Long-term memory (LTM) depends on the synthesis of new proteins. Using a temperature-sensitive ribosome-inactivating toxin to acutely inhibit protein synthesis, we screened individual neurons making new proteins after olfactory associative conditioning in Drosophila. Surprisingly, LTM was impaired after inhibiting protein synthesis in two dorsal-anterior-lateral (DAL) neurons but not in the mushroom body (MB), which is considered the adult learning and memory center. Using a photoconvertible fluorescent protein KAEDE to report de novo protein synthesis, we have directly visualized cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-dependent transcriptional activation of calcium/calmodulin-dependent protein kinase II and period genes in the DAL neurons after spaced but not massed training. Memory retention was impaired by blocking neural output in DAL during retrieval but not during acquisition or consolidation. These findings suggest an extra-MB memory circuit in Drosophila: LTM consolidation (MB to DAL), storage (DAL), and retrieval (DAL to MB).

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of intermediate-term memories: anesthesia-sensitive memory, which requires the amnesiac gene, and anesthesiaresistant memory (ARM), which requires the radish gene. Here, we report that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This result suggests that 5HT and Radish protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two dorsal paired medial (DPM) neurons onto the mushroom bodies (MBs), the olfactory learning and memory center in Drosophila: (i) DPM neurons were 5HT-antibody immunopositive; (ii) temporal inhibition of 5HT synthesis or release from DPM neurons, but not from other serotonergic neurons, impaired ARM formation; (iii) knocking down the expression of d5HT1A serotonin receptors in α/β MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the Amnesiac peptide required for anesthesia-sensitive memory formation, the two DPM neurons also release 5HT acting on MB neurons for ARM formation.brain | olfaction | aversive conditioning | neurotransmitter P avlovian olfactory learning in Drosophila involves coincidence detection of a conditioned stimulus (CS), an odor, and an unconditioned stimulus (US), an electric shock (1). After one session of aversive olfactory conditioning, flies can form short-and intermediate-term memories but not long-term memory (LTM), which requires repetitive spaced training and is dependent on protein synthesis (2, 3). The intermediate-term memory has been dissected further into an anesthesia-sensitive form (ASM) that requires a gene called "amnesiac" and an anesthesia-resistant form (ARM) that requires the radish gene (4-6). Three hours after one session of training, ARM and ASM contribute equally to performance and can be distinguished by application of a short cold shock-induced anesthesia that impairs ASM but not ARM. Importantly, although ARM is consolidated, in the sense that it is resistant to cold shock, it is not thought to be a protein synthesisdependent memory because it is resistant to cycloheximide (CXM) (2). One day after repetitive spaced training, both ARM and LTM are thought to contribute to memory performance. In contrast, repetitive massed training without rest intervals induces only radish-dependent ARM without detectable cAMP response element binding protein-dependent LTM measured 1 d after training (2, 3) (but see ref.2 for an alternative model). Thus, more than one genetically distinct memory-storage system contributes to performance both at intermediate time points (e.g., 3 h after one training session) and at later time points (e.g., 24 h after repetitive training). Given the complexity of memory consolidation observed at the genetic level, much effor...

After Drosophila males are rejected by mated females, their subsequent courtship is inhibited even when encountering virgin females. Molecular mechanisms underlying courtship conditioning in the CNS are unclear. In this study, we find that tyramine ␤ hydroxylase (T␤H) mutant males unable to synthesize octopamine (OA) showed impaired courtship conditioning, which could be rescued by transgenic T␤H expression in the CNS. Inactivation of octopaminergic neurons mimicked the T␤H mutant phenotype. Transient activation of octopaminergic neurons in males not only decreased their courtship of virgin females, but also produced courtship conditioning. Single cell analysis revealed projection of octopaminergic neurons to the mushroom bodies. Deletion of the OAMB gene encoding an OA receptor expressed in the mushroom bodies disrupted courtship conditioning. Inactivation of neurons expressing OAMB also eliminated courtship conditioning. OAMB neurons responded robustly to male-specific pheromone cis-vaccenyl acetate in a dose-dependent manner. Our results indicate that OA plays an important role in courtship conditioning through its OAMB receptor expressed in a specific neuronal subset of the mushroom bodies.

Mapping the connectome, a wiring diagram of the entire brain, requires large-scale imaging of numerous single neurons with diverse morphology. It is a formidable challenge to reassemble these neurons into a virtual brain and correlate their structural networks with neuronal activities, which are measured in different experiments to analyze the informational flow in the brain. Here, we report an in situ brain imaging technique called Fly Head Array Slice Tomography (FHAST), which permits the reconstruction of structural and functional data to generate an integrative connectome in Drosophila. Using FHAST, the head capsules of an array of flies can be opened with a single vibratome sectioning to expose the brains, replacing the painstaking and inconsistent brain dissection process. FHAST can reveal in situ brain neuroanatomy with minimal distortion to neuronal morphology and maintain intact neuronal connections to peripheral sensory organs. Most importantly, it enables the automated 3D imaging of 100 intact fly brains in each experiment. The established head model with in situ brain neuroanatomy allows functional data to be accurately registered and associated with 3D images of single neurons. These integrative data can then be shared, searched, visualized, and analyzed for understanding how brain-wide activities in different neurons within the same circuit function together to control complex behaviors.