Flying insects exhibit remarkable navigational abilities controlled by their compact nervous systems. Optic flow, the pattern of changes in the visual scene induced by locomotion, is a crucial sensory cue for robust self-motion estimation, especially during rapid flight. Neurons that respond to specific, large-field optic flow patterns have been studied for decades, primarily in large flies, such as houseflies, blowflies, and hover flies. The best-known optic-flow sensitive neurons are the large tangential cells of the dipteran lobula plate, whose visual-motion responses, and to a lesser extent, their morphology, have been explored using single-neuron neurophysiology. Most of these studies have focused on the large, Horizontal and Vertical System neurons, yet the lobula plate houses a much larger set of 'optic-flow' sensitive neurons, many of which have been challenging to unambiguously identify or to reliably target for functional studies. Here we report the comprehensive reconstruction and identification of the Lobula Plate Tangential Neurons in an Electron Microscopy (EM) volume of a whole Drosophila brain. This catalog of 58 LPT neurons (per brain hemisphere) contains many neurons that are described here for the first time and provides a basis for systematic investigation of the circuitry linking self-motion to locomotion control. Leveraging computational anatomy methods, we estimated the visual motion receptive fields of these neurons and compared their tuning to the visual consequence of body rotations and translational movements. We also matched these neurons, in most cases on a one-for-one basis, to stochastically labeled cells in genetic driver lines, to the mirror-symmetric neurons in the same EM brain volume, and to neurons in an additional EM data set. Using cell matches across data sets, we analyzed the integration of optic flow patterns by neurons downstream of the LPTs and find that most central brain neurons establish sharper selectivity for global optic flow patterns than their input neurons. Furthermore, we found that self-motion information extracted from optic flow is processed in distinct regions of the central brain, pointing to diverse foci for the generation of visual behaviors.

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.

Similar to many insect species, Drosophila melanogaster is capable of maintaining a stable flight trajectory for periods lasting up to several hours. Because aerodynamic torque is roughly proportional to the fifth power of wing length, even small asymmetries in wing size require the maintenance of subtle bilateral differences in flapping motion to maintain a stable path. Flies can even fly straight after losing half of a wing, a feat they accomplish via very large, sustained kinematic changes to both the damaged and intact wings. Thus, the neural network responsible for stable flight must be capable of sustaining fine-scaled control over wing motion across a large dynamic range. In this study, we describe an unusual type of descending neuron (DNg02) that projects directly from visual output regions of the brain to the dorsal flight neuropil of the ventral nerve cord. Unlike many descending neurons, which exist as single bilateral pairs with unique morphology, there is a population of at least 15 DNg02 cell pairs with nearly identical shape. By optogenetically activating different numbers of DNg02 cells, we demonstrate that these neurons regulate wingbeat amplitude over a wide dynamic range via a population code. Using two-photon functional imaging, we show that DNg02 cells are responsive to visual motion during flight in a manner that would make them well suited to continuously regulate bilateral changes in wing kinematics. Collectively, we have identified a critical set of descending neurons that provides the sensitivity and dynamic range required for flight control.

Precise, repeatable genetic access to specific neurons via GAL4/UAS and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which generally lack the single-cell resolution required for reliable cell type identification. Here we use stochastic GAL4 labeling with the MultiColor FlpOut approach to generate cellular resolution confocal images at large scale. We are releasing aligned images of 74,000 such adult central nervous systems. An anticipated use of this resource is to bridge the gap between neurons identified by electron or light microscopy. Identifying individual neurons that make up each GAL4 expression pattern improves the prediction of split-GAL4 combinations targeting particular neurons. To this end we have made the images searchable on the NeuronBridge website. We demonstrate the potential of NeuronBridge to rapidly and effectively identify neuron matches based on morphology across imaging modalities and datasets.

Aggressive social interactions are used to compete for limited resources and are regulated by complex sensory cues and the organism's internal state. While both sexes exhibit aggression, its neuronal underpinnings are understudied in females. Here, we identify a population of sexually dimorphic aIPg neurons in the adult central brain whose optogenetic activation increased, and genetic inactivation reduced, female aggression. Analysis of GAL4 lines identified in an unbiased screen for increased female chasing behavior revealed the involvement of another sexually dimorphic neuron, pC1d, and implicated aIPg and pC1d neurons as core nodes regulating female aggression. Connectomic analysis demonstrated that aIPg neurons and pC1d are interconnected and suggest that aIPg neurons may exert part of their effect by gating the flow of visual information to descending neurons. Our work reveals important regulatory components of the neuronal circuitry that underlies female aggressive social interactions and provides tools for their manipulation.

The mating decisions of Drosophila melanogaster females are primarily revealed through either of two discrete actions: opening of the vaginal plates to allow copulation, or extrusion of the ovipositor to reject the male. Both actions are triggered by the male courtship song, and both are dependent upon the female's mating status. Virgin females are more likely to open their vaginal plates in response to song; mated females are more likely to extrude their ovipositor. Here, we examine the neural cause and behavioral consequence of ovipositor extrusion. We show that the DNp13 descending neurons act as command-type neurons for ovipositor extrusion, and that ovipositor extrusion is an effective deterrent only when performed by females that have previously mated. The DNp13 neurons respond to male song via direct synaptic input from the pC2l auditory neurons. Mating status does not modulate the song responses of DNp13 neurons, but rather how effectively they can engage the motor circuits for ovipositor extrusion. We present evidence that mating status information is mediated by ppk sensory neurons in the uterus, which are activated upon ovulation. Vaginal plate opening and ovipositor extrusion are thus controlled by anatomically and functionally distinct circuits, highlighting the diversity of neural decision-making circuits even in the context of closely related behaviors with shared exteroceptive and interoceptive inputs.

Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways – one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these “when” and “what” descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

Understanding the circuit mechanisms behind motion detection is a long-standing question in visual neuroscience. In , recent synapse-level connectomes in the optic lobe, particularly in ON-pathway (T4) receptive-field circuits, in concert with physiological studies, suggest an increasingly intricate motion model compared with the ubiquitous Hassenstein-Reichardt model, while our knowledge of OFF-pathway (T5) has been incomplete. Here we present a conclusive and comprehensive connectome that for the first time integrates detailed connectivity information for inputs to both T4 and T5 pathways in a single EM dataset covering the entire optic lobe. With novel reconstruction methods using automated synapse prediction suited to such a large connectome, we successfully corroborate previous findings in the T4 pathway and comprehensively identify inputs and receptive fields for T5. While the two pathways are likely evolutionarily linked and indeed exhibit many similarities, we uncover interesting differences and interactions that may underlie their distinct functional properties.

Inside the cell, proteins essential for signaling, morphogenesis, and migration navigate complex pathways, typically via vesicular trafficking or microtubule-driven mechanisms 1-3. However, the process by which soluble cytoskeletal monomers maneuver through the cytoplasm’s ever-changing environment to reach their destinations without using these pathways remains unknown. 4-6 Here, we show that actin cytoskeletal treadmilling leads to the formation of a semi-permeable actin-myosin barrier, creating a specialized compartment separated from the rest of the cell body that directs proteins toward the cell edge by advection, diffusion facilitated by fluid flow. Contraction at this barrier generates a molecularly non-specific fluid flow that transports actin, actin-binding proteins, adhesion proteins, and even inert proteins forward. The local curvature of the barrier specifically targets these proteins toward protruding edges of the leading edge, sites of new filament growth, effectively coordinating protein distribution with cellular dynamics. Outside this compartment, diffusion remains the primary mode of protein transport, contrasting sharply with the directed advection within. This discovery reveals a novel protein transport mechanism that redefines the front of the cell as a pseudo-organelle, actively orchestrating protein mobilization for cellular front activities such as protrusion and adhesion. By elucidating a new model of protein dynamics at the cellular front, this work contributes a critical piece to the puzzle of how cells adapt their internal structures for targeted and rapid response to extracellular cues. The findings challenge the current understanding of intracellular transport, suggesting that cells possess highly specialized and previously unrecognized organizational strategies for managing protein distribution efficiently, providing a new framework for understanding the cellular architecture’s role in rapid response and adaptation to environmental changes.

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of “glitter bomb” labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho-carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.