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.

<ol>
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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...

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。

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			<td style="width:341px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Scanning confocal microscope</td>
			<td style="width:91px;">Leica</td>
			<td style="width:119px;">DM IRE2 with&#160; TCS SP2 system</td>
			<td>Most scanning confocal microscopes should be suitable.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:91px;">Zeiss</td>
			<td>LSM 510 Meta&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:91px;">Leica</td>
			<td>SPE-II</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Alexa555-a-bungarotoxin (red-BGT)</td>
			<td style="width:91px;">Life technologies</td>
			<td style="width:119px;">B35451</td>
			<td>Used for labelling AChRs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Alexa647-&#945;-bungarotoxin (far-red-BGT)</td>
			<td style="width:91px;">Life technologies</td>
			<td>B35450</td>
			<td>Far red fluorescence: barely visible through the eyepiece&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rabbit anti-synaptophysin</td>
			<td style="width:91px;">Life technologies</td>
			<td>18-0130</td>
			<td>Different batches of primary antibody differ in effective working dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FITC-anti-rapsyn mab1234</td>
			<td>Milipore</td>
			<td style="width:119px;">FCMAB134F</td>
			<td>Monoclonal antibody conjugated to FITC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FITC-donkey anti-rabbit IgG</td>
			<td style="width:91px;">Jackson</td>
			<td>711-095-152</td>
			<td>Polyclonal secondary antibodies can vary in quality according to source and batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimal Cutting Temperature compound (O.T.C.)</td>
			<td style="width:91px;">ProSciTech</td>
			<td>IA018</td>
			<td>Cryostat embedding matrix for freezing&#160; muscles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DABCO</td>
			<td>Sigma</td>
			<td align="right">10981</td>
			<td>Mounting medium that slows photobleaching of fluorophors</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。

NMJでのシナプス面積の測定 エリアの任意の推定値は、シナプスの専門分野の範囲を定義するために、境界の描画に依存している。健康な若い成人の筋肉のNMJの画像がのAChRとシナプトフィジン染色（ 図2AおよびB）の両方のために明確に定義された境界線を表示する必要があります。 AChRの及びシナプトフィジンの両方の蛍光強度は、...

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

Research

JoVE Journal

Neuroscience

21.4K 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

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

スチリル染料の光変換を使用して、シナプス小胞プールの勉強

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

神経科学

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

で、アクティブなゾーンの解剖とイメージングショウジョウバエ神経筋接合部

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

培養神経細胞における膜タンパク質の側方拡散とエキソサイトーシスは、蛍光回復と蛍光損失光退色を用いて評価

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

軟体​​動物細胞内カルシウム濃度とシナプス伝達効率での変更のモニタ<em&gt;アメフラシ</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

の検出<em&gt;その場で</emで&gt;タンパク質 - タンパク質複合体<em&gt;ショウジョウバエ</em近接ライゲーションアッセイの使用&gt;幼虫神経筋接合部

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

カエルの神経筋終末へのカルシウム色素の神経切片への積載：カエル神経筋接合部におけるカルシウムの一時的な位置合わせ

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

神経筋接合部機能を測定

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海馬の齧歯動物の脳スライスにおけるシナプス密度の評価

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

ショウジョウバエ幼虫神経筋接合部のシナプス電流の焦点 Macropatch 録音

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...