Transgenic manipulation of subsets of brain cells is increasingly used for studying behaviors and their underlying neural circuits. In Drosophila, the GAL4-upstream activating sequence (UAS) binary system is powerful for gene manipulation, but GAL4 expression is often too broad for fine mapping of neural circuits. Here, we describe the development of unique molecular genetic tools to restrict GAL4 expression patterns. Building on the GAL4-UAS system, our method adds two components: a collection of enhancertrap recombinase, Flippase (ET-FLP), transgenic lines that provide inheritable, reproducible, and tissue-specific FLP and an FRT-dependent GAL80 "flip-in" construct that converts FLP expression into tissue-specific repression of GAL4 by GAL80. By including a UASencoded fluorescent protein, circuit morphology can be simultaneously marked while the circuit function is assessed using another UAS transgene. In a proof-of-principle analysis, we applied this ET-FLP-induced intersectional GAL80/GAL4 repression (FINGR) method to map the neural circuitry underlying fly wing inflation. The FINGR system is versatile and powerful in combination with the vast collection of GAL4 lines for neural circuit mapping as well as for clonal analysis based on the infusion of the yeast-derived FRT/FLP system of mitotic recombination into Drosophila. The strategies and tactics underlying our FINGR system are also applicable to other genetically amenable organisms in which transgenes including the GAL4, UAS, GAL80, and FLP factors can be applied.U nderstanding the neural substrates underlying behavior remains a major goal in neuroscience. One effective approach is altering behavioral outputs in animals through genetic manipulation of neurons or neural circuits. This approach is exemplified by numerous studies carried out in mice, zebrafish, Caenorhabditis elegans, and Drosophila (1). In Drosophila, extensive studies of learning, memory, circadian rhythm, courtship, wing inflation, and climbing have provided valuable insights into the neural substrates of these behaviors (2-8). These fly activities are easily observed, are amenable to genetic perturbations, and can be quantitatively analyzed to correlate manipulations of circuits with changes in behavior.Manipulating neural structures underlying these behaviors has principally depended on imaginative designs and applications of molecular-genetic entities. The yeast-derived GAL4-upstream activating sequence (UAS) binary transgene expression system laid the foundation for many of these manipulations (reviewed in ref. 9). In this system, GAL4 can be expressed in a tissue-specific manner via either enhancer-traps or gene-specific promoters. Wherever GAL4 is made active, a protein-coding sequence under the control of UAS is also expressed (10, 11) (Fig. 1A). Thousands of tissue-specific GAL4 enhancer-trap lines have been created.Despite the enormous diversity of available tissue-specific lines, GAL4 expression patterns are rarely restrictive enough to map key elements of neural circuitry. Hen...

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UASShi ts , regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tub P

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