Ex vivo Culturing of Whole, Developing Drosophila Brains
Ex vivo Culturing of Whole, Developing Drosophila Brains
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DOI：
10.3791/4270
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发表时间：
2012-07-01
影响因子：
1.2
通讯作者：
Giniger, Edward
中科院分区：
文献类型：
--
作者：
Prithviraj, Ranjini;Trunova, Svetlana;Giniger, Edward
我们描述了一种对整个果蝇脑进行体外培养的方法。这可以用来作为慢性遗传操作的对立面，通过允许急性药物干预和细胞过程的实时成像来研究细胞生物学和中央大脑结构的发育。作为这项技术的一个例子，我们实验室的先前工作(1)表明，在果蝇中央大脑轴突的轴突和躯体树突间存在一个以前未被识别的亚细胞间隔。这个被称为轴突初始段(AIS)(2)的间隔室的发育被证明在遗传学上依赖于神经元特异性细胞周期蛋白依赖性激酶CDK5。我们在这里表明，体外处理野生型果蝇幼虫的大脑，用CDK5特异性的药物抑制剂rocovitine和olomouine(3)可以引起肌动蛋白组织和细胞表面蛋白Fasciclin 2的定位的急剧变化，这模仿了在遗传上缺乏CDK5活性的突变体中看到的变化。体外培养技术的第二个例子是在幼虫到成虫的变态过程中重建胚胎蘑菇体(MB)伽马神经元的连接。蘑菇体是苍蝇嗅觉学习和记忆的中心(4)，这些伽马神经元在幼虫发育期间修剪它们的轴突和树突分支，然后在以后的时间点重新延伸分支，以建立成年神经支配模式(5)。已经证明，MB的这些神经元的修剪是通过轴突分支(6)的局部退化发生的，通过作用于蜕皮激素受体B1(7)的蜕皮激素(一种类固醇激素)触发的机制，并依赖于泛素-蛋白酶体系统(6)的活性。我们的体外培养方法可以用来进一步探讨发育重塑的机制。我们发现，在体外培养环境中，MB的伽马神经元以与体内相似的时间进程重现了发育修剪的过程。然而，必须等到蚕蛹形成后1.5小时才移植组织，以便细胞不可逆转地进行变态；在蚕蛹开始时解剖动物导致在培养中很少或根本没有变态。因此，经过适当的修改，体外培养方法可以应用于研究中枢脑生物学的动态和稳态方面。
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 lab(1) 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 olomoucine(3) 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 fly(4), 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 pattern(5). Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches(6), by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1(7), and that is dependent on the activity of the ubiquitin-proteasome system(6). 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.