Name: ex vivo culturing of whole, developing drosophila brains
Uploaded: 2012-07-27T13:00:00
Description: Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

हम उनकी सलाह उपयोगी पांडुलिपि पर और चर्चा टिप्पणी के लिए हमारी प्रयोगशाला के सभी सदस्यों को धन्यवाद देना चाहूंगा. हम भी विशेष रूप से मार्ता कोच और Bassem हसन उत्कृष्ट सुझाव है कि हम remodeling के प्रयोगों में संवर्धन के लिए दिमाग विदारक से पहले 1.5 घंटे APF जब तक प्रतीक्षा के लिए धन्यवाद देना चाहूंगा. हम भी अपने confocal सूक्ष्मदर्शी का उपयोग NHGRI इमेजिंग सुविधा के लिए धन्यवाद. इस काम NINDS अंदर का रिसर्च प्रोग्राम एनआईएच (NS003106 Z01) के मूल तंत्रिका विज्ञान प्रोग्राम द्वारा समर्थित किया गया.

ए मीडिया की तैयारी <ol><li> फिल्टर स्टरलाइज़ Schneider ड्रोसोफिला मीडिया 10% भ्रूण गोजातीय सीरम और 1% पेनिसिलिन / स्ट्रेप्टोमाइसिन के साथ पूरक पूरी मीडिया की तैयारी. </li><li> मीडिया हर बार नए सिरे से तैयार किया जाना ?...

<ol>
	<li>Trunova, S., Baek, B., Giniger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdk5+regulates+the+size+of+an+axon+initial+segment-like+compartment+in+mushroom+body+neurons+of+the+Drosophila+central+brain.">Cdk5 regulates the size of an axon initial segment-like compartment in mushroom body neurons of the Drosophila central brain.</a> J. Neurosci. 31 (29), 10451-10462 (2011).</li><li>Nakada, C., 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=Accumulation+of+anchored+proteins+forms+membrane+diffusion+barriers+during+neuronal+polarization.">Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.</a> Nat. Cell Biol. 5 (7), 626-632 (2003).</li><li>Schmid, G., Strosznajder, J. B., Wesierska-Gadek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+the+p53+tumor+suppressor+protein+family+and+Cdk5:+novel+therapeutic+approaches+for+the+treatment+of+neurodegenerative+disease+using+selective+Cdk+inhibitors.">Interplay between the p53 tumor suppressor protein family and Cdk5: novel therapeutic approaches for the treatment of neurodegenerative disease using selective Cdk inhibitors.</a> Mol Neurobiol. 34 (1), 27-50 (2006).</li><li>Kahsai, L., Zars, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+and+memory+in+Drosophila:+behavior,+genetics,+and+neural+system.">Learning and memory in Drosophila: behavior, genetics, and neural system.</a> Int. Rev. Neurobiol. 99, 139-167 (2011).</li><li>Lee, T., Lee, A., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+mushroom+bodies:+sequential+generation+of+three+distinct+types+of+neurons+from+a+neuroblast.">Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.</a> Development. 126, 4065-4076 (1999).</li><li>Watts, R. J., Hoopfer, E. D., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+pruning+during+Drosophila+metamorphosis:+evidence+for+local+degeneration+and+requirement+of+the+ubiquitin-proteasome+system.">Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system.</a> Neuron. 38 (6), 871-885 (2003).</li><li>Lee, T., Marticke, S., Sung, C., Robinow, S., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+requirement+of+the+USP/EcR-B+ecdysone+receptor+for+mushroom+body+neuronal+remodeling+in+Drosophila.">Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila.</a> Neuron. 28 (3), 807-818 (2000).</li><li>Connell-Crowley, L., Le Gall, M., Vo, D. J., Giniger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cyclin-dependent+kinase+Cdk5+controls+multiple+aspects+of+axon+patterning+in+vivo.">The cyclin-dependent kinase Cdk5 controls multiple aspects of axon patterning in vivo.</a> Curr. Biol. 10 (10), 599-602 (2000).</li><li>Truman, J. W., Riddiford, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+insights+into+the+evolution+of+metamorphosis+in+insects.">Endocrine insights into the evolution of metamorphosis in insects.</a> Annu. Rev. Entomol. 47, 467-500 (2002).</li><li>Noguchi, T., Miller, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+actin+dynamics+in+individualization+during+spermatogenesis+in+Drosophila+melanogaster.">A role for actin dynamics in individualization during spermatogenesis in Drosophila melanogaster.</a> Development. 130, 1805-1816 (2003).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Mol. Biol. Cell. 16 (11), 5127-5140 (2005).</li><li>Brown, H. L. D., Cherbas, L., Cherbas, P., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+time-lapse+imaging+and+dominant+negative+receptors+to+dissect+the+steroid+receptor+control+of+neuronal+remodeling+in+Drosophila.">Use of time-lapse imaging and dominant negative receptors to dissect the steroid receptor control of neuronal remodeling in Drosophila.</a> Development. 133, 275-288 (2006).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Developing+and+Adult+Drosophila+Brains+and+Retinae+for+Live+Imaging.">Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging.</a> J. Vis. Exp. (37), e1936 (2010).</li></ol>

<html> ऊतक संरचना और समारोह म्यूटेशनों और transgenes की अभिव्यक्ति सहित, की पुरानी आनुवंशिक जोड़तोड़, आम तौर पर तत्काल प्रभाव और देर से अप्रत्यक्ष परिणाम है कि अलग करने के लिए बहुत मुश्किल हो सकता है की एक संयोजन ?...

<table border="1"><tbody><tr><td> अभिकर्मक का नाम </td><td> कंपनी </td><td> कुल संख्या </td><td> अंतिम एकाग्रता </td></tr><tr><td> Schneider ड्रोसोफिला मीडिया </td><td> Invitrogen </td><td> 21720-024 </td><td></td></tr><tr><td> भ्रूण गोजातीय सीरम (गर्मी निष्क्रिय) </td><td> Invitrogen </td><td> 10082-147 </td><td> 10% </td></tr><tr><td> पेनिसिलिन स्ट्रेप्टोमाइसिन / </td><td> Invitrogen </td><td> 15140-122 </td><td> 1% </td></tr><tr><td> इन्सुलिन </td><td> Invitrogen </td><td> 12585-014 </td><td> 10μg/ml </td></tr><tr><td> Ecdysone </td><td> सिग्मा Aldrich </td><td> H5142 </td><td> 2μg/ml </td></tr><tr><td> Roscovitine </td><td> सिग्मा Aldrich </td><td> R7772 </td><td> 100μM </td></tr><tr><td> Olomoucine </td><td> सिग्मा Aldrich </td><td> O0886 </td><td> 100μM </td></tr><tr><td> सामान्य बकरी सीरम </td><td> Invitrogen </td><td> 50062Z </td><td> 1% </td></tr><tr><td> गोजातीय Albumin सीरम </td><td> फिशर साइंटिफिक </td><td> M9501 </td><td> 1% </td></tr><tr><td> Formaldehyde </td><td> सिग्मा Aldrich </td><td> D4551 </td><td> 4% </td></tr><tr><td> ट्राइटन - एक्स 100 </td><td> फिशर साइंटिफिक </td><td> BP151 </td><td> 0.5% </td></tr><tr><td> फास्फेट खारा बफर </td><td> Invitrogen </td><td> 70011-044 </td><td> 1X </td></tr><tr><td> Vectashield </td><td> वेक्टर प्रयोगशालाओं </td><td> H1000 </p&gt; <td></td></tr><tr><td> Millicell सेल संस्कृति आवेषण </td><td> Millipore </td><td> PI8P01250 </td><td></td></tr><tr><td> Multidish, चार अच्छी तरह से </td><td> थर्मो वैज्ञानिक </td><td> 176,740 </td><td></td></tr><tr><td> # 5 संदंश डुमोंट </td><td> ठीक विज्ञान उपकरण </td><td> 11251-20 </td><td></td></tr><tr><td> खुर्दबीन स्लाइड </td><td> फिशर साइंटिफिक </td><td> 12-544-7 </td><td></td></tr><tr><td> ग्लास कवर </td><td> फिशर साइंटिफिक </td><td> 12-548-5M </td><td></td></tr></tbody></table>

ex vivo culturing of whole, developing drosophila brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Watch this Scientific Journal Video about Ex vivo Culturing of Whole, Developing Drosophila Brains at JoVE.com

Ex vivo Culturing of Whole, Developing Drosophila Brains

यह लेख एक विधि है जिसके द्वारा एक नकल कर सकते हैं का वर्णन<em&gt; Vivo में</em&gt; विकास<em&gt; ड्रोसोफिला</em&gt; एक शरीर में मशरूम<em&gt; पूर्व vivo</em&gt; संस्कृति प्रणाली.

पूर्व पूरे के vivo संवर्धन, विकास ड्रोसोफिला दिमाग

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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Neuroscience

Ex vivo Culturing of Whole, Developing Drosophila Brains

In vivo Electroporation of Developing Mouse Retina

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विकास माउस रेटिना के vivo electroporation में

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

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समकालिक<em&gt; पूर्व vivo</emदो Retinas की&gt; कार्यात्मक परीक्षण से<em&gt; Vivo</em&gt; Electroretinogram सिस्टम

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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एक साधारण एक कदम प्रौढ़ की साबुत माउंट तैयारी के लिए विच्छेदन प्रोटोकॉल<em&gt; ड्रोसोफिला</em&gt; दिमाग

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

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

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माजी Utero Electroporation और Organotypic स्लाइस भ्रूण माउस दिमाग के लाइव के लिए संस्कृतियों-GABAergic न्यूरॉन्स पलायन की इमेजिंग

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

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पूर्व वीवो Drosophila में अंत-स्रावी संकेत करने के लिए मस्तिष्क प्रतिक्रियाओं visualizing के लिए कैल्शियम इमेजिंग

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

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This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

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Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment (Video) | JoVE

एक ही माउस रेटिना की कॉन्फोकल छवियों के साथ दृश्य-प्रकाश ऑप्टिकल समेकन टोमोग्राफी फाइबरग्राम का संरेखण

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

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रीढ़ की हड्डी उत्तेजना के दौरान मोटर न्यूरॉन्स में पूरे सेल पैच-क्लैंप रिकॉर्डिंग के लिए रीढ़ की हड्डी के टुकड़े की पूर्व विवो तैयारी

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...

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पूर्व वीवो मिर्गी के ड्रोसोफिला मॉडल के लिए कैल्शियम इमेजिंग

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...