The larval 4D standard brain of Drosophila melanogaster on a single cell level

大脑中的行为输出是如何根据外部感觉输入、内部动机状态，甚至是通过先前经验获得的知识来组织的？了解这些过程是神经科学领域最重要的问题。几个世纪以来，脑功能科学的研究人员一直在将行为的特征映射到大脑的各个区域。然而，从简单的地图到普遍接受的模型，事实证明是极其困难的。在极度减少的模式生物(如线虫或海兔)中，只有在有限的水平上才能与人类进行有意义的比较，以及更复杂的脊椎动物系统(如老鼠、斑马鱼)，它们有数字、伦理和技术上的限制，经典的遗传模式生物果蝇已经获得了重要的中间地位。最近的方法要么试图通过连续透射电子显微镜(TEM)建立幼虫大脑的完整连接体，要么基于不同GAL4驱动线的共聚焦显微镜堆叠，为成虫建立3D脑图谱。然而，这两种方法都达到了生物学上的极限；而TEM方法仅限于单个大脑的解剖水平，但允许基于其高分辨率建立连接组，而3D配准允许将解剖数据与功能研究联系起来。然而，到目前为止，由于其约200.000个神经元的数值复杂性，它仅限于成年果蝇大脑的约10%。因此，我们提出了一种联合方法来建立基于Gal4、LexA和Split-Gal4系的幼虫大脑约10.000个神经元的综合4D重建。该项目将由在神经科学和信息学领域工作的三个小组的独特专业知识来实现：1)THUM实验室将通过免疫组织化学技术和共聚焦显微镜对这些神经元进行可视化。2)梅尔霍夫实验室将根据其解剖信息将这些神经元注册到标准大脑上。3)Bühler实验室将在一个开放访问数据库中处理、组织和可视化处理后的数据。此外，B.Dickson和J.Truman的实验室都位于HHMI Janelia Farm Research Campus(美国)，他们将通过提供世界上最大的一套遗传工具和有关幼虫大脑解剖的解剖原始数据来支持这一努力。综上所述，我们的方法将在神经元水平上重建4D幼虫大脑；同时，它还将定义一组新的注释(在开放获取数据库中)和可自由访问的神经遗传学工具，以研究整个大脑中单个神经元的特定功能。到那时--这将是第一次--有可能建立一个共同的资源基础，为欧洲迅速增长的研究果蝇幼虫的神经生物学社区提供支持。

How is behavior output organized in a brain based on external sensory inputs, internal motivational states or even knowledge gained through prior experience? Understanding these processes is the most essential issue in the field of neuroscience. For centuries, researchers in the functional brain sciences have been mapping properties of behavior to areas of the brain. Yet, the step from simple maps to a generally accepted model has proven exceedingly difficult. Among the extremely reduced model organisms (e.g. C.elegans or Aplysia) that only on a constricted level allow for meaningful comparisons with humans, and more complex vertebrate systems (e.g. mouse, zebra fish) that have numerical, ethical and technical constraints, the classical genetic model organism Drosophila has acquired an important intermediate position. Recent approaches either try to establish the complete connectome of the larval brain by serial transmission electron microscopy (TEM) or work on a 3D brain atlas for its adult counterpart based on confocal microscopy stacks of different GAL4 driver lines. However, both approaches have reached biological limits; whereas the TEM method is restricted to the anatomical level of a single brain but allows for the establishment of the connectome based on its high resolution, the 3D registration allows linking anatomical data to functional studies. However, so far it is restricted to about 10% of the adult Drosophila brain due to its numerical complexity of about 200.000 neurons.Therefore, we propose a joined approach to establish a comprehensive 4D reconstruction of the about 10.000 neurons of the larval brain based on GAL4, LexA and split-GAL4 lines. The project will be realized by the unique expertise of three groups working in the field of neuroscience and informatics; 1) The Thum lab will visualize these neurons by immunohistochemical techniques and confocal microscopy. 2) The Merhof lab will register these neurons based on its anatomical information onto a standard brain. 3) The Bühler lab will process, organize and visualize the processed data in an open access database. In addition, the labs of B. Dickson and J. Truman, both located at the HHMI Janelia Farm Research Campus (USA) will support the endeavor by offering access to the world-wide largest set of genetic tools and anatomical raw data on larval brain anatomy. In summary, our approach will reconstruct the larval brain in 4D on the neuronal level; at the same time it will also define a new set of annotated (in an open access database) and freely accessible neurogenetic tools to investigate the particular function of single neurons within an entire brain. By that - for the first time - it will be possible to establish a common resource base that offers support for the rapidly growing neurobiology community in Europe working on the Drosophila larva.