Animals retain some but not all experiences in long-term memory (LTM). Sleep supports LTM retention across animal species. It is well established that learning experiences enhance post-learning sleep. However, the underlying mechanisms of how learning mediates sleep for memory retention are not clear. Drosophila males display increased amounts of sleep after courtship learning. Courtship learning depends on Mushroom Body (MB) neurons, and post-learning sleep is mediated by the sleep-promoting ventral Fan-Shaped Body neurons (vFBs). We show that post-learning sleep is regulated by two opposing output neurons (MBONs) from the MB, which encode a measure of learning. Excitatory MBONs-γ2α'1 becomes increasingly active upon increasing time of learning, whereas inhibitory MBONs-β'2mp is activated only by a short learning experience. These MB outputs are integrated by SFS neurons, which excite vFBs to promote sleep after prolonged but not short training. This circuit may ensure that only longer or more intense learning experiences induce sleep and are thereby consolidated into LTM.

Color and polarization provide complementary information about the world and are detected by specialized photoreceptors. However, the downstream neural circuits that process these distinct modalities are incompletely understood in any animal. Using electron microscopy, we have systematically reconstructed the synaptic targets of the photoreceptors specialized to detect color and skylight polarization in Drosophila, and we have used light microscopy to confirm many of our findings. We identified known and novel downstream targets that are selective for different wavelengths or polarized light, and followed their projections to other areas in the optic lobes and the central brain. Our results revealed many synapses along the photoreceptor axons between brain regions, new pathways in the optic lobes, and spatially segregated projections to central brain regions. Strikingly, photoreceptors in the polarization-sensitive dorsal rim area target fewer cell types, and lack strong connections to the lobula, a neuropil involved in color processing. Our reconstruction identifies shared wiring and modality-specific specializations for color and polarization vision, and provides a comprehensive view of the first steps of the pathways processing color and polarized light inputs.

Choosing a mate is one of the most consequential decisions a female will make during her lifetime. A female fly signals her willingness to mate by opening her vaginal plates, allowing a courting male to copulate. Vaginal plate opening (VPO) occurs in response to the male courtship song and is dependent on the mating status of the female. How these exteroceptive (song) and interoceptive (mating status) inputs are integrated to regulate VPO remains unknown. Here we characterize the neural circuitry that implements mating decisions in the brain of female Drosophila melanogaster. We show that VPO is controlled by a pair of female-specific descending neurons (vpoDNs). The vpoDNs receive excitatory input from auditory neurons (vpoENs), which are tuned to specific features of the D. melanogaster song, and from pC1 neurons, which encode the mating status of the female. The song responses of vpoDNs, but not vpoENs, are attenuated upon mating, accounting for the reduced receptivity of mated females. This modulation is mediated by pC1 neurons. The vpoDNs thus directly integrate the external and internal signals that control the mating decisions of Drosophila females.

Mating and egg laying are tightly cooordinated events in the reproductive life of all oviparous females. Oviposition is typically rare in virgin females but is initiated after copulation. Here we identify the neural circuitry that links egg laying to mating status in Drosophila melanogaster. Activation of female-specific oviposition descending neurons (oviDNs) is necessary and sufficient for egg laying, and is equally potent in virgin and mated females. After mating, sex peptide-a protein from the male seminal fluid-triggers many behavioural and physiological changes in the female, including the onset of egg laying. Sex peptide is detected by sensory neurons in the uterus, and silences these neurons and their postsynaptic ascending neurons in the abdominal ganglion. We show that these abdominal ganglion neurons directly activate the female-specific pC1 neurons. GABAergic (γ-aminobutyric-acid-releasing) oviposition inhibitory neurons (oviINs) mediate feed-forward inhibition from pC1 neurons to both oviDNs and their major excitatory input, the oviposition excitatory neurons (oviENs). By attenuating the abdominal ganglion inputs to pC1 neurons and oviINs, sex peptide disinhibits oviDNs to enable egg laying after mating. This circuitry thus coordinates the two key events in female reproduction: mating and egg laying.

Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. Here, we show that consolidation of courtship long-term memory in is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. We identify specific fan-shaped body neurons that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, we provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation.

The behavioral state of an animal can dynamically modulate visual processing. In flies, the behavioral state is known to alter the temporal tuning of neurons that carry visual motion information into the central brain. However, where this modulation occurs and how it tunes the properties of this neural circuit are not well understood. Here, we show that the behavioral state alters the baseline activity levels and the temporal tuning of the first directionally selective neuron in the ON motion pathway (T4) as well as its primary input neurons (Mi1, Tm3, Mi4, Mi9). These effects are especially prominent in the inhibitory neuron Mi4, and we show that central octopaminergic neurons provide input to Mi4 and increase its excitability. We further show that octopamine neurons are required for sustained behavioral responses to fast-moving, but not slow-moving, visual stimuli in walking flies. These results indicate that behavioral-state modulation acts directly on the inputs to the directionally selective neurons and supports efficient neural coding of motion stimuli.

Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. We reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB’s α lobe, using a dataset of isotropic 8-nm voxels collected by focused ion-beam milling scanning electron microscopy. We found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only six percent of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). We identified two unanticipated classes of synapses, KC>DAN and DAN>MBON. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall.

We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).

The perception of visual motion is critical for animal navigation, and flies are a prominent model system for exploring this neural computation. In Drosophila, the T4 cells of the medulla are directionally selective and necessary for ON motion behavioral responses. To examine the emergence of directional selectivity, we developed genetic driver lines for the neuron types with the most synapses onto T4 cells. Using calcium imaging, we found that these neuron types are not directionally selective and that selectivity arises in the T4 dendrites. By silencing each input neuron type, we identified which neurons are necessary for T4 directional selectivity and ON motion behavioral responses. We then determined the sign of the connections between these neurons and T4 cells using neuronal photoactivation. Our results indicate a computational architecture for motion detection that is a hybrid of classic theoretical models.

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons—the TN1A neurons—that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. We find that doublesex is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.

•Doublesex-expressing TN1 neurons are necessary and sufficient for the male sine song•A subclass of TN1 neurons, TN1A, contributes to the sine song•TN1A neurons are functionally coupled to a sine song motoneuron, hg1•Doublesex regulates the connectivity between the TN1A and hg1 neurons

It is unclear how developmental regulatory genes specify sex-specific behaviors. Shirangi et al. demonstrate that the Drosophila sexual differentiation gene doublesex encodes a sex-specific behavior—male song—by promoting the connectivity between the male-specific TN1A neurons and the sex-non-specific hg1 neurons, which are required for production of the song.