This work was supported by the National Health and Medical Research Council [570930]. Imaging was carried out at the Bosch Institute Advanced Microscopy Facility. Former members of the lab, whose work is cited, are thanked for their contributions to developing these methods.

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The authors declare they have no competing financial interests.

ここで説明するプロトコルは、確実に測定し、正常な老化と疾病状態を含む条件の範囲にわたって、NMJの特性の変化を定量化するために私たちを有効にしている。顔NMJ画像専用のために記載された方法は、研究者が前と後シナプスの専門分野の面積とシナプス重なり/位置合わせの面積を比較することができるようになります。前およびシナプス後のタンパク質の相対的強度を比較するために横方向の光学切片を使用する第2のプロトコルは、好ましい。第三のプロトコルは、具体的にはシナプス後膜における性AChRのパッキングの近接の変化をテストします。 特異コントロールは、免疫蛍光顕微鏡で不可欠です。間接的免疫蛍光のために、任意の一次抗体を使用する場合は、最初に、筋肉切片におけるその標的タンパク質に特異的に結合することを保証する必要がある。組織処理および固定異なる種類の差次の特異性を変化させることができる抗体。それは本当に運動終板でのAChRに集中している（たとえばのrapsyn用）その免疫蛍光染色を確認することが重要です。陰性対照セクションはまた、抗体結合が特異的であることを確実にするために検査する必要があります。 - / -マウス例えば、rapsyn免疫蛍光のための最高の負の制御はrapsynからのセクションになります。これらは、抗rapsynとのエンドプレート染色を示さないはずである。非特異的蛍光は、組織（自家蛍光）または蛍光二次抗体コンジュゲートにより、非特異的結合からの内因性蛍光化学物質から生じ得る。このような蛍光は、多くの場合、アルデヒド固定によって悪化する。また、エンドプレートのTRITC-BGT染色は時々FITC蛍光チャネルで検出することができ、この蛍光ブリードスルーは、特定のFITC免疫蛍光と混同されるかもしれない。非特異的蛍光の後者の三つの形式を防ぐため、染色されたスライドのすべてのバッチは、いくつかの &#39;Nを含むべきであるO-一次抗体コントロール」セクション（3.7および4.6ステップ）。これらの制御部からエンドプレートの画像は、のNMJの間接的な免疫蛍光染色が本当に一次抗体の結合を反映していることを確認するために実験的なスライドからのものと比較すべきである。 横方向の共焦点切片はシナプスにおける免疫染色の相対強度の違いを評価するために特に有用である。横断共焦点セクションでは、シナプスタンパク質の正確な共局在を判断する方が簡単です。三日月形のエンドプレートプロファイルは、単にサンプルカットスルーNMJ問題になっているを表します。しかし、バックグラウンド（シナプス）の蛍光は、一般的に下面をz投影画像専用と比較される。このように、「本当の」（特定の）免疫染色を識別し、固定された共焦点ゲインを確立し、横方向の光学切片13-15,18を使用して値を相殺するために容易にすることができます。例えば、重症筋無力症のマウスモデルにおいて（wher無力E端板のAChR染色は著しく減少する）エンドプレートは明らかに横断光学切片18,21-に描写された。 NMJでの蛍光の平均強度の差は、シナプスの専門内の標的タンパク質の改変された密度を反映する可能性がある。注意点は、いくつかの状況では、隣接するタンパク質による抗体結合の標的タンパク質または閉塞の構造変化は、改変された染色強度を説明するかもしれない、ということである。 実験のデザインは、いくつかの検討が必要です。多くの場合、実験は、NMJのサイズに導入遺伝子、遺伝子ノックダウン、または疾患状態の影響をテストすることを目指す。実験サンプルのグループは、同じ性別と遺伝的背景の健康な若い（野生型）マウスと比較されることがあります。終板シナプトフィジン、AChRのいくつかの筋シナプスの重なり部分の面積の基準値を表1に示した。サンプルサイズは、動物性の程度に依存する治療群内の-to-動物変動と効果の大きさ（標準偏差あたりの実験対対照群のための平均の差）。分析は、品質の良い画像に制限されると一貫性の公正度は、試料中に発見された健康的な2ヶ月の雌のC57BL6Jマウス（ 図6AおよびB）間のエンドプレート領域ための手段。このように、3匹のマウス17,20,32のサンプルサイズが、対照と比較して、患者を無力抗MuSKの陽性重症筋からのIgGを注射したマウスにおけるシナプス領域において有意な30〜40％の減少を実証することができた。高齢マウスは、若いマウス22よりエンドプレートパラメータのより大きな動物·ツー·動物の変動を示した。結果的に老齢マウスを含む実験は、より大きなサンプルサイズが必要になる場合があります。 主な関心事は、専用顔終板のサイズを測定することである場合、ゲインとオフセットレベルの設定は、すべての個々のNMJのために最適化されるべきである。枚ずつ指定idualのNMJは、疾患状態が検査される場合は特に、のAChRとシナプトフィジン染色の明るさがかなり変化することができる。また余分なシナプス（非特異的）蛍光の強度は、健康な若い動物（ 図5CおよびD）のものと比較して、老化した動物の筋肉の多くの場合より高く、より可変である。最終画像内に保持される色調情報を最大にするように、1〜256の階調を十分に活用すべきである。これは、ゲインを調整伴い、zスタックを収集する対象となるすべてのNMJためのレベルを相殺する。 図5Dは、色調情報の前および後の領域の境界を定義する上で重要である可能性がNMJ画像の一例を示しているシナプスの専門分野。 シナプス領域の測定は、異なる筋肉製剤および実験に適用することができる。シナプス領域の我々の測定のほとんどは、スナップから縦凍結切片を採用してきた<html>凍結した筋肉。固定前に筋肉をフリーズすると、広範囲のタンパク質の抗原性を維持します。ときは、抗原との互換性、事前の凍結切片にパラホルムアルデヒド固定およびショ糖浸潤（ステップ2.1）はNMJ構造の良好な保存を提供することができます。最適な構造的な保存はパラホルムアルデヒドで心臓灌流によって得られる可能性があります。凍結切片のアーティファクトは、完全に固定された筋肉21からからかわ分冊に無傷で筋肉やイメージングのNMJの表面上の標識のエンドプレートによって回避することができる。関係なく、準備の、サンプリング、画像処理およびエリア定量のための手順は、（プロトコル手順4〜5）変わらない。ブラインドサンプリング、イメージングおよび分析プロトコルの一貫した適用は、かなり再現性の平均値（ 表1のチェンらとツェ結果を比較）をもたらすことができる、（異なるオペレータ、マウスおよび異なる時間の異なる試料を用いて）。 &quot;&gt;エンドプレートは、種々の疾患状態における「断片化」になっとして記載されている。例えば、マウスの筋肉、筋線維の散発変性老化にプレッツェルのようなエンドプレートのAChRプラークのリモデリングをもたらした（その再生が続く）フォームに複数の小さいのAChRクラスター6。抗ムスク重症筋無力患者を無力からのIgGを注射したマウスでは、エンドプレートの断片化はかなり異なっていた。大部分が分散したエンドプレートのAChRのプレッツェル、小さな（&lt;4メートル2）のAChR「微小凝集の星座を残し「20,21。これらの2つの例は、実験動物に対する制御21の終板でのAChRクラスタのサイズ分布を比較する必要性を強調している。 他の方法は、NMJでのシナプス領域または染色強度を評価するために報告されている。ここで使用される2次元のz投影画像がSYNを過小評価する可能性がありますように、モータのエンドプレートは、時には折り畳むことができるaptic地域。絶対的なシナプスの面積が33を定義する必要があります場合は、三次元共焦点再構成は、より正確な措置を提供することがあります。ここで説明するz軸投影プロトコルの重要な利点は、しかしながら、複数の治療群と電位の変化の信頼できる同定から測定されるエンドプレートの多数の許可された比較的単純である。終板の染色強度を比較するためのプロトコルは、多くの異なるシナプスタンパク質のレベルの変化を研究するために適合させることができる。この方法は、全てのサンプルは、同じ共焦点セッション中に撮像された免疫蛍光のために処理される要求によって、しかし、限られている。ヤンポリスキーら 5による最近の研究では、この制限を克服するに役立つかもしれないエンドプレートのAChR密度を測定するための方法を説明した。本研究では、終板の同じフィールドは、いくつかの異なるレーザパワー設定で画像化した。レーザパワーとrhodami関係の傾きね-BGT蛍光強度が異なるマウス5におけるエンドプレートでのAChR密度の相対的変化を評価するために使用した。この方法は、長期間の研究の過程で異なる時間で撮像されたサンプル中のAChRの強度を比較するのに有用かもしれない。 のAChR-AChRのFRETは、終板性AChRの組織について具体的かつ補完的な情報を提供します。 125を使用して電子顕微鏡オートラジオグラフィー。隣接する膜infoldingsはAChRの34の非常に低い密度を含んでいながら、I-α-BGTは、10 4メートルの平面密度-2直ちに伝達物質放出の各シナプス前サイトの下で密に充填することが性AChRを示している。 AChRの-AChRのFRETは、比較的簡単なAChR包装で（超顕微鏡的）の変化を検出することができる。 FRET効率の低下は、平均BGT蛍光強度の変化によって検出されないことがありシナプス後膜における性AChRの超顕微鏡的再配分を反映する。複数のF俳優はFRETの効率の変化を引き起こす可能性があります。これらは、ドナー·アクセプターの間隔と相対的な向きだけでなく、分子環境35,36が含まれています。終板FRET効率の低下は、おそらくのAChR格子の幾何学の変化から生じる可能性がある。しかし、ほとんどの場合、それは、ナノスケールのシナプス後分子格子14にパックされ性AChRの割合の減少によるものであろう。 運動ニューロン及び筋線維の間の接続の損失は、運動ニューロン疾患および定住老化9,22,23における筋力低下の直接の原因であると思われる。のNMJを測定するための共有メソッドとパラメータは、簡単に別の研究グループは、公開調査結果を比較対照するために作る必要があります。詳細なプロトコル（およびそれらの将来の改善）の共有は、それが疾患状態に影響を受けることができるかNMJメンテナンスのメカニズムを理解する上で進歩を促進し、助けることができる。 </P&gt;

神経筋接合部（NMJ）は、神経系および骨格筋との間の通信を仲介する重要な中継シナプスである。これは、すべての自発的な運動のために必要とされる。蛍光顕微鏡は、長いNMJ 1-3マウスの導入遺伝子の影響を研究するために、またはげっ歯類のNMJ 4-11時の年齢、食事、運動、および疾患の影響を比較するために使用されてきた。このような研究は私たちにNMJの生理学および病態生理学について多くを教えてきましたが、（ 例えば、AChRのエリア、終板面積、周囲長、断片化指数）が報告多様なパラメータは、しばしば、それが難しいこれらの研究の調査結果を比較することにする。前臨床研究者が特に疾患12の齧歯類モデルを用いた研究では、再現性を発揮できるようにするための増加期待があります。ここで説明するプロトコルは、発達生理学的および病態生理学的CHを調査の一連の研究を通して洗練されたNMJにアンジェス。このような研究は、マウスの運動終板と後シナプスの専門分野13-15内のシナプスタンパク質のパッキングの相対密度でシナプスの専門分野の面積の測定を必要とする。 これらの方法の有用性は、重症筋無力症、抗MuSKのマウスモデルにおける最近の研究によって示されている。抗MuSKの陽性重症筋からのIgGの毎日の注射は成体マウスに、患者は彼らが2週間以内に16弱になった原因無力。 （神経端末で）シナプトフィジンのための二重標識された筋肉切片の共焦点最大投影画像とシナプス後性AChRは、一次変化としてのAChR染色の分野で進行性の低下を明らかにした。重要なことには減少率は、同等のシナプス電位の振幅の減少、シナプス伝達の障害及び筋衰弱17,18を説明するのに十分であった。定性的に類似の所見は他の研究グループにより報告された10,19。同じNMJ測定方法ので、このマウスモデル20,21に無力抗MuSKの重症筋を治療するための3種の薬物の影響を評価するために使用されている。 座りがちな高齢化は、神経筋の​​接続の損失につながることができます。マウスは古い時代に進行としてここに記載されているプロトコルは、モータのエンドプレートでの神経終末シナプトフィジンのエリアの加齢に伴う減少を明らかにした。同じ方法は、自発的な運動は、主に他のグループ4による以前の研究と一致して、シナプス前神経末端領域22の低下を防ぐことができることを明らかにした。神経接続の損失はまた、筋萎縮性側索硬化症9,23のSOD1G93Aマウスモデルにおいて発生する。 上記の研究では、健康状態の様々なNMJでの前または後シナプスの専門分野のいずれかの面積の削減につながる可能性があることを示している。これは損なわれ、シナプス楽しいことがありctionまたは神経筋の接続の完全な喪失を告げることがあります。 3つのプロトコルは、シナプスの専門分野の面積と密度の定量化を可能にすることが記載されている。第一のプロトコルの目的は、蛍光顕微鏡を用いて、シナプス前および後の専門および哺乳類のNMJでのその配列の領域の実用的かつ再現可能な測定を提供することにある。二次元の最大投影共焦点画像およびNIH ImageJを用いて画像解析をシナプトフィジン染色（シナプス小胞）、シナプス後性AChRおよびシナプス重なり領域の面積の変化を検出するために使用される。シナプスの専門分野を識別するために使用される視覚情報が最大となるように共焦点撮像パラメータ（ゲイン、オフセット·レベル）は、各NMJのために最適化される。神経筋障害はまた、シナプス後のAChRおよび/または他のシナプスタンパク質の密度の変化に起因することができます。第二のプロトコルは、シナプス後のタンパク質の相対密度の変化を検出するために適用することができるムスク、rapsyn、ジストログリカン、リン酸化されたSrcキナーゼおよびリン酸化のAChR 18,21-として。 重症筋無力症では、シナプス後膜内のAChRの減少密度は、シナプス障害や筋力低下の直接の原因である。第三のプロトコルは、シナプス後膜14,15内の隣接性AChRの近接性の変化を評価するための蛍光共鳴エネルギー移動（FRET）法が記載されている。この方法は、蛍光αブンガロトキシン（BGT）で標識された隣接性AChRの間のエネルギー移動を検出する。 FRETは、蛍光ドナーとアクセプタープローブが離れて10nm未満である場合にのみ発生します。これは、直接シナプス電位の振幅に関連することができるのAChRパッキングの気密性で（超顕微鏡）の変化を明らかにすることができます。 過去10年間で洗練されたこれらの3つのプロトコルは、一貫して再現可能な方法で、NMJ整合性の補完的な対策を提供する。標準化されたプロトコルを使用してA番目のパラメータは、哺乳類のNMJの際の遺伝子と環境介入の効果の比較を容易にするはずである。

<table><tbody><tr><td>Scanning confocal microscope</td><td>Leica</td><td>DM 2000 with&amp;nbsp; TCS SP2 system</td><td>Most scanning confocal microscopes should be suitable.&amp;nbsp;</td></tr><tr><td></td><td>Zeiss</td><td>LSM 510 Meta&amp;nbsp;</td><td></td></tr><tr><td></td><td>Leica</td><td>SPE-II</td><td></td></tr><tr><td>Alexa555-a-bungarotoxin (red-BGT)</td><td>Life technologies</td><td>B35451</td><td>Used for labelling AChRs</td></tr><tr><td>Alexa647-&amp;alpha;-bungarotoxin (far-red-BGT)</td><td>Life technologies</td><td>B35450</td><td>Far red fluorescence: barely visible through the eyepiece&amp;nbsp;</td></tr><tr><td>rabbit anti-synaptophysin</td><td>Life technologies</td><td>18-0130</td><td>Different batches of primary antibody differ in effective working dilution</td></tr><tr><td>FITC-anti-rapsyn mab1234</td><td>Milipore</td><td>FCMAB134F</td><td>Monoclonal antibody conjugated to FITC</td></tr><tr><td>FITC-donkey anti-rabbit IgG</td><td>Jackson</td><td>711-095-152</td><td>Polyclonal secondary antibodies can vary in quality according to source and batch</td></tr><tr><td>Optimal Cutting Temperature compound (O.T.C.)</td><td>ProSciTech</td><td>IA018</td><td>Cryostat embedding matrix for freezing&amp;nbsp; muscles</td></tr><tr><td>DABCO</td><td>Sigma</td><td>10981</td><td>Mounting medium that slows photobleaching of fluorophores</td></tr></tbody></table>

the neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy

神経筋接合部（NMJ）は、哺乳動物、運動ニューロンは、自発的な筋収縮を制御を通じて大、コリン作動性シナプスのリレーです。 NMJでの構造変化は、弱さ、萎縮と筋線維の死で、その結果、神経伝達障害が発生することができます。多くの研究は、遺伝子改変または疾患マウスNMJの構造を変化させることができる方法を検討した。残念ながら、それは直接、彼らはしばしば、異なるパラメータおよび分析方法を採用しているため、これらの研究からの知見を比較することは困難であることができる。 3つのプロトコルは、ここで説明されている。最初は、アセチルコリン受容体（AChRの）の面積を測定するために最大値投影共焦点画像を使用して終板とその上のシナプス前神経終末におけるシナプス小胞の染色の領域におけるリッチシナプス後膜ドメイン。第二のプロトコルは、シナプス後膜におけるシナプス蛋白質の免疫染色の相対強度を比較します。第三のPRotocolはエンドプレートでのシナプス後性AChRのパッキングの変化を検出するために蛍光共鳴エネルギー移動（FRET）を使用します。プロトコルが開発され、一連の研究の上に洗練されてきた。品質と結果の一貫性に影響を与える要因を議論すると基準データは健康な若い成体マウスのNMJで提供される。

Watch this Scientific Journal Video about The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy at JoVE.com

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神経筋接合部：測定シナプスサイズ、断片化および共焦点蛍光顕微鏡を用いてシナプスタンパク質の密度の変化

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

the-neuromuscular-junction-measuring-synapse-size-fragmentation

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

21.6K ビュー.  University of Sydney. The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

ビデオ: The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

定量シナプス：シナプス数を定量する免疫細胞ベースのアッセイ

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

神経科学

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

培養中枢神経系ニューロンの樹状突起スパイン形態の解析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

シナプス蛍光の自動定量<em&gt; C.エレガンス</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn:シナプス密度の変化を検出するための迅速で偏りのない蛍光法

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

生化学

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

高スループットとのマルチパラメトリック定量化のための2個のアルゴリズム<em&gt;ショウジョウバエ</em&gt;神経筋接合部形態

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

全山神経筋接合部の免疫蛍光解析と形態測定解析のための単一骨格筋線維の解剖

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

筋萎縮性側索硬化症のモデルにおけるシナプス機能のリアルタイム蛍光測定

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

共焦点顕微鏡と超解像顕微鏡の組み合わせによるマウスの神経筋接合部のキャラクタリゼーション

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

共焦点顕微鏡を用いたマウス神経筋接合部におけるカルシウム過渡現象の高時間分解能による登録

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

ラット内側腓腹筋における神経筋接合部の形態学的特徴の可視化

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

神経筋接合部：測定シナプスサイズ、断片化および共焦点蛍光顕微鏡を用いてシナプスタンパク質の密度の変化

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