Name: dissection and imaging of active zones in the drosophila neuromuscular junction
Uploaded: 2011-04-27T16:00:00
Description: Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

हम इस पांडुलिपि के बारे में अपनी सहायक टिप्पणियों के लिए डा. Nael Alami और डॉ. वियतनाम चुल किम धन्यवाद.

1. Immunofluorescence के लिए तैयार: <ol><li> विदारक सतह बनाने के लिए, एक छोटे से टिशू कल्चर थाली में Sylgard 184 सिलिकॉन Elastomer बेस डालना. नहीं थाली पूरी तरह से इतना है कि विच्छेदन क्षेत्र रिम से कम है भरने के लिए सुनिश्चित करें. </li><li> ?...

<ol>
	<li>Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse+maturation+and+structural+plasticity+at+Drosophila+neuromuscular+junctions.">Synapse maturation and structural plasticity at Drosophila neuromuscular junctions.</a> Curr Opin Neurobiol. 6, 858-867 (1996).</li><li>Fouquet, W., Owald, D., Wichmann, C., Mertel, S., Depner, H., Dyba, M., Hallermann, S., Kittel, R. J., Eimer, S., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+active+zone+assembly+by+Drosophila+Bruchpilot.">Maturation of active zone assembly by Drosophila Bruchpilot.</a> J Cell Biol. 186, 129-145 (2009).</li><li>Hoang, B., Chiba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+Analysis+of+Drosophila+Larval+Neuromuscular+Synapses.">Single-Cell Analysis of Drosophila Larval Neuromuscular Synapses.</a> Developmental Biology. 229, 55-70 (2001).</li><li>Keshishian, H., Kim, Y. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orchestrating+development+and+function:+retrograde+BMP+signaling+in+the+Drosophila+nervous+system.">Orchestrating development and function: retrograde BMP signaling in the Drosophila nervous system.</a> Trends in Neurosciences. 27, 143-147 (2004).</li><li>Kittel, R. J., Wichmann, C., Rasse, T. M., Fouquet, W., Schmidt, M., Schmid, A., Wagh, D. A., Pawlu, C., Kellner, R. R., Willig, K. I., Hell, S. W., Buchner, E., Heckmann, M., Sigrist, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot+Promotes+Active+Zone+Assembly,+Ca2++Channel+Clustering,+and+Vesicle+Release.">Bruchpilot Promotes Active Zone Assembly, Ca2+ Channel Clustering, and Vesicle Release.</a> Science. 312, 1051-1054 (2006).</li><li>Koh, Y. H., Gramates, L. S., Budnik, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+neuromuscular+junction:+Molecular+components+and+mechanisms+underlying+synaptic+plasticity.">Drosophila larval neuromuscular junction: Molecular components and mechanisms underlying synaptic plasticity.</a> Microscopy Research and Technique. 49, 14-25 (2000).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann N Y Acad Sci. 1184, e1-e20 (2010).</li><li>Ratnaparkhi, A., Lawless, G. M., Schweizer, F. E., Golshani, P., Jackson, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+of+ALS:+human+ALS-associated+mutation+in+VAP33A+suggests+a+dominant+negative+mechanism.">A Drosophila model of ALS: human ALS-associated mutation in VAP33A suggests a dominant negative mechanism.</a> PLoS One. 3, e2334-e2334 (2008).</li><li>Wagh, D. A., Rasse, T. M., Asan, E., Hofbauer, A., Schwenkert, I., D&#252;rrbeck, H., Buchner, S., Dabauvalle, M. -. C., Schmidt, M., Qin, G., Wichmann, C., Kittel, R., Sigrist, S. J., Buchner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bruchpilot,+a+Protein+with+Homology+to+ELKS/CAST,+Is+Required+for+Structural+Integrity+and+Function+of+Synaptic+Active+Zones+in+Drosophila.">Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila.</a> Neuron. 49, 833-844 (2006).</li><li>Zweier, C., Jong, E. K. d. e., Zweier, M., Orrico, A., Ousager, L. B., Collins, A. L., Bijlsma, E. K., Oortveld, M. A., Ekici, A. B., Reis, A., Schenck, A., Rauch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTNAP2+and+NRXN1+are+mutated+in+autosomal-recessive+Pitt-Hopkins-like+mental+retardation+and+determine+the+level+of+a+common+synaptic+protein+in+Drosophila.">CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt-Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila.</a> Am J Hum Genet. 85, 655-666 (2009).</li></ol>

न्यूरॉन्स के लिए, synaptic टर्मिनल क्षेत्र के महत्व का है, और के बाद और पूर्व synaptic कोशिकाओं के बीच समुचित संचार के लिए पुल है. रोग मॉडल में न्यूरॉन के स्वास्थ्य की जांच के लिए एक शक्तिशाली तरीका immunofluorescence द्वारा syna...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियाँ </td></tr><tr><td> Sylgard 184 सिलिकॉन Elastomer बेस </td><td> डॉव Corning </td><td> 68037-59-2 </td><td> मिश्रण के बाद बुलबुले धीमी अंग को घुमानेवाली पेशी पर डाल या 30 मिनट या उससे अधिक के लिए बैठने की अनुमति धीरे धीरे बाहर वृद्धि के लिए अनुमति देते हैं. </td></tr><tr><td> स्टेनलेस स्टील Minutien पिंस </td><td> ललित विज्ञान उपकरण </td><td> 26002-10 </td><td> लगभग छाँटो. नियमित रूप से कैंची से लंबाई में 3-4mm </td></tr><tr><td> Laminectomy संदंश (ब्लंट पिन लोभी के लिए प्रयुक्त) </td><td> ललित विज्ञान उपकरण </td><td> 11223-20 </td><td> पिन लोभी के लिए कुंद संदंश के रूप में प्रयोग करें </td></tr><tr><td> विच्छेदन संदंश </td><td> विश्व परिशुद्धता उपकरण </td><td> 501985 </td><td></td></tr><tr><td> सर्वोत्तम Vannas कैंची, लंबे 8cm </td><td> विश्व परिशुद्धता उपकरण </td><td> 501778 </td><td></td></tr><tr><td> माउस प्रतिरक्षी विरोधी - BRP </td><td> DSHB </td><td> NC82 </td><td> 1:50 कमजोर पड़ने का उपयोग करें </td></tr><tr><td> Cy3 Affinipure बकरी विरोधी सहिजन peroxidase </td><td> जैक्सन Immunoresearch </td><td> 123-165-021 </td><td> 1:200 कमजोर पड़ने पर प्रयोग </td></tr><tr><td> एलेक्सा Fluor 488 बकरी आईजीजी विरोधी माउस </td><td> Invitrogen </td><td> A11001 </td><td> लगभग का प्रयोग करें. 1:200 मन्दन </td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction at JoVE.com

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

neuromuscular जंक्शन (NMJ)<em&gt; ड्रोसोफिला मेलानोगास्टर</em&gt; सामान्य synaptic के रूप में अच्छी तरह के रूप में समारोह में synaptic कुछ स्नायविक रोगों में पाया समारोह के लिए perturbations के अध्ययन के लिए एक महत्वपूर्ण मॉडल प्रणाली है. हम के विच्छेदन के लिए एक प्रोटोकॉल प्रस्तुत<em&gt; ड्रोसोफिला</em&gt; लार्वा मोटर NMJ भीतर सक्रिय क्षेत्र प्रोटीन के लिए सिस्टम और immunostaining.

विच्छेदन में सक्रिय जोन इमेजिंग ड्रोसोफिला Neuromuscular जंक्शन

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

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Neuroscience

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Single Drosophila Ommatidium Dissection and Imaging

Single Drosophila Ommatidium Dissection and Imaging

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The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

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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 ...

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

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A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

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There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

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

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

Drosophila लार्वा Neuromuscular जंक्शन से Synaptic धाराओं की फोकल Macropatch रिकॉर्डिंग

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

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