Dopamine (DA) is synonymous with reward and motivation in mammals1,2. However, only recently has dopamine been linked to motivated behavior and rewarding reinforcement in fruit flies3,4. Instead octopamine (OA) has historically been considered the signal for reward in insects5–7. Here we show using temporal control of neural function in Drosophila that only short-term appetitive memory is reinforced by OA. Moreover, OA-dependent memory formation requires signaling through DA neurons. Part of the OA signal requires the α-adrenergic like OAMB receptor in an identified subset of mushroom body (MB)-targeted DA neurons. OA triggers an increase in intracellular calcium in these DA neurons and their direct activation can substitute for sugar to form appetitive memory, even in flies lacking OA. Analysis of the β-adrenergic like Octβ2R receptor reveals that OA-dependent reinforcement also requires an interaction with DA neurons that control appetitive motivation. These data suggest that sweet taste engages a distributed OA signal that reinforces memory through discrete subsets of MB-targeted DA neurons. In addition, they reconcile prior findings with OA and DA and suggest that reinforcement systems in flies are more similar to mammals than previously envisaged.

The function of a complex nervous system depends on an intricate interplay between neuronal and glial cell types. One of the many functions of glial cells is to provide an efficient insulation of the nervous system and thereby allowing a fine tuned homeostasis of ions and other small molecules. Here, we present a detailed cellular analysis of the glial cell complement constituting the blood-brain barrier in Drosophila. Using electron microscopic analysis and single cell-labeling experiments, we characterize different glial cell layers at the surface of the nervous system, the perineurial glial layer, the subperineurial glial layer, the wrapping glial cell layer, and a thick layer of extracellular matrix, the neural lamella. To test the functional roles of these sheaths we performed a series of dye penetration experiments in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants allowed to conclude that most of the barrier function is mediated by the septate junctions formed by the subperineurial cells, whereas the perineurial glial cell layer and the neural lamella contribute to barrier selectivity against much larger particles (i.e., the size of proteins). We further compare the requirements of different septate junction components for the integrity of the blood-brain barrier and provide evidence that two of the six Claudin-like proteins found in Drosophila are needed for normal bloodbrain barrier function.

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Tissue-specific gene expression using the UAS/GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we have developed a convertible genetic platform, called the Integrase Swappable In vivo Targeting Element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient, in vivo manipulations, making it possible to generate many swaps in parallel. Furthermore, this system is entirely modular, allowing future genetic tools to be easily incorporated into the existing framework.

SUMMARY In the visual system, peripheral processing circuits are often tuned to specific stimulus features. How this selectivity arises and how these circuits are organized to inform specific visual behaviors is incompletely understood. Using forward genetics and quantitative behavioral studies, we uncover a new input channel to motion detecting circuitry in Drosophila. The second order neuron L3 acts combinatorially with two previously known inputs, L1 and L2, to inform circuits specialized to detect moving light and dark edges. In vivo calcium imaging of L3, combined with neuronal silencing experiments, suggests a neural mechanism to achieve selectivity for moving dark edges. We further demonstrate that different innate behaviors, turning and forward movement, can be independently modulated by visual motion. These two behaviors make use of different combinations of input channels. Such modular use of input channels to achieve feature extraction and behavioral specialization likely represents a general principle in sensory systems.