The fruit fly can evaluate its energy state and decide whether to pursue food-related cues. Here, we reveal that the mushroom body (MB) integrates hunger and satiety signals to control food-seeking behavior. We have discovered five pathways in the MB essential for hungry flies to locate and approach food. Blocking the MB-intrinsic Kenyon cells (KCs) and the MB output neurons (MBONs) in these pathways impairs food-seeking behavior. Starvation bi-directionally modulates MBON responses to a food odor, suggesting that hunger and satiety controls occur at the KC-to-MBON synapses. These controls are mediated by six types of dopaminergic neurons (DANs). By manipulating these DANs, we could inhibit food-seeking behavior in hungry flies or promote food seeking in fed flies. Finally, we show that the DANs potentially receive multiple inputs of hunger and satiety signals. This work demonstrates an information-rich central circuit in the fly brain that controls hunger-driven food-seeking behavior.

Significance The adult Drosophila mushroom body (MB) is one of the most extensively studied neural circuits. However, how its circuit organization is established during development is unclear. In this study, we provide an initial characterization of the assembly process of the extrinsic neurons (dopaminergic neurons and MB output neurons) that target the vertical MB lobes. We probe the cellular mechanisms guiding the neurite targeting of these extrinsic neurons and demonstrate that Semaphorin 1a is required in several MB output neurons for their dendritic innervations to three specific MB lobe zones. Our study reveals several intriguing molecular and cellular principles governing assembly of the MB circuit.

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14The Drosophila mushroom body (MB) is a learning and memory center in the fly brain. It is the most 15 extensively studied brain structure in insects, but we know little about the molecular and cellular 16 mechanisms underlying assembly of its neural circuit. The MB is composed of around 2200 intrinsic 17 Kenyon cells (KCs), whose axons are bundled to form multiple MB lobes. The MB lobes are 18 innervated by a large number of extrinsic neurons. Twenty types of dopaminergic neurons (DANs) 19 and 21 types of MB output neurons (MBONs) have been identified. Each type of these extrinsic 20 neurons innervates specific compartments or zones in the MB lobes. Here, we characterize the 21 assembly of the MB circuit and reveal several intriguing features of the process. The DANs and 22 31 to those zones, demonstrating a potential to rewire the MB circuit. Taken together, our work 32 provides an initial characterization of the cellular and molecular mechanisms underlying MB circuit 33 assembly. 34 35 127 128 129 DANs and MBONs independently innervate MB zones 130 131Given that the axons of PPL1-αʹ2α2 DANs precede the MBON-αʹ2 dendrites in the αʹ2 zone, next we 132 examined the possibility that DAN axons provide guidance cues for MBON dendrites. We genetically 133 ablated PPL1-αʹ2α2 DANs by ectopically expressing the apoptotic genes head involution defective 134 (hid) and reaper (rpr) (Grether et al., 1995; White et al., 1994) in these neurons using MB058B-135 splitGAL4. No MB058B-positive cells were seen in PPL1-αʹ2α2 DAN-ablated adult brains, and the αʹ2 136 and α2 MB lobe zones lacked anti-TH staining signal ( Fig. 2A-A' and B-B'). Ablation of PPL1-αʹ2α2 137 DANs occurred quite early during development and all MB058B-positive neurites were eliminated 138 before 24 h APF, i.e., by the time PPL1-αʹ2α2 DANs start to innervate MB lobes in wild-type brains 139 ( Fig. S3). TH-negative zones in the PPL1-αʹ2α2 DAN-ablated brains suggest that axon-axon contacts 140 between neighboring DANs have a minimal effect in determining the borders of DAN axon zonal 141 networks in MB lobes. Ablation of PPL1-αʹ2α2 DANs did not affect the dendritic targeting of MBON-142 αʹ2 (labeled with R20G03-LexA::P65-driven rCD2-RFP), suggesting that PPL1-αʹ2α2 DANs do not