この作品は、放射線腫瘍学、アラバマバーミンガム大学総合がんセンター、ファイティングがんの子供を守る財団、ガブリエルのエンジェル財団（ESYへ。）の部門からインパクト賞によってサポートされていました。

1。細胞培養<ol><li>培養神経細胞および必要に応じてそれらを扱う。 </li><li> 15 mlチューブに細胞を回収します：吸引メディアは、リン酸でリンス液（PBS、カルシウム及びマグネシウムを含まない）緩衝生理食塩水、15 mlチューブに収集し、適切な血清含有培地にトリプシンを中和し、トリプシンを追加します。 </li><li> 5分間、1,000×gで遠心する。 </li><li>メディアを吸引します。 </li><li> PBSに細胞を再懸濁する。 </li><li> 5分間、1,000×gで遠心する。 </li><li>新鮮なPBSでメディアを再懸濁した細胞を吸引します。 </li><li>血球計算盤またはお好みのセルカウンターを用いて細胞をカウントする。 </li><li>細胞サンプルは、アッセイを開始する前に、すぐに準備する必要があります。 </li><li>すべてのサンプルは、紫外線からのDNA損傷を防止するために暗いまたは黄色の光で処理する必要があります。 </li></ol> 2。コメット解析 1。スライドの準備<ol><li> 1％アガロースを（1Xトリス塩基、ホウ酸、EDTA、TBで1グラム/ 100ミリリットル溶かすE）の3分間電子レンジですべての顆粒が消えるまで。 </li><li>溶融アガロースにスライドを浸し、キムワイプできれいな側を拭いてください。透明フィルムに空気乾燥にアガロースを許可します。これは、事前に行うことができ、スライドを保存することができます。 </li></ol> 2。中性コメットアッセイ<ol><li>使用前に少なくとも1時間、4°Cで一チル溶解液（2.5MのNaCl、100mMのEDTA pHが10、10mMトリスベース、1％ラウリルサルコシン、および1％Triton X-100、pHは10）。 </li><li> 2.2.2。すべての顆粒が消えるまで3分間電子レンジで1％低融点アガロース（1Xトリスベースで1グラム/ 100ミリリットル、ホウ酸、EDTA）を溶かす。アガロースは、彗星の尾の人工的な誘導を避けるために、℃の水浴で37に冷却する必要があります。理想的には、アガロースは、使用前に半時間のために冷却する必要がある。 </li><li> 2.2.3。 ml当たり100,000個の細胞があるように細胞懸濁液を希釈します。低融点アガロース（37℃）で一緒に細胞懸濁液を組み合わせる直ちに1:10（v / v）で、渦簡潔に、との比率は、コメットスライド上に50μlを移します。サンプルエリアに均等に細胞懸濁液を広めるために、ピペットチップのサイドを使用しています。各治療群は、少なくとも三連でなければなりません。 </li><li>円はスライドの周囲に表示されるまで、場所は、30分間冷蔵庫でフラットスライドします。 </li><li> Prechill 4℃、暗所で30分間この溶液中の溶解液と水没スライド℃（または冷蔵庫で）。 </li><li>捨てや溶解液を吸引し、1Xニュートラル電気泳動バッファー（トリス塩基、ホウ酸、EDTA、1×TBE）を追加します。冷蔵庫で半時間のために、このバッファにスライドしたままにします。 </li><li>電気泳動槽内の予冷1Xニュートラル電気泳動バッファー（TBE）、電気泳動スライドトレイ内のプレーススライドを追加します。彼らは、電極から等距離にあるように、スライドの位置を合わせます。 </li><li>スライド上0.2インチに1Xニュートラル電気泳動バッファーを注ぐ。過剰なバッファはインター意志電気泳動でFERE。 </li><li> cmあたり1 V（電極に電極を測定）に電源電圧を設定し、暗所で4℃（または寒い部屋）で30分間実行します。 </li></ol> 3。アルカリコメットアッセイ<ol><li>使用前に少なくとも1時間、4°Cで一チル溶解液（2.5MのNaCl、100mMのEDTA pHが10、10mMトリスベース、1％ラウリルサルコシン、および1％Triton X-100、pHは10）。 </li><li>すべての顆粒が消えるまで3分間電子レンジで1％低融点アガロース（1Xトリスベースで1グラム/ 100ミリリットル、ホウ酸、EDTA）を溶かす。その後、少なくとも30分間37℃の水浴中で冷却する。 </li><li>コメットスライド上1:10（v / v）で、渦簡単に、すぐにピペットを50μlの割合で溶融した低融点アガロース（37℃）で1×105個/ mlの細胞を組み合わせています。サンプルエリアに均等に細胞懸濁液を広めるために、ピペットチップのサイドを使用しています。各治療群は、少なくとも三連でなければなりません。 </li><li>場所は、スライドESフラ​​ットは、30分間冷蔵庫で円はスライドの周囲に表示されるまで。 </li><li>予冷した溶解液中で、4℃のまま℃に暗闇の中で一晩に1時間浸しスライド。 </li><li>溶解液からスライドを外し、スライドを排出し、アルカリ巻き戻しステップの前に残留洗剤と塩を除去するために5分間冷中和バッファーで1回すすいでください。 </li><li>スライドよりも0.5センチメートルを超えないように予冷した作りたての電気泳動バッファー（300 mMのNaOHを、1mMのEDTA、pH値&gt; 13）を充填したゲル電気泳動槽に入れ、スライド。彼らは、電極から等距離にあるように、スライドの位置を合わせます。 </li><li>スライドはDNAやアルカリ責任損傷の発現の巻き戻しを可能にするために暗所で30分間アルカリ性緩衝液中で座ってみましょう。 </li><li> cmあたり1 V（電極に電極を測定）に電源電圧を設定して、4℃で30分間実行℃（または寒い部屋）。 </li></ol> 4。固定と駅iningセル<ol><li>過剰な電気泳動バッファをドレイン</li><li>室温で5分間予備冷却スライドを蒸留水で浸す。 </li><li>室温で5分間予備冷却70％エタノールに浸しスライド。 </li><li>一晩乾燥したサンプル。明るい光にスライドを公開しないでください。サンプルはこの段階で得点する前に室温で数ヶ月の​​ために保存することができる。 </li><li>冷蔵庫で20分間のSYBR Green（PBSで希釈1X）に浸漬することにより、スライドを染色する。 </li><li>スライドを削除し、それらは、暗闇の中で完全に乾燥させます。ときに完全に乾いたアガロースは透明になります。 </li></ol> 4。画像の収集と解析<ol><li>緑色フィルタに設定された蛍​​光顕微鏡（Zeiss AxioVisionの）を使って画像を取得し、コメットアッセイソフトウェア（パーセプティブInstruments）を用いて分析する。 </li><li> &quot;彗星の頭部&quot;（核）をクリックすると、ソフトウェアは、平均テールモーメントおよびDNAの量などのパラメータを計算核。 </li><li>治療ごとに少なくとも200個の細胞を分析します。 </li><li> Microsoft Excelにデータをエクスポートします。 </li><li>各治療群及び必要に応じてプロットデータのテールモーメントの平均を計算。 </li></ol> 5。代表的な結果神経細胞上のコメットアッセイ分析の一例を図2及び図3に示します。このケースでは、神経細胞の照射はDNA損傷を誘導する。細胞が電界にさらされるように、DNAは、その後コメットアッセイソフトウェアを使用して分析されている大きさの違いにより、異なるレートで移行されます。より多くのDNA損傷は、遠いDNAが核の外に移行されます。これは、その後高いテールモーメントのソフトウェアおよび結果によってピックアップされ、核内蛍光強度が低下します。 表1は、図4 represenコメットアッセイ分析や図から代表的なテーブルを示すtativeグラフは、コメットアッセイによって測定された放射線に続く神経細胞におけるDNA損傷を比較する。 <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="図1"/> 図1。コメットアッセイのフローチャート。 <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="図2"/> 図2：神経細胞の代表的なイメージ（）なしと（B）の彗星の尾を持つ。 <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="図3"/> コメットアッセイソフトウェアを使用して、 図3コメットアッセイ分析。 （A）画像の代表的なスクリーンショットでは、カールツァイス蛍光顕微鏡を用いて取得し、Cometを使用して分析アッセイソフトウェア。 （B）は、核または&quot;彗星の頭部&quot;（緑の円内）をクリックすると、蛍光灯、地図とグラフ（黄色の丸で囲んだ）と（C）データテーブル（赤丸部分）を生成します。 <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="図4"/> 図4中性コメットアッセイを用いて、照射と非照射神経細胞におけるDNA損傷を解析して得られた平均テイルモーメントをプロットして得られた代表的なグラフ。予想されるように照射された神経細胞においてより高い平均のテールモーメントによって示されるように、3Gyの放射線（X線）は、DNA損傷を誘導する。 、** P &lt;0.01 - 平均テールモーメントは（標準誤差+ / - ）も示されている。 <img src="/files/ftp_upload/50049/50049table1.jpg" alt="表1"/> 表1。 </strong&gt;の照射と非照射神経細胞にコメットアッセイ解析によって得られた代表的なテーブル。 <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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特別な利害関係は宣言されません。

コメットアッセイは、個々の細胞を分析するユニークな能力を持っています。これは、細胞毒性薬に反応差を実証する細胞の亜集団の同定に有利である。いくつかの実用的な制限は考慮に入れなければならない。個別に評価することができる細胞の数は、個々によって異なります。集団内のDNA損傷のばらつきがある場合にはサンプルサイズを増やす必要があります。壊死またはアポトーシス?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> 1.5 mlチューブ</td><td> Santa Cruz Biotechnology社株式会社</td><td> SC-200271 </td><td></td></tr><tr><td> 10X TBE（トリス塩基、ホウ酸、EDTA） </td><td>フィッシャー·サイエンティフィック</td><td> BP13331 </td><td></td></tr><tr><td> 15mlチューブ</td><td>フィッシャー·サイエンティフィック</td><td> 0553851 </td><td></td></tr><tr><td>アガロース</td><td>シグマ</td><td> A5093 </td><td></td></tr><tr><td>アルミ箔</td><td>フィッシャー·サイエンティフィック</td><td> 01213101 </td><td></td></tr><tr><td>ビーカー</td><td>フィッシャー·サイエンティフィック</td><td> FB102300 </td><td></td></tr><tr><td>遠心</td><td>サーモサイエンティフィック</td><td> 75004261PR </td><td></td></tr><tr><td>コメットアッセイソフトウェア</td><td>コメットアッセイIVの画像解析システム</td><td></td><td></td></tr><tr><td>シリンダー</td><td>フィッシャー·サイエンティフィック</td><td> 08555F </td><td></td></tr><tr><td> EDTAを</td><td> GIBCO </td><td> 15575 </td><td></td></tr><tr><td>電気泳動槽</td><td>サーモサイエンティフィック</td><td> 09528101 </td><td></td></tr><tr><td>エタノール</td><td>フィッシャー·サイエンティフィック</td><td> A407P4 </td><td></td></tr><tr><td>蛍光顕微鏡</td><td>ツァイスアクシオビジョン</td><td></td><td>緑色のフィルタを使用して、任意の蛍光顕微鏡で十分でしょう</td></tr><tr><td>血球計</td><td>フィッシャー·サイエンティフィック</td><td> 0267152 </td><td></td></tr><tr><td>低融点アガロース</td><td>プロメガ</td><td> V2111 </td><td></td></tr><tr><td>マイクロ波</td><td>シアーズ</td><td></td><td></td></tr><tr><td>リン酸緩衝生理食塩水、カルシウムフリー、マグネシウムを含まないバッファ付き</td><td> Hyclone社</td><td> SH3025601 </td><td></td></tr><tr><td>パワーサプライ</td><td> BioRad社</td><td> 1645050 </td><td></td></tr><tr><td>冷蔵庫</td><td>シアーズ</td><td></td><td></td></tr><tr><td>定規</td><td>ステープルズ</td><td></td><td></td></tr><tr><td>スライド</td><td>フィッシャー·サイエンティフィック</td><td> 12550143 </td><td></td></tr><tr><td>塩化ナトリウム</td><td>シグマ</td><td> S7653 </td><td></td></tr><tr><td>水酸化ナトリウム</td><td>シグマ</td><td> S5881 </td><td></td></tr><tr><td>ナトリウムラウリルサルコシン</td><td>フィッシャー·サイエンティフィック</td><td> S529 </td><td></td></tr><tr><td> SYBRグリーン</td><td>インビトロジェン</td><td> S7585 </td><td></td></tr><tr><td>トレイ</td><td> FIS彼女の科学</td><td> 15242B </td><td></td></tr><tr><td>トリス塩基</td><td>シグマ</td><td> T6066 </td><td></td></tr><tr><td>トリトンX-100 </td><td>シグマ</td><td> T8787 </td><td></td></tr><tr><td>トリプシン</td><td> Hyclone社</td><td> SH3023601 </td><td></td></tr><tr><td>渦</td><td>フィッシャー·サイエンティフィック</td><td> 02216108 </td><td></td></tr><tr><td>水浴</td><td>フィッシャー·サイエンティフィック</td><td> 154622Q </td><td></td></tr></tbody></table>

assaying dna damage in hippocampal neurons using the comet assay

多くの薬剤は、DNA修復経路を標的とし、細胞がDNA損傷を作成することによって殺す誘発する。したがって、直接DNA損傷を測定するためのプロセスが広く評価されている。従来の方法は時間がかかり、高価な、リソースを大量に消費され、複製細胞を必要とする。これとは対照的に、コメットアッセイ、単一細胞ゲル電気泳動アッセイは、DNA損傷と修復の高速化、非侵襲的、安価、直接的かつ敏感な尺度である。 DNA損傷だけでなく、DNA修復のすべてのフォームは、この強力な技術を用いて、単一細胞レベルで可視化することができる。 コメットアッセイの基礎となる原理は、DNA損傷がこの組織を混乱させるのに対し、無傷のDNAが高度に秩序化されています。損傷したDNAをアガロースマトリックスに汚水を垂れ流すと、電界にさらされたとき、負に荷電したDNAは正に帯電している陰極に向かって移動する。大きな損傷を受けていないDNA鎖は核から遠くに移行することはできません。 DNA損傷遠く無傷のDNAより旅行小さなDNA断片を作成します。コメットアッセイでは、画像解析ソフト、対策や核外移行したDNAを用いて核内のDNAの全体的な蛍光強度を比較します。移行されたDNAからの蛍光シグナルは、DNA損傷に比例します。長く明るいDNAテールは、増加したDNA損傷を意味します。測定されるパラメータの一部は、尾部のDNA量と尾、尾の長さのDNAの分布とDNAのパーセンテージの両方の尺度であるテールモーメントです。このアッセイは、DNA損傷の解像度は修理が行われたことを表しますので、同様にDNA修復を測定することができます。感度の限界は、二倍体の哺乳類細胞1,2あたり約50鎖の切断である。例えば、エトポシドなどの任意のDNA損傷剤で処理した細胞を、ポジティブコントロールとして使用することができる。したがって、コメットアッセイ、DNA損傷を測定するための迅速かつ効果的な手順です。

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

コメットアッセイは、アルカリに不安定なサイトや海馬ニューロンを含むすべての細胞のDNAにDNA-DNA/DNA-proteinクロスリンクを含むシングルと二重鎖切断を検出する効率的な方法です。この方法は、DNA損傷の量の違いに起因する電界中のDNAの差のマイグレーションを活用しています。

コメットアッセイを用いて海馬神経細胞のDNA損傷を測定する

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.1K ビュー.  University of Alabama-Birmingham. コメットアッセイは、アルカリに不安定なサイトや海馬ニューロンを含むすべての細胞のDNAにDNA-DNA/DNA-proteinクロスリンクを含むシングルと二重鎖切断を検出する効率的な方法です。この方法は、DNA損傷の量の違いに起因する電界中のDNAの差のマイグレーションを活用しています。

ビデオ: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

トランスジェニックを用いてアッセイするβ-アミロイドの毒性 C.エレガンスモデル

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

神経科学

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

ニューロモデュレーションとミトコンドリア輸送：長時間にわたって海馬ニューロンのライブイメージング

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

胎児マウスから海馬ニューロンの単離および培養

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

初代海馬ニューロンのリン酸カルシウムトランスフェクション

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

構造化照明顕微鏡を用いてラット初代海馬ニューロンの樹状突起棘を画像化

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

細胞内のCaの蛍光と生物発光イメージング<sup&gt; 2+</sup&gt;高齢者における海馬ニューロン

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

ストライプアッセイは、解離海馬ニューロンを用いたタンパク質基質の魅力や反発作用を検討します

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Golgi-Cox法を用いた老齢マウスにおける海馬の樹状突起の複雑さの評価

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

培養海馬神経細胞のシナプス小胞エンドサイトーシスを測定

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人海馬神経前駆細胞のアッセイ回路特異的調節

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...