Our focus has evolved, partly in response to the recommendations made by our advisory committee to focus on the production and characterization of “cell-type-specific” split-GAL4 lines for the larval and adult nervous systems. We have begun with areas of the fly brain that are under active study at Janelia and are now widening our efforts to the complete nervous system, with the involvement of a set of international collaborators. We anticipate hosting between ten and twelve such collaborators at any given time over the next four years. A number of individuals have already joined us, but we are seeking additional collaborators with the required anatomical expertise who are willing to spend one to two years in residence at Janelia working with us.
Mushroom Body Output Neurons
Figure 1: The morphologies of the individual mushroom body output neurons in the adult are shown (Aso et al.)
Characterizing the morphology of cells from a split-GAL4 line
Figure 2: (Left) image of a membrane-targeted reporter construct. (Center) The two cells in this field of view are labeled in different colors using the MCFO method. The cells have indistinguishable morphologies. (Right) Double labeling using reporters targeted either to the cell membrane or synaptic terminals reveals the polarity of the neurons. (See Aso et al. for more detail).
Stochastic labeling distinguishes similar cell types
Figure 3: (Top) membrane labeling. (Bottom) labeling of individual cells in difernet colors reveals that these two cells have distinguishable morphologies and thus we consider them two cell types. (See Aso et al. for more detail.)
We increasingly view the role of the FlyLight project as providing a critical bridge between the detailed wiring diagrams generated by EM-level connectomics and the physiological and behavioral experiments that are needed to understand the function of such neural networks. For example, we would like to be able to select a specific cell (or cell type) in an EM-level connectome of the fly brain and, based on matching light and EM level morphology, identify a specific GAL4 driver line for that cell type. In the central brain, we are finding that such “cell type-specific” GAL4 lines generally drive expression in less than ten, and in many cases only one, cell per brain hemisphere. Such a driver line can then be used to: (1) stochastically label single cells to determine details of morphology; (2) express a sensor for functional imaging; (3) examine the behavioral effect —either on the animal or the neural network—of activating or inactivating that cell type (or an individual protein within it); and (4) to isolate that cell type for RNAseq analysis.
Light level anatomy allows us to look at a much larger sample size than is possible with EM, providing insights into stereotypy. The individual cell morphologies determined by light microscopy also provide a useful check on the accuracy of certain aspects of EM reconstructions. We feel that a major strength of the FlyLight project is the close working relationship between the project and the labs of the steering committee members. First, most of the genetic and imaging methods used by the project were originally developed, at least to establishment of proof-of-principle, in one of these labs. Second, the production of the GAL4 and split-GAL4 lines was carried out (and funded by) these laboratories (either at Janelia or at the Institute for Molecluar Pathology in Vienna). Finally, for each of the brain areas we are studying there is an individual in one of the participating labs who is a world class expert on that region’s anatomy and who devotes significant time (generally more than 25% time) to working with FlyLight to analyze data, select lines for use in generating split-GAL4 intersections and to prepare data for dissemination (on the web and by traditional publications). In return, the project enables the work of those labs, and many others, by providing the capability to do brain dissections, histology and imaging at scale and with effective quality control.
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