पूर्व पूरे के vivo संवर्धन, विकास ड्रोसोफिला दिमाग
Summary
यह लेख एक विधि है जिसके द्वारा एक नकल कर सकते हैं का वर्णन<em> Vivo में</em> विकास<em> ड्रोसोफिला</em> एक शरीर में मशरूम<em> पूर्व vivo</em> संस्कृति प्रणाली.
Abstract
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.
Protocol
Discussion
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 ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of Reagent | Company | Catalog Number | Final Concentration |
| Schneider’s Drosophila Media | Invitrogen | 21720-024 | |
| Fetal Bovine Serum (heat-inactivated) | Invitrogen | 10082-147 | 10% |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | 1% |
| Insulin | Invitrogen | 12585-014 | 10μg/ml |
| Ecdysone | Sigma-Aldrich | H5142 | 2μg/ml |
| Roscovitine | Sigma-Aldrich | R7772 | 100μM |
| Olomoucine | Sigma-Aldrich | O0886 | 100μM |
| Normal Goat Serum | Invitrogen | 50062Z | 1% |
| Bovine Serum Albumin | Fisher Scientific | M9501 | 1% |
| Formaldehyde | Sigma-Aldrich | D4551 | 4% |
| Triton-X 100 | Fisher Scientific | BP151 | 0.5% |
| Phosphate Buffered Saline | Invitrogen | 70011-044 | 1X |
| Vectashield | Vector Laboratories | H1000 | |
| Millicell Cell Culture Inserts | Millipore | PI8P01250 | |
| Multidish, 4 well | Thermo Scientific | 176740 | |
| Dumont #5 Forceps | Fine Science Tools | 11251-20 | |
| Microscope Slides | Fisher Scientific | 12-544-7 | |
| Cover Glass | Fisher Scientific | 12-548-5M |
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