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.

הפרוטוקולים שתוארו כאן, אפשרו לנו למדוד באופן מהימן ולכמת שינויים במאפיינים של NMJ על פני מגוון מצבים, כולל מדינות הזדקנות ומחלות נורמליות. השיטות שתוארו עבור en פנים תמונות NMJ תאפשר לחוקרים להשוות את השטח של התמחויות טרום postsynaptic ואזור חפיפה / יישור הסינפטי. כדי להשוות ?...

הצומת העצבית-שהרירית (NMJ) היא סינפסה הממסר הקריטית שמתווכת תקשורת בין מערכת העצבים ושרירי שלד. היא נדרשת לכל התנועה רצונית. מיקרוסקופ פלואורסצנטי משמש כבר זמן רב כדי לחקור את ההשפעות של transgenes על העכבר NMJ 1-3 או להשוות את ההשפעות של גיל, תזונה, פעילות גופנית ומחלות על NMJs מכרסמים 4-11. מחקרים מסוג זה לימדו אותנו הרבה על הפיסיולוגיה ופתופיזיולוגיה של NMJ, אבל הפרמטרים המגוונים דיווחו (למשל, אזור AChR, אזור endplate, אורך היקפי, מדדי פיצול) לעתים קרובות לעשות את זה קשה להשוות את הממצאים של מחקרים אלה. יש ציפייה גוברת לחוקרים פרה-קליניים כדי להיות מסוגלים להפגין שחזור, במיוחד במחקרים עם מודלים של מכרסמים של מחלה 12. הפרוטוקולים שתוארו כאן היו מעודנים באמצעות סדרה של מחקרים שחקרה ch התפתחותית, פיסיולוגי וpathophysiologicalAnges לNMJ. מחקרים מסוג זה דורשים מדידה של השטח של התמחויות הסינפטי בendplate מנוע עכבר והצפיפות היחסית של אריזה של חלבונים סינפטיים בתוך התמחויות postsynaptic 13-15. השירות של שיטות אלה בא לידי ביטוי במחקרים שנעשה לאחרונה במודל של עכברים של אנטי-מושק מיאסטניה גרביס. זריקות יומיות של IgG ממיאסטניה אנטי-מושק חיובית גרביס חולים לעכברים בוגרים שנגרמו להם להיות חלשים בתוך 2 שבועות 16. תמונות מרבי הקרנת Confocal סעיפים שריר שכפול שכותרתו עבור synaptophysin (בעצבי מסופים) וAChRs postsynaptic הציגו ירידה הדרגתית באזור של מכתים AChR כשינוי היסודי. חשוב לציין את קצב הירידה היה די כדי להסביר ירידות דומות במשרעת של פוטנציאלים סינפטיים, כישלון של העברת הסינפטית וחולשת השרירים 17,18. איכותי ממצאים דומים דווחו על ידי קבוצות מחקר אחרות10,19. באותן שיטות מדידת NMJ ומאז נהגו להעריך את ההשפעה של שלוש תרופות לטיפול במיאסטניה גרביס אנטי-המושק ב20,21 מודל העכבר הזה. הזדקנות כרוך בישיבה יכולה להוביל לאובדן של קשרים עצביים-שרירית. הפרוטוקולים שתוארו כאן חשפו ירידת גיל הקשורים בתחום synaptophysin מסוף העצב בendplates המוטורי כמו עכברים להתקדם בגיל מבוגר. באותן השיטות גילו כי פעילות גופנית במידה רבה מרצון יכולה למנוע את הירידה בשטח מסוף עצב presynaptic 22, עולה בקנה אחד עם מחקרים קודמים על ידי קבוצות אחרות 4. אובדן של קשרים עצביים-שרירית מתרחש גם במודל עכבר SOD1G93A של טרשת לרוחב amyotrophic 9,23. המחקרים שהוזכרו לעיל מראים כי מגוון רחב של מצבים בריאותיים עלול להוביל לירידה בשטח של שני התמחויות לפני או אחרי-סינפטי בNMJ. הדבר עלול לגרום כיף הסינפטי לקויction או עשוי לבשר אובדן הקשר העצבי-שהרירית מלא. שלושה פרוטוקולים מתוארים המאפשרים לכמת את האזור והצפיפות של התמחויות הסינפטי. המטרה של הפרוטוקול הראשון היא לספק מידה מעשית ושחזור של התחומים לפני והתמחויות פוסט-סינפטי ויישורם בNMJs היונקים, באמצעות מיקרוסקופ פלואורסצנטי. תמונות דו-ממדיות המרביות confocal ההקרנה וניתוח תמונה עם NIH ImageJ משמש כדי לזהות שינויים באזור של מכתים synaptophysin (שלפוחית ​​סינפטית), AChRs postsynaptic ואזור חפיפה הסינפטי. פרמטרים Confocal הדמיה (רווח לקזז רמה) מותאמים לכל NMJ על מנת למקסם את המידע החזותי המשמש להבחין תחום התמחות הסינפטי. כישלון Neuromuscular יכול לנבוע גם משינויים בצפיפות של AChR postsynaptic ו / או חלבונים סינפטיים אחרים. הפרוטוקול השני ניתן ליישם כדי לזהות שינויים בצפיפות היחסית של חלבוני postsynaptic כזהכמושק, rapsyn, dystroglycan, קינאז Src phosphorylated וAChR phosphorylated 18,21. במיאסטניה גרביס, צפיפות מופחתת של AChR בתוך הקרום postsynaptic היא הגורם המיידי לכישלון הסינפטי וחולשת שרירים. הפרוטוקול השלישי מתאר שיטת העברת הקרינה תהודה אנרגיה (סריג) כדי להעריך את השינויים בקרבת AChRs הסמוך בתוך ממברנות postsynaptic 14,15. שיטה זו מזהה העברת אנרגיה בין AChRs השכן שכותרתו עם הניאון-α-bungarotoxin (BGT). סריג מתרחש רק כאשר בדיקות תורם acceptor ניאון בנפרד פחות מ -10 ננומטר. זה יכול לגלות שינויים (תת-מיקרוסקופים) באטימות של אריזת AChR שעשוי להיות קשור ישירות להמשרעת של פוטנציאל הסינפטי. שלושת פרוטוקולים אלה, מעודנים בעשור האחרון, מספקים אמצעי משלימים של יושרת NMJ באופן עקבי לשחזור. שימוש בפרוטוקולים סטנדרטייםפרמטרים nd צריכים לאפשר השוואה של ההשפעות של גנים והתערבויות סביבתיות על NMJ של היונקים.

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			<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>
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			<td>Used for labelling AChRs</td>
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			<td height="42" style="height:42px;width:315px;">Alexa647-&#945;-bungarotoxin (far-red-BGT)</td>
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			<td>B35450</td>
			<td>Far red fluorescence: barely visible through the eyepiece&#160;</td>
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			<td>Different batches of primary antibody differ in effective working dilution</td>
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			<td>Monoclonal antibody conjugated to FITC</td>
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			<td>Cryostat embedding matrix for freezing&#160; muscles</td>
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			<td>Sigma</td>
			<td align="right">10981</td>
			<td>Mounting medium that slows photobleaching of fluorophors</td>
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the neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy

הצומת העצבית-שהרירית (NMJ) היא סינפסה הגדולה, כולינרגית הממסר שדרכו הנוירונים מוטוריים יונקים לשלוט התכווצות שרירים מרצון. שינויים מבניים בNMJ יכולים לגרום לכישלון עצבית, וכתוצאה מכך חולשה, ניוון ואף למוות של סיבי השריר. מחקרים רבים בחנו כיצד שינויים או מחלה גנטיים יכולים לשנות את המבנה של NMJ העכבר. למרבה הצער, זה יכול להיות קשה כדי להשוות ישירות ממצאים ממחקרים אלה כי לעתים קרובות הם מועסקים פרמטרים שונים ושיטות אנליטיות. שלושה פרוטוקולים מתוארים כאן. הראשון משתמש בתמונות confocal הקרנת עוצמה מקסימלית כדי למדוד את השטח של קולטן האצטילכולין (AChR) -rich תחומים קרום postsynaptic בendplate והאזור של מכתים שלפוחית ​​הסינפטית במסוף עצב presynaptic שמעליה. הפרוטוקול השני משווה את העוצמות היחסית של immunostaining לחלבונים סינפטיים בקרום postsynaptic. יחסי הציבור השלישיotocol משתמש העברת הקרינה תהודה אנרגיה (סריג) כדי לזהות שינויים באריזה של AChRs postsynaptic בendplate. הפרוטוקולים פותחו ושוכללו על סדרה של מחקרים. גורמים המשפיעים על האיכות ועקביות של תוצאות נדונים ונתונים נורמטיביים מסופקים NMJs בעכברים צעירים למבוגרים בריאים.

מדידה של שטח Synaptic בNMJ כל הערכה של האזור מסתמכת על הציור של גבול כדי להגדיר את היקף התמחויות הסינפטי. בשרירים צעירים מבוגרים בריאים תמונות NMJ צריכים להציג גבולות מוגדרים היטב עבור שני AChR וצביעת synaptophysin (איור 2 א &#39;וב&#39;...

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.

Neuromuscular צומת: גודל סינפסה מדידה, שינויי פיצול ובצפיפות חלבון Synaptic שימוש Confocal מיקרוסקופ פלואורסצנטי

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

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21.4K Views.  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.

Video: 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.

לימוד בריכות שלפוחיות Synaptic באמצעות photoconversion של צבעים Styryl

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

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.

Dissection והדמיה של אזורי פעיל תסיסנית Neuromuscular צומת

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.

דיפוזיה Exocytosis לרוחב של חלבונים בממברנה של נוירונים בתרבית הוערכה באמצעות שחזור פלואורסצנציה Fluorescence מסופקים Photobleaching

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.

מעקב אחר שינויים בריכוז הסידן התאיים ויעילות הסינפטית ברכיכות<em&gt; Aplysia</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.

תמונה רציפה-הלבנה להתוות תאי שווא יחידים בצומת Neuromuscular

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.

זיהוי של<em&gt; באתר</em&gt; מכלולי חלבון-חלבון ב<em&gt; דרוזופילה</em&gt; הזחל Neuromuscular צומת שימוש סמיכות קשירת 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 ...

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.

טעינת צבע סידן לתוך צפרדע עצב צפרדע דרך גדם עצב: סידן חולף הרשמה צפרדע Neuromuscular צפרדע

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.

מדידת פונקציונליות צומת עצב-שריר

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.

הערכה של צפיפות סינפסה פרוסות המוח מכרסמים בהיפוקמפוס

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.

מוקד Macropatch הקלטות של זרמי סינפטית מצומת דרוזופילה Neuromuscular זחל

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