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.

وقد مكنت البروتوكولات الموصوفة هنا لنا لقياس بشكل موثوق وقياس التغيرات في خصائص NMJ عبر مجموعة من الشروط، بما في ذلك الشيخوخة والمرض الدول العادية. الطرق الموضحة للأون تواجه والصور NMJ تسمح للباحثين لمقارنة مجال التخصصات قبل وبعد المشبكي ومنطقة متشابك التداخل / المحاذ...

الوصل العصبي العضلي (NMJ) هو المشبك تتابع الأهمية بمكان أن يتوسط التواصل بين الجهاز العصبي والعضلات والهيكل العظمي. هو مطلوب منها لجميع الحركات الإرادية. منذ فترة طويلة تستخدم مضان المجهر لدراسة آثار الجينات المحورة على الماوس NMJ 1-3 أو لمقارنة آثار السن، والنظام الغذائي وممارسة الرياضة والمرض على لل NMJs القوارض 4-11. لقد علمتنا هذه الدراسات لنا الكثير عن علم وظائف الأعضاء والفيزيولوجيا المرضية من NMJ، ولكن ذكرت المعلمات المتنوعة (على سبيل المثال، منطقة الاستشاري لحقوق الإنسان، منطقة endplate، وطول محيط، مؤشرات تجزئة) غالبا ما تجعل من الصعب مقارنة النتائج التي توصلت إليها هذه الدراسات. وهناك توقعات متزايدة للباحثين ما قبل السريرية لتكون قادرة على إثبات استنساخ، لا سيما في الدراسات مع نماذج القوارض من المرض 12. تم تنقيح البروتوكولات المذكورة هنا من خلال سلسلة من الدراسات التي حققت الفصل التنموي، الفسيولوجية والمرضية في جسم المريضفي نفس الفئة إلى NMJ. وتتطلب مثل هذه الدراسات قياس مجال التخصصات متشابك في endplate الماوس السيارات والكثافة النسبية من التعبئة والتغليف من البروتينات متشابك ضمن التخصصات بعد المشبكي 13-15. وتتجلى فائدة هذه الأساليب التي كتبها الدراسات الحديثة في نموذج الفأر المضادة للالمسك الوهن العضلي الوبيل. حقن يومية من مفتش من الوهن العضلي مكافحة المسك إيجابية الوهن تسبب المرضى إلى فئران بالغة لهم أن يصبحوا ضعيفة في حدود 2 أسابيع 16. وكشفت الصور الأقصى الإسقاط مبائر من أقسام العضلات التي وصفت نقرا مزدوجا لsynaptophysin (في النهايات العصبية) ومركز عمان بعد المشبكي في الانخفاض التدريجي في مجال الاستشاري لحقوق الإنسان تلطيخ كما تغير الابتدائي. كان الأهم معدل الانخفاض كافية لشرح انخفاضات مماثلة في السعة من إمكانات متشابك، فشل انتقال متشابك وضعف العضلات 17،18. نوعيا تم الإبلاغ عن نتائج مماثلة من قبل مجموعات بحثية أخرى10،19. ومنذ ذلك الحين تم استخدام طرق القياس NMJ نفسها لتقييم تأثير ثلاثة عقاقير لعلاج مضاد للالمسك الوهن العضلي الوبيل في هذا النموذج الماوس 20،21. الشيخوخة المستقرة يمكن أن يؤدي إلى فقدان الاتصالات العصبية والعضلية. وقد كشفت البروتوكولات الموصوفة هنا تراجع المرتبط بالعمر في مجال الأعصاب الطرفية synaptophysin في endplates السيارات مع تقدم الفئران في سن الشيخوخة. كشفت الأساليب نفسها التي يمكن أن يمنع ممارسة الطوعية إلى حد كبير في الحد من العصبية قبل المشبكي منطقة محطة 22، بما يتفق مع الأعمال السابقة من قبل مجموعات أخرى 4. فقدان اتصالات العصبية والعضلية يحدث أيضا في نموذج SOD1G93A الماوس من التصلب الجانبي الضموري 9،23. وتظهر الدراسات المذكورة أعلاه أن مجموعة متنوعة من الظروف الصحية قد يؤدي إلى تخفيضات في مجال التخصصات إما قبل أو بعد متشابك في NMJ. هذا قد يؤدي إلى متعة متشابك ضعفction أو قد تبشر خسارة كاملة للاتصال العصبي العضلي. ووصف ثلاثة بروتوكولات الكميات التي تسمح للمنطقة وكثافة التخصصات متشابك. الغرض من البروتوكول الأول هو توفير قدر عملي وقابلة للتكرار للمناطق قبل والتخصصات ما بعد متشابك واتساقها في لل NMJs الثدييات، وذلك باستخدام المجهر مضان. يستخدم ثنائي الأبعاد القصوى الصور الإسقاط متحد البؤر وتحليل الصور مع المعاهد الوطنية للصحة يماغيج للكشف عن تغيرات في مجال synaptophysin تلطيخ (الحويصلات متشابك)، مركز عمان بعد المشبكي ومنطقة تداخل متشابك. هي الأمثل المعلمات مبائر التصوير (الربح وتعويض المستوى) لكل NMJ وذلك لتحقيق أقصى قدر من المعلومات البصرية تستخدم لتمييز مجال التخصص متشابك. يمكن فشل العصبي العضلي أيضا نتيجة من التغيرات في كثافة الاستشاري لحقوق الإنسان بعد المشبكي و / أو البروتينات متشابك الأخرى. ويمكن تطبيق البروتوكول الثاني للكشف عن تغيرات في الكثافة النسبية للبروتينات بعد المشبكي هذهكما المسك، rapsyn، dystroglycan، فسفرته SRC كيناز وفسفرته الاستشاري لحقوق الإنسان 18،21. في الوهن العضلي الوبيل، كثافة انخفاض الاستشاري لحقوق الإنسان داخل الغشاء بعد المشبكي هي السبب المباشر لفشل متشابك وضعف العضلات. ويصف البروتوكول الثالث طريقة نقل الإسفار الرنين الطاقة (الحنق) لتقييم التغيرات في المناطق القريبة من مركز عمان المجاورة داخل الأغشية بعد المشبكي 14،15. هذه الطريقة بالكشف عن نقل الطاقة بين مركز عمان المجاورة المسمى مع فلوري-α-بنغاروتوكسين (BGT). الحنق يحدث فقط عندما يكون تحقيقات الفلورسنت المانحة ومتقبل أقل من 10 نانومتر إربا. هذا يمكن أن تكشف (دون المجهرية) التغيرات في ضيق من التعبئة الاستشاري لحقوق الإنسان التي قد تتصل مباشرة إلى السعة من إمكانات متشابك. هذه البروتوكولات الثلاثة، والمكرر على مدى العقد الماضي، توفر تدابير تكميلية النزاهة NMJ بطريقة متسقة وقابلة للتكرار. استخدام بروتوكولات موحدة ليجب المعلمات ND تسهيل المقارنة بين تأثيرات الجينات والتدخلات البيئية على NMJ الثدييات.

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

الوصل العصبي العضلي (NMJ) هو كبير، والتتابع الكوليني المشبك من خلالها الخلايا العصبية الحركية الثدييات تتحكم تقلص العضلات الطوعية. التغييرات الهيكلية في NMJ يمكن أن يؤدي إلى فشل العصبي، مما يؤدي إلى ضعف، وضمور وحتى الموت من الألياف العضلية. حققت العديد من الدراسات كيف تعديلات وراثية أو أمراض يمكن أن يغير هيكل NMJ الماوس. لسوء الحظ، يمكن أن يكون من الصعب مقارنة النتائج مباشرة من هذه الدراسات لأنها غالبا ما تستخدم معايير مختلفة وأساليب التحليلية. وصفت ثلاثة بروتوكولات هنا. أول يستخدم أقصى الإسقاط كثافة الصور مبائر لقياس مجال أستيل مستقبلات (الاستشاري لحقوق الإنسان) الغنية المجالات الغشاء بعد المشبكي في endplate ومنطقة متشابك حويصلة تلطيخ في المغطي الأعصاب الطرفية قبل المشبكي. بروتوكول الثاني يقارن كثافة نسبية للالمناعية للبروتينات متشابك في الغشاء بعد المشبكي. العلاقات العامة الثالثةotocol يستخدم نقل الإسفار الرنين الطاقة (الحنق) للكشف عن تغيرات في التعبئة من مركز عمان بعد المشبكي في endplate. وقد تم تطوير البروتوكولات وصقلها خلال سلسلة من الدراسات. وتناقش العوامل التي تؤثر على جودة واتساق النتائج ويتم توفير البيانات المعيارية للل NMJs في صحية الفئران الشباب البالغين.

قياس المساحة متشابك في NMJ يعتمد أي تقدير للمنطقة على رسم حدود لتحديد مدى التخصصات متشابك. في عضلات صحية الشباب البالغين يجب عرض الصور NMJ حدود واضحة المعالم لكلا الاستشاري لحقوق الإنسان وتلطيخ synaptophysin (الشكل 2A وباء).</st...

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.

على الموصل العصبي العضلي: قياس المشبك الحجم، التغييرات تجزئة ومتشابك الكثافة البروتين عن طريق متحد البؤر مضان المجهر

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.

دراسة تجمعات الحويصلة متشابك من الأصباغ باستخدام Photoconversion 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.

تشريح والتصوير من المناطق النشطة في ذبابة الفاكهة الوصل العصبي العضلي

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.

المقررة الإنتشار الأفقي والإيماس من غشاء بروتينات في الخلايا العصبية مثقف باستخدام الاسترداد الإسفار وPhotobleaching الإسفار الخسارة

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.

رصد التغيرات في تركيز الكالسيوم داخل الخلايا وفعالية متشابك في الرخويات<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.

صور متتابعة تبيض لتحديد خلايا شوان واحدة عند تقاطع الاعصاب

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.

الكشف عن<em&gt; في الموقع</em&gt; مجمعات البروتين البروتين في<em&gt; ذبابة الفاكهة</em&gt; اليرقية الموصل العصبي العضلي عن طريق القرب ربط الفحص

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.

تحميل صبغ الكالسيوم إلى نهايات العصب الضفدع من خلال الجذع العصبي: تسجيل الكالسيوم العابر في الضفدع العصبي العضلي تقاطع

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.

قياس الأداء الوظيفي الوصلات العصبية العضلية

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.

تقييم كثافة المشبك في الدماغ القوارض هيبوكامبال شرائح

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.

تسجيلات ماكروباتش المحورية للتيارات متشابك من الوصلات العصبية العضلية اليرقات المورفولوجية

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