に密封保管することができます。 </li><li>細菌の増殖を避けるために、-20℃に保たれた株式のグルコース溶液、1モル/ L、。 </li><li>実験日に、5ミリモル/ Lの最終濃度にリンゲル液にブドウ糖を追加する一日の終わりには、リンゲルそれは細菌の成長可能性があるため、グルコースを含むことを捨てる。 </li></ol> 3。網膜の単離<ol><li>網膜の適切な切除のためには、動物を犠牲にする前に少なくとも2-3時間のために暗順応することが重要です。動物は暗い部屋で、適切な換気コンテナに暗順応してください。 </li><li>ハーフウェイリンゲル液を持つ2つの35ミリメートルのペトリ皿を埋める。 </li><li>暗赤色灯の下で動物を犠牲にして目を削除する。その後、すべての手続きは、解剖顕微鏡またはビデオモニターとカメラを用いて赤外光下で実施されています。 </li><li>眼の外表面から、残った組織を取り除きます。前部をカットして削除し、リンゲルで満たされたペトリ皿の一つにアイカップを移す。 </li><li>硝子体を取り外し、慎重にオフはさんだり、任意の添付ファイルをカットすることでアイカップの残りの部分から網膜を分離。ゆっくりと網膜を持ち上げ、アイカップから完全に分離する。 </li><li>プラスチック製のトランスファーピペットで2ペトリ皿に網膜を転送する。遮光ボックスで網膜を含む料理をしてください。 </li></ol> 4。単一視細胞の分離<ol><li>すべての手順は、赤外光下で実施されています。網膜の小片を切り取り、Sylgard覆われた皿にプラスチック製のピペットでそれを転送する。網膜の一部を含む溶液の体積は約250μLにする必要があります。 </li><li>ブレードホルダーとカミソリの刃の小片をつかむ - ブレードのエッジがホルダーに約45 °の角度になっているはず。 Sylgard層上に網膜の部分を平らにして、Sylgard層に付着保ちながら、ブレードが細かく網膜の一部を切る使用。 </li><li>実験室に細胞を含む溶液200μLを転送する - Sylgardに覆われた皿に網膜の残りの部分を残す。遮光ボックスに単離された細胞でチャンバーを保つ。 </li><li>リンゲルの2-3 mLを追加し、解決するために細胞を10分間待ちます。この段階で、分離した細胞を色素ローディングプロトコルに応じて特定の蛍光色素（例えばフラ-2の場合）をロードすることができます。 </li><li>細胞は、現在の実験のために顕微鏡のステージに撮影することができます。 </li></ol> 5。蛍光イメージング<ol><li>落射蛍光顕微鏡のステージにチャンバーを転送する。液灌流、温度プローブ、赤外線照明、等を調整する</li><li>カーテンを閉じて、実験を開始。顕微鏡ケージの中にある赤外光をオンにする、実験室の底部に焦点を当て、そして細胞を探してステージを移動します。 </li></ol> 6。代表結果S： 図。 1は、サンショウウオ（Ambystoma tigrinum）網膜からこのプロトコルを用いて得られた健康な分離株桿体と錐体の光受容体の形態を示しています。サンショウウオの細胞は、サイズが大きいためと網膜から単離後数時間のために生き残るために彼らの能力の単一細胞蛍光イメージング研究のために広く使用されている。さらに、サンショウウオの網膜から一は、定期的にロッドと錐体光受容体の両方を取得することができます。 細胞の健康のための一つの重要な基準はそのまま楕円体（ 図1）、ミトコンドリアが密集している細胞の一部の存在です。細胞はDAPI光学系の下に表示されているときに、ミトコンドリアのこの濃度は、NADHの存在に起因する強い蛍光シグナル（ 図2）を与える。無傷の楕円体の欠如は、実験のための一般的に不適当損傷した細胞のサインです。 図。図3は、腫れ細胞体と凝縮核に、損傷したサンショウウオ桿体光受容器を示しています。このような細胞は、はるかに低い蛍光DA​​PI光学系の下が表示されますが、FITC光学系で観察楕円領域に強いFADの信号を（酸化フラビンモノヌクレオチドとflavoproteinsから発信される）を示す。分離された光受容体の健康のためのもう一つの基準は、光による刺激の際、外側のセグメント中の全トランスレチノール（ビタミンA）を生成する能力です。ビタミンの世代は、無傷の代謝機構に依存するNADPH、かなりの量を必要とします。図4および図5は、それぞれそのままカエルやマウスの桿体の外節にビタミンの形成を示しています。 <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="図1"/> 図1。健康なシングル桿体と錐体光受容体。細胞は、トラフサンショウウオ網膜から単離した。光情報伝達は、外側のセグメントで行われ、楕円体は、密にミトコンドリアが詰まっています。ロッドは、薄暗い光のビジョン、明るい光のビ​​ジョンのための円錐形のための責任があります。 <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="図2"/> 図2。生活サンショウウオロッドとコーンの蛍光。これらは暗順応細胞である、それぞれの楕円体とは有意なビタミンで、その外側のセグメントで蛍光を強いNADHの蛍光を示す。蛍光画像の取り込みは、暗順応の期間後の可視光への最初の暴露を表しています。 <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="図3"/> 図3。損傷したサンショウウオ桿体光受容器。腫れ細胞体と凝縮核が損傷を示すものである。細胞が酸化し、最小限のNADHの​​信号（DAPI光学系）があるが、はるかに強いFADの信号（FITC光）される。 <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="図4"/> 図4。 NADHとレチノールとカエルロッド。これは、楕円体領域で強力なNADHの蛍光を示す健康なカエルの棒の光受容体である。露光する前に外側のセグメントで最小の蛍光がある。露光に続いて、ためにビタミンAの形成に外側のセグメントの蛍光の顕著な増加がある<img src="/files/ftp_upload/2789/2789fig5.jpg" alt="図5"/> 図5。レチノールとマウスロッド。これは、健康なマウスの桿体光受容器は、マウスの桿体の楕円の領域は、強い蛍光シグナルを表示しないため、ビタミンAの形成に露光した後、かなりの外側のセグメントの蛍光を示している。

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利害の衝突は宣言されません。

健康な単離された細胞が得られない場合、問題は、網膜の分離や健康状態、またはそのチョッピングのいずれかである。一般的に、眼と硝子体の前部を除去した後、網膜は容易に色素上皮をオフに持ち上げる。表示されない場合は、アイカップの周囲から始まるoffにはがしてみてください。それは分離することは依然として困難である場合は、おそらく可能性は、動物が時間の十分な期間の?...

<table border="1"><tbody><tr><th>の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>暗い部屋（100〜150フィート2） </td><td></td><td></td><td></td></tr><tr><td>レッドライト19 </td><td></td><td></td><td>オンラインストア</td></tr><tr><td>赤外線光源と赤外線画像ビューア</td><td> FJW光システム、（株） </td><td></td><td></td></tr><tr><td>解剖顕微鏡19 </td><td></td><td></td><td>赤外線視聴者が装備</td></tr><tr><td>遮光ケージ19に囲まれた落射蛍光顕微鏡</td><td></td><td></td><td></td></tr><tr><td>解剖ツール（はさみ、ピンセット、ブレードホルダー） </td><td> Robozまたはファイン科学ツール</td><td></td><td></td></tr><tr><td> Sylgardエラストマー</td><td>エセックス（シャーロット、ノースカロライナ州） </td><td> Sylgard 184エラストマーキット</td><td></td></tr><tr><td>ポリ- L -オルニチン（0.01％） </td><td>シグマアルドリッチ</td><td> P4957 </td><td></td></tr><tr><td>ポリ- L -リジン（0.1％） </td><td>シグマアルドリッチ</td><td> P8920 </td><td> 0.01％に希釈する</td></tr><tr><td>実験チャンバー</td><td>ワーナーインスツルメンツ（ハムデン、コネチカット州） </td><td> D3512P </td><td></td></tr><tr><td>ペトリ皿、プラスチックピペット</td><td>フィッシャーサイエンティフィック</td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

脊椎動物の網膜では、光伝達、電気信号に光の変換は、桿体と錐体視細胞1〜4で行われている。桿体は薄暗い光のビジョン、明るい光の円錐形のための責任があります。光伝達は光受容細胞、視色素、一次光検出器の高濃度が含まれている特殊なコンパートメントの外側の部分で行われます。視覚的な色素は、発色団、11で構成されて-タンパク質、オプシンに添付された、 シスレチナール。すべてのトランスへのシス -視覚色素によって吸収された光子は、11から発色団を異性化。この光異性化は、膜電位の変化で絶頂に達すると電気信号に光刺激の伝達をもたらす反応のカスケードを開始する視覚色素のコンフォメーション変化をもたらす。光刺激から細胞の回復は、光によって活性化中間体の不活性化、および膜電位の再確立を行います。のCa 2 +は、光情報伝達に関与する酵素のいくつかの活性を変調し、その濃度は、光刺激により低減されます。この方法では、Ca 2 +は背景光の光刺激とその適応からの細胞の回復において重要な役割を果たしている。 全トランス 5月7日にシス発色団-回復のプロセスの別の本質的な部分はその11の光異性化による光検出の際に破棄されている視覚色素の再生です。この再生は、アポタンパク質のオプシンを残し、光活性化顔料によって全トランスレチナールのリリースから始まります。リリースされたすべてのトランスレチナールは急速に全- トランスレチノールにNADPHを利用する反応で減少し、オプシンは新鮮な11で結合している-視覚的な色素を改革するために外側のセグメントに持ち込まれた網膜シス 。全- トランスレチノールは、専門のキャリアInterphotoreceptorレチノイド結合タンパク質（IRBP）で外側のセグメントで構成し、隣接する細胞へ搬出される。 単一視細胞の蛍光イメージングは​​、それらの生理学および細胞生物学を研究するために使用することができます。 のCa 2 +感受性蛍光色素8月12日だけでなく、内側のセグメントのCa 2の役割のCa 2 +ストアを明るみに詳細に外側のセグメントのCa 2 +の変化と応答の間の相互作用を調べるために使用することができます+ホメオスタシス13,14 。蛍光染料はまたのMg 2を測定するために使用することができます+濃度15、pHを、水性および膜のコンパートメント16のトレーサーとして。最後に、全- トランスレチノール（ビタミンA）の固有蛍光が単一の視細胞17から19で、その形成と除去の速度を監視するために使用することができます。

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

方法は、蛍光イメージングのための別の脊椎動物種から単生きた光受容細胞の調製のために説明されています。方法は、画像にそのようなNADHやビタミンAなどの内因性蛍光体の蛍光を使用し、又はCaに敏感な外因的に添加蛍光色素のことができる<sup&gt; 2 +</sup&gt;または他の要因。

蛍光イメージングのための生活絶縁脊椎動物視細胞の調製

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

preparation-living-isolated-vertebrate-photoreceptor-cells-for

Research

JoVE Journal

Neuroscience

12.2K ビュー.  Medical University of South Carolina. 方法は、蛍光イメージングのための別の脊椎動物種から単生きた光受容細胞の調製のために説明されています。方法は、画像にそのようなNADHやビタミンAなどの内因性蛍光体の蛍光を使用し、又はCaに敏感な外因的に添加蛍光色素のことができる 2 +または他の要因。

ビデオ: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

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 ...

神経科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

内在性NADH蛍光の二光子イメージングによるマウス大脳皮質におけるMicroregional低酸素の検出

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

カルシウムイメージングの急性脳室下帯スライスの調製

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

4次元蛍光イメージングと住んでいる神経末端におけるエンドソームダイナミクスの可視化

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

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

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

ビブラトームセクショニングマウス網膜は、感光体文化を準備する

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

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

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

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

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

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

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

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Sod1 突然変異体蛋白質集計における細胞の高いコンテンツ定量化のための分析法の開発

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Reliable Isolation of Central Nervous System Microvessels Across Five Vertebrate Groups

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often rodents. This approach limits the interrogation of blood-brain barrier (BBB) properties to the cortex and does not allow for individual comparison. This project focuses on the development of an isolation method that allows for the comparison of the neurovascular unit (NVU) from multiple CNS regions: cortex, cerebellum, optic lobe, hypothalamus, pituitary, brainstem, and spinal cord. Moreover, this protocol, originally developed for murine samples, was successfully adapted for use on CNS tissues from small and large vertebrate species from which we are also able to isolate microvessels from brain hemisphere white matter. This method, when paired with immunolabeling, allows for quantitation of protein expression and statistical comparison between individuals, tissue type, or treatment. We proved this applicability by evaluating changes in protein expression during experimental autoimmune encephalomyelitis (EAE), a murine model of a neuroinflammatory disease, multiple sclerosis. Additionally, microvessels isolated by this method could be used for downstream applications like qPCR, RNA-seq, and Western blot, among others. Even though this is not the first attempt to isolate CNS microvessels without the use of ultracentrifugation or enzymatic dissociation, it is unique in its adeptness for the comparison of single individuals and multiple CNS regions. Therefore, it allows for investigation of a range of differences that may otherwise remain obscure: CNS portions (cortex, cerebellum, optic lobe, brainstem, hypothalamus, pituitary, and spinal cord), CNS tissue type (gray or white matter), individuals, experimental treatment groups, and species.

The goal of this protocol is to isolate microvessels from multiple regions of the central nervous system of lissencephalic and gyrencephalic vertebrates.

5つの脊椎動物群にわたる中枢神経系微小血管の信頼性の高い絶縁

Isolation of microvessels from the central nervous system (CNS) is commonly performed by combining cortical tissue from multiple animals, most often ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

生理学的条件下でのα-シヌクレイン構造ダイナミクスの研究のためのミリ秒水素/重水素交換質量分析

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...