Yazarların laboratuvar çalışmalarında Fondazione Roma ve televizyon dizisi tarafından (Hayır ver. desteklenmiştir GGP14066).

<ol>
	<li>Dadon-Nachum, M., Melamed, E., Offen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;dying-back&amp;#34;+phenomenon+of+motor+neurons+in+ALS.">The &amp;#34;dying-back&amp;#34; phenomenon of motor neurons in ALS.</a> J Mol Neurosci. 43 (3), 470-477 (2011).</li><li>Dobrowolny, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+Muscle+Is+a+Primary+Target+of+SOD1G93A-Mediated+Toxicity.">Skeletal Muscle Is a Primary Target of SOD1G93A-Mediated Toxicity.</a> Cell Metab. 8 (5), 425-436 (2008).</li><li>Mantilla, C. B., Zhan, W. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+improve+neuromuscular+transmission+in+the+adult+rat+diaphragm.">Neurotrophins improve neuromuscular transmission in the adult rat diaphragm.</a> Muscle Nerve. 29 (3), 381-386 (2004).</li><li>Prakash, Y. S., Miyata, H., Zhan, W. Z., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivity-induced+remodeling+of+neuromuscular+junctions+in+rat+diaphragmatic+muscle.">Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle.</a> Muscle Nerve. 22 (3), 307-319 (1999).</li><li>Sieck, D. C., Zhan, W. Z., Fang, Y. H., Ermilov, L. G., Sieck, G. C., Mantilla, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-activity+relationships+in+rodent+diaphragm+muscle+fibers+vs.+neuromuscular+junctions.">Structure-activity relationships in rodent diaphragm muscle fibers vs. neuromuscular junctions.</a> Respir Physiol Neurobiol. 180 (1), 88-96 (2012).</li><li>Van Lunteren, E., Moyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+DAP+on+diaphragm+force+and+fatigue,+including+fatigue+due+to+neurotransmission+failure.">Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure.</a> J Appl Physiol. 81 (5), 2214-2220 (1996).</li><li>Van Lunteren, E., Moyer, M., Kaminski, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+effects+of+myasthenia+gravis+on+rat+phrenic+diaphragm+contractile+performance.">Adverse effects of myasthenia gravis on rat phrenic diaphragm contractile performance.</a> J Appl Physiol. 97 (3), 895-901 (2004).</li><li>Lee, Y., Mikesh, M., Smith, I., Rimer, M., Thompson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+a+mouse+model+of+spinal+muscular+atrophy+show+profound+defects+in+neuromuscular+development+even+in+the+absence+of+failure+in+neuromuscular+transmission+or+loss+of+motor+neurons.">Muscles in a mouse model of spinal muscular atrophy show profound defects in neuromuscular development even in the absence of failure in neuromuscular transmission or loss of motor neurons.</a> Dev Biol. 356 (2), 432-444 (2011).</li><li>Personius, K. E., Sawyer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+and+failure+of+neurotransmission+in+the+diaphragm+of+mdx+mice.">Variability and failure of neurotransmission in the diaphragm of mdx mice.</a> Neuromuscular Disord. 16 (3), 168-177 (2006).</li><li>R&#246;der, I. V., Petersen, Y., Choi, K. R., Witzemann, V., Hammer, J. A., Rudolf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+myosin+Va+in+the+plasticity+of+the+vertebrate+neuromuscular+junction+in+vivo.">Role of myosin Va in the plasticity of the vertebrate neuromuscular junction in vivo.</a> PLoS ONE. 3 (12), e3871 (2008).</li><li>Chevessier, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+congenital+myasthenic+syndrome+due+to+MuSK+mutations+reveals+defects+in+structure+and+function+of+neuromuscular+junctions.">A mouse model for congenital myasthenic syndrome due to MuSK mutations reveals defects in structure and function of neuromuscular junctions.</a> Hum Mol Genet. 17 (22), 3577-3595 (2008).</li><li>Farchi, N., Soreq, H., Hochner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acetylcholinesterase+overexpression+induces+multilevelled+aberrations+in+mouse+neuromuscular+physiology.">Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology.</a> J Physiol. 546 (1), 165-173 (2002).</li><li>Kuei, J. H., Shadmehr, R., Sieck, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+contribution+of+neurotransmission+failure+to+diaphragm+fatigue.">Relative contribution of neurotransmission failure to diaphragm fatigue.</a> J Appl Physiol (Bethesda, Md: 1985). 68 (1), 174-180 (1990).</li><li>Del Prete, Z., Musar&#242;, A., Rizzuto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mechanical+properties,+including+isotonic+fatigue,+of+fast+and+slow+MLC/mIgf-1+transgenic+skeletal+muscle.">Measuring mechanical properties, including isotonic fatigue, of fast and slow MLC/mIgf-1 transgenic skeletal muscle.</a> Ann Biomed Eng. 36 (7), 1281-1290 (2008).</li><li>Lynch, G. S., Hinkle, R. T., Chamberlain, J. S., Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+fast+and+slow+skeletal+muscles+from+mdx+mice+6-28+months+old.">Force and power output of fast and slow skeletal muscles from mdx mice 6-28 months old.</a> J Physiol. 535 (2), 591-600 (2001).</li><li>Brooks, S. V., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+properties+of+skeletal+muscles+from+young,+adult+and+aged+mice.">Contractile properties of skeletal muscles from young, adult and aged mice.</a> J Physiol. 404, 71-82 (1988).</li><li>Lynch, G. S., Hinkle, R. T., Faulkner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+and+power+output+of+diaphragm+muscle+strips+from+mdx+and+control+mice+after+clenbuterol+treatment.">Force and power output of diaphragm muscle strips from mdx and control mice after clenbuterol treatment.</a> Neuromuscul Disord. 11 (2), 192-196 (2001).</li><li>Rizzuto, E., Pisu, S., Musar, A., Del Prete, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Neuromuscular+Junction+Functionality+in+the+SOD1+G93A+Animal+Model+of+Amyotrophic+Lateral+Sclerosis.">Measuring Neuromuscular Junction Functionality in the SOD1 G93A Animal Model of Amyotrophic Lateral Sclerosis.</a> Ann Biomed Eng . 43, (2015).</li><li>Soliani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manuale di statistica per la ricerca e la professione statistica univariata e bivariata parametrica e non-parametrica per le discipline ambientali e biologiche. , (2004).</li><li>Gurney, M. E., Pu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+degeneration+in+mice+that+express+a+human+Cu,Zn+superoxide+dismutase+mutation.">Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.</a> Science (New York, N.Y). 264 (5166), 1772-1775 (1994).</li></ol>

Yazarlar ifşa gerek yok.

Yukarıda açıklanan deneysel protokol ölçme ve doğrudan kas veya meydana gelen herhangi bir işlevsel değişiklikler ayrımcılık dolaylı olarak nöromüsküler kavşak düzeyinde ideal bir yol sağlar. Bu teknik NMJ işlevselliği dolaylı bir ölçüm üzerinde dayalı olduğundan, herhangi bir kusur morfolojik değişiklikler veya biyokimyasal değişiklikler ile ilişkili Eğer kurmak için kullanılamaz. Buna karşılık, herhangi bir morfolojik ya da biyokimyasal değişiklikler işlevselliği sinyal neurot...

Nöromüsküler kavşak (NMJ) kas fiber motor endplate ve motor nöron axon terminal arasında bağlantı oluşturduğu bir kimyasal synapse var. Kas ve sinir arasındaki iletişim, yaşlanma veya amyotrofik lateral skleroz (ALS) gerçekleştiği sırada bozulmuş olduğunda çok önemli bir rol oynamaya NMJ gösterilmiştir. Kas ve sinir bir çift yönlü yol1,2' olarak, NMJ kusurları kas defektleri dan ayrı olarak ölçmek için güçlü olmak onların Fizyopatolojik Interplay yeni görüşler sağlayabilir. Gerçekten de, bu işlevsel değerlendirme morfolojik ya da biyokimyasal değişiklikler işlevselliği sinyal neurotransmission azaltmak değerlendirmek için yardımcı olabilir.
Kas contractile yanıt sinir stimülasyonu ve onun membran doğrudan uyarılması tarafından uyarılmış aynı kas yanıt elde edildi karşılaştırılması NMJ işlevselliği dolaylı bir ölçüm evlenme teklif etti. Gerçekten de, sinyal membran stimülasyon geçer tarafından neurotransmission beri iki contractile yanıt-e doğru tüm farklılıkları NMJ değişikliklere atfedilen. Bu yaklaşım yaygın olarak fareler3,4,5,6,7için önerilen ve ayrıca fare modelleri8tarihinde,9,10,11,12bilgi toplamak için kullanılır.
Burada, tüketim ve soleus siyatik ve diyafram frenik hazırlıkları iki kas-sinir hazırlıklar, yani sınamak için bir yordam ayrıntılı olarak açıklanmaktadır. Bir özel kullanarak yazılım, biz tasarlanmış işlevsellik, NMJ ve kas karakterize birkaç parametre ölçümü birleştirir sürekli bir test Protokolü böylece NMJ hasar kapsamlı bir değerlendirme ayrı ayrı bu kas verimli. Özellikle, protokol seğirme gücü, kas Kinetik, kuvvet-frekans eğrisi için doğrudan ve sinir elektrodlar, hem bir ateş ve tetanic sıklık ve intratetanic yorgunluk7neurotransmission başarısızlık13 ölçer.

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			<td>Processor 3.50 Ghz with Windows 7</td>
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			<td>National Instruments</td>
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			<td height="21" style="height:21px;width:283px;">Krebs-Ringer Bicarbonate Buffer&#160;</td>
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measuring neuromuscular junction functionality

Nöromüsküler kavşak (NMJ) işlevselliği motor nöron ve kas arasındaki iletişimi, yaşlanma ve amyotrofik lateral skleroz (ALS) gibi Engelli olduğu hastalıklar okurken çok önemli bir rol oynar. Burada iki tür elektriksel stimülasyon birleştirerek NMJ işlevselliği ölçmek için kullanılan bir deneysel protokol tarif: doğrudan kas zarı stimülasyon ve stimülasyon sinir aracılığıyla. Bu iki farklı elektrodlar kas cevaben karşılaştırılması işlev düzeyinde fonksiyonel kas düşüş yol olası değişiklikler NMJ içinde tanımlamak için yardımcı olabilir.
Ex vivo hazırlıkları iyi kontrollü çalışmalar için uygundur. Burada tarif biz kas ve NMJ işlevselliği soleus siyatik sinir hazırlık ve diyafram frenik sinir hazırlık birkaç parametrelerini ölçmek için yoğun bir iletişim kuralı. Protokol yaklaşık 60 dakika sürer ve kesintisiz bir ısmarlama aracılığıyla yapılır seğirme Kinetik özellikleri, sinir ve kas elektrodlar için güç frekanslı ilişki ve iki parametre NMJ işlevselliği için belirli Yani neurotransmission yetmezliği ve intratetanic yorgunluk ölçer yazılım. Bu metodoloji hasar soleus ve diyafram kas-sinir hazırlıkları SOD1G93A transgenik fare, ubiquitously mutant antioksidan enzim süperoksit dismutaz 1 (SOD1) overexpresses ALS deneysel bir model kullanarak bulmak için kullanıldı.

Biz açıklanan protokol fonksiyonel denervation birkaç nöromüsküler hastalıklar veya yaşlanma-mücadele hakkında bilgiler sağlar. Bu iletişim kuralı olup olmadığını (ve eğer öyleyse, hangi düzeyde) belirlemek için kullanılan kas değişiklikler çoğu kas kendisi veya nöromuskuler iletim oluşan seçici değişiklikleri nedeniyle. Aşağıda gösterilen veri amyotrofik lateral skleroz hastalığı20son aşamasında SOD1G93A transgeni...

Watch this Scientific Journal Video about Measuring Neuromuscular Junction Functionality at JoVE.com

Measuring Neuromuscular Junction Functionality

Nöromüsküler kavşak (NMJ) işlevsel bir değerlendirilmesi sinir ve kas arasındaki iletişimi ilgili gerekli bilgileri sağlar. Burada biz kapsamlı bir şekilde kullanarak iki farklı kas-sinir hazırlıklar, yani soleus siyatik ve diyafram frenik NMJ ve kas işlevselliğini değerlendirmek için bir protokol tanımlamak.

Nöromüsküler kavşak işlevleri ölçme

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

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17.9K Görüntüleme.  Sapienza University of Rome. Nöromüsküler kavşak (NMJ) işlevsel bir değerlendirilmesi sinir ve kas arasındaki iletişimi ilgili gerekli bilgileri sağlar. Burada biz kapsamlı bir şekilde kullanarak iki farklı kas-sinir hazırlıklar, yani soleus siyatik ve diyafram frenik NMJ ve kas işlevselliğini değerlendirmek için bir protokol tanımlamak.

Video: Measuring Neuromuscular Junction Functionality

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.

Aktif Bölgeleri Diseksiyon ve Görüntüleme Drosophila Nöromüsküler Junction

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

Nörobilim

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.

Sıralı Nöromusküler Kavşağı&#39;nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

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 ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Tüm Montajlı Nöromüsküler Kavşak Analizi için Enine Abdominis Kasının Diseksiyonu

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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.

Algılama<em&gt; In Situ</emEn&gt; Protein-protein kompleksleri<em&gt; Drosophila</emProximity ligasyon Testi Kullanımı&gt; Larva Nöromüsküler Junction

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

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

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

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.

Nöromusküler Kavşak: Ölçme Synapse Boyut, Konfokal Floresan Mikroskobu kullanılarak sinaptik Protein Yoğunluk Parçalanma ve Değişiklikler

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

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

En fenotip Karakterizasyonu: Niceleme Matters Neden<em&gt; Drosophila</em&gt; Larva Nöromüsküler Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

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.

Kurbağa Sinir Uçlarına Sinir Kütüğü Vasıtasıyla Kalsiyum Boyası Yükleme: Kurbağa Nöromüsküler Kavşağında Kalsiyum Geçici Kayıt

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

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

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

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

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

Yüksek throughput ve Çok parametrik Nicelik için iki Algoritmalar<em&gt; Drosophila</em&gt; Nöromusküler Junction Morfoloji

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

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.

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

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

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Tanımlanmış Bir Motor Nöron için Nöromüsküler Kavşaktaki Değişiklikleri Tespit Etmek için Drosophila Yetişkin Bacağının Diseksiyonu ve İmmünhistokimyası

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Nöromüsküler kavşak işlevleri ölçme

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Aktif Bölgeleri Diseksiyon ve Görüntüleme Drosophila Nöromüsküler Junction

Sıralı Nöromusküler Kavşağı'nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

Tüm Montajlı Nöromüsküler Kavşak Analizi için Enine Abdominis Kasının Diseksiyonu

Algılama

Nöromusküler Kavşak: Ölçme Synapse Boyut, Konfokal Floresan Mikroskobu kullanılarak sinaptik Protein Yoğunluk Parçalanma ve Değişiklikler

En fenotip Karakterizasyonu: Niceleme Matters Neden

Kurbağa Sinir Uçlarına Sinir Kütüğü Vasıtasıyla Kalsiyum Boyası Yükleme: Kurbağa Nöromüsküler Kavşağında Kalsiyum Geçici Kayıt

Yüksek throughput ve Çok parametrik Nicelik için iki Algoritmalar

Drosophila larva nöromüsküler kavşak sinaptik akımlardan Fokal Macropatch kayıtları

Tanımlanmış Bir Motor Nöron için Nöromüsküler Kavşaktaki Değişiklikleri Tespit Etmek için Drosophila Yetişkin Bacağının Diseksiyonu ve İmmünhistokimyası