We would like to thank all the members of our lab for their advice, useful discussions and comments on the manuscript. We would also particularly like to thank Marta Koch and Bassem Hassan for the excellent suggestion that we wait until 1.5 hours APF before dissecting brains for culturing in remodeling experiments. We also thank the NHGRI imaging facility for the use of their confocal microscope. This work was supported by the Basic Neuroscience Program of the NINDS Intramural Research Program, NIH (Z01 NS003106).

A. Preparation of Media
<ol>
	<li>Prepare complete media by filter-sterilizing Schneider's Drosophila Media supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin.</li>
	<li>Media should be prepared fresh each time. Alternatively, a fresh aliquot of pre-frozen media can be thawed before each use.</li>
	<li>Prior to culturing, add 10 &mu;g/ml insulin and 2 &mu;g/ml ecdysone to the media. Drugs were prepared as concentrated stocks and frozen in small aliquots. Thawed aliquots were not refrozen.<...

<ol>
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Chronic genetic manipulations of tissue structure and function, including mutations and expression of transgenes, typically produce a combination of immediate effects and late indirect consequences that can be very difficult to separate. Complementing genetic methods with pharmacology can facilitate interrogation of the time course behind a phenotype. For example, this has been a powerful approach for dissecting the contributions of different cytoskeletal components during spermatogenesis in Drosophila <s...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Schneider's Drosophila Media</td>
 <td>Invitrogen</td>
 <td>21720-024</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (heat-inactivated)</td>
 <td>Invitrogen</td>
 <td>10082-147</td>
 <td>10% </td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 <td>1% </td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Invitrogen</td>
 <td>12585-014</td>
 <td>10&mu;g/ml </td>
 </tr>
 <tr>
 <td>Ecdysone</td>
 <td>Sigma-Aldrich</td>
 <td>H5142</td>
 <td>2&mu;g/ml </td>
 </tr>
 <tr>
 <td>Roscovitine</td>
 <td>Sigma-Aldrich</td>
 <td>R7772</td>
 <td>100&mu;M </td>
 </tr>
 <tr>
 <td>Olomoucine</td>
 <td>Sigma-Aldrich</td>
 <td>O0886</td>
 <td>100&mu;M </td>
 </tr>
 <tr>
 <td>Normal Goat Serum</td>
 <td>Invitrogen</td>
 <td>50062Z</td>
 <td>1% </td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Fisher Scientific</td>
 <td>M9501</td>
 <td>1% </td>
 </tr>
 <tr>
 <td>Formaldehyde</td>
 <td>Sigma-Aldrich</td>
 <td>D4551</td>
 <td>4% </td>
 </tr>
 <tr>
 <td>Triton-X 100</td>
 <td>Fisher Scientific</td>
 <td>BP151</td>
 <td>0.5% </td>
 </tr>
 <tr>
 <td>Phosphate Buffered Saline</td>
 <td>Invitrogen</td>
 <td>70011-044</td>
 <td>1X </td>
 </tr>
 <tr>
 <td>Vectashield</td>
 <td>Vector Laboratories</td>
 <td>H1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Millicell Cell Culture Inserts</td>
 <td>Millipore</td>
 <td>PI8P01250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Multidish, 4 well</td>
 <td>Thermo Scientific</td>
 <td>176740</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumont #5 Forceps</td>
 <td>Fine Science Tools</td>
 <td>11251-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope Slides</td>
 <td>Fisher Scientific</td>
 <td>12-544-7</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cover Glass</td>
 <td>Fisher Scientific</td>
 <td>12-548-5M</td>
 <td>&nbsp;</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

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

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 ...

ex-vivo-culturing-of-whole-developing-drosophila-brains

Research

JoVE Journal

Neuroscience

13.0K visualizações.  National Institute of Neurological Disorders and Stroke. Este artigo descreve um método pelo qual se pode imitar In vivo Desenvolvimento do Drosophila Corpo cogumelo em uma Ex vivo Sistema de cultura.