Precise, repeatable genetic access to specific neurons via the GAL4/UAS system and related methods is a key advantage of Drosophila neuroscience. Neuronal targeting is typically documented using light microscopy of full GAL4 expression patterns, which mostly 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 27,000 such adult central nervous systems.An anticipated use of this resource is to bridge the gap between electron microscopyidentified neurons and light microscopy-based intersectional genetic approaches such as the split-GAL4 system. Identifying the individual neurons that make up each GAL4 expression pattern improves the prediction of which GAL4 enhancer fragments best combine via split-GAL4 to target neurons of interest. To this end we have developed the NeuronBridge search tool, which matches these light microscope neuronal images to neurons in the recently published FlyEM hemibrain. This work thus provides a resource and search tool that will significantly enhance both the efficiency and efficacy of split-GAL4 targeting of EM-identified neurons and further advance Drosophila neuroscience.Meissner, et al., 2020Gen1 MCFO Phase 1 release

The olfactory system of male moths has an extreme sensitivity with the capability to detect and recognize conspecific pheromones dispersed and greatly diluted in the air. Just 170 molecules of the silkmoth (Bombyx mori) sex pheromone bombykol are sufficient to induce sexual behavior in the male. However, it is still unclear how the sensitivity of olfactory receptor neurons (ORNs) is relayed through the brain to generate high behavioral responsiveness. Here, we show that ORN activity that is subthreshold in terms of behavior can be amplified to suprathreshold levels by temporal integration in antennal lobe projection neurons (PNs) if occurring within a specific time window. To control ORN inputs with high temporal resolution, channelrhodopsin-2 was genetically introduced into bombykol-responsive ORNs. Temporal integration in PNs was only observed for weak inputs, but not for strong inputs. Pharmacological dissection revealed that GABAergic mechanisms inhibit temporal integration of strong inputs, showing that GABA signaling regulates PN responses in a stimulus-dependent fashion. Our results show that boosting of the PNs' responses by temporal integration of olfactory information occurs specifically near the behavioral threshold, effectively defining the lower bound for behavioral responsiveness.optogenetics | pheromone orientation behavior | transgenic silkmoth | olfaction O lfaction is a key element in many aspects of animal behavior, such as foraging, oviposition, and mate recognition. In many moth species, a special class of odorants called sex pheromones plays a critical role for identification of and orientation to potential mates. Because sex pheromones emitted by females are greatly diluted and dispersed in the air, sophisticated olfactory systems to detect minute amounts of sex pheromones and processing systems to translate subtle peripheral sensory responses into appropriate behavioral responses have evolved in male moths. Theoretical calculations have shown that the detection of 170 molecules of the sex pheromone bombykol [(E,Z)-10,12-hexadecadienol] can trigger sexual behavioral responses in males of the silkmoth Bombyx mori (1). The physical and chemical mechanisms in the antennae, the sensilla, and the olfactory receptor neurons (ORNs) responsible for this remarkably high sensitivity, which can detect even a single molecule, are well understood (2, 3). However, it is unclear how a small number of spikes from a small number of ORNs is processed centrally to allow for high behavioral responsiveness.In moths, pheromone molecules are detected by specialized antennal ORNs that express particular pheromone receptor genes (4-10). The axons of ORNs convey pheromone information to the first olfactory center, the antennal lobe (AL; an analog of the vertebrate olfactory bulb). The AL is composed of a number of glomeruli where ORNs establish connections with two types of neurons: projection neurons (PNs), which relay olfactory information to higher brain regions, and local interneurons (LNs), which are involved ...

How do descending inputs from the brain control leg motor circuits to change how an animal walks? Conceptually, descending neurons are thought to function either as command-type neurons, in which a single type of descending neuron exerts a high-level control to elicit a coordinated change in motor output, or through a population coding mechanism, whereby a group of neurons, each with local effects, act in combination to elicit a global motor response. The Drosophila Moonwalker Descending Neurons (MDNs), which alter leg motor circuit dynamics so that the fly walks backwards, exemplify the command-type mechanism. Here, we identify several dozen MDN target neurons within the leg motor circuits, and show that two of them mediate distinct and highly-specific changes in leg muscle activity during backward walking: LBL40 neurons provide the hindleg power stroke during stance phase; LUL130 neurons lift the legs at the end of stance to initiate swing. Through these two effector neurons, MDN directly controls both the stance and swing phases of the backward stepping cycle. These findings suggest that command-type descending neurons can also operate through the distributed control of local motor circuits.

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.

Knowledge of one's own behavioral state---whether one is walking, grooming, or resting---is critical for contextualizing sensory cues including interpreting visual motion and tracking odor sources. Additionally, awareness of one's own posture is important to avoid initiating destabilizing or physically impossible actions. Ascending neurons (ANs), interneurons in the vertebrate spinal cord or insect ventral nerve cord (VNC) that project to the brain, may provide such high-fidelity behavioral state signals. However, little is known about what ANs encode and where they convey signals in any brain. To address this gap, we performed a large-scale functional screen of AN movement encoding, brain targeting, and motor system patterning in the adult fly, Drosophila melanogaster. Using a new library of AN sparse driver lines, we measured the functional properties of 247 genetically-identifiable ANs by performing two-photon microscopy recordings of neural activity in tethered, behaving flies. Quantitative, deep network-based neural and behavioral analyses revealed that ANs nearly exclusively encode high-level behaviors---primarily walking as well as resting and grooming---rather than low-level joint or limb movements. ANs that convey self-motion---resting, walking, and responses to gust-like puff stimuli---project to the brain's anterior ventrolateral protocerebrum (AVLP), a multimodal, integrative sensory hub, while those that encode discrete actions---eye grooming, turning, and proboscis extension---project to the brain's gnathal ganglion (GNG), a locus for action selection. The structure and polarity of AN projections within the VNC are predictive of their functional encoding and imply that ANs participate in motor computations while also relaying state signals to the brain. Illustrative of this are ANs that temporally integrate proboscis extensions over tens-of-seconds, likely through recurrent interconnectivity. Thus, in line with long-held theoretical predictions, ascending populations convey high-level behavioral state signals almost exclusively to brain regions implicated in sensory feature contextualization and action selection.

To perform most behaviors, animals must send commands from higher-order processing centers in the brain to premotor circuits that reside in ganglia distinct from the brain, such as the mammalian spinal cord or insect ventral nerve cord. How these circuits are functionally organized to generate the great diversity of animal behavior remains unclear. An important first step in unraveling the organization of premotor circuits is to identify their constituent cell types and create tools to monitor and manipulate these with high specificity to assess their function. This is possible in the tractable ventral nerve cord of the fly. To generate such a toolkit, we used a combinatorial genetic technique (split-GAL4) to create 195 sparse driver lines targeting 198 individual cell types in the ventral nerve cord. These included wing and haltere motoneurons, modulatory neurons, and interneurons. Using a combination of behavioral, developmental, and anatomical analyses, we systematically characterized the cell types targeted in our collection. Taken together, the resources and results presented here form a powerful toolkit for future investigations of neural circuits and connectivity of premotor circuits while linking them to behavioral outputs.

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Knowing one’s own behavioral state has long been theorized as critical for contextualizing dynamic sensory cues and identifying appropriate future behaviors. Ascending neurons (ANs) in the motor system that project to the brain are well positioned to provide such behavioral state signals. However, what ANs encode and where they convey these signals remains largely unknown. Here, through large-scale functional imaging in behaving animals and morphological quantification, we report the behavioral encoding and brain targeting of hundreds of genetically identifiable ANs in the adult fly, Drosophila melanogaster. We reveal that ANs encode behavioral states, specifically conveying self-motion to the anterior ventrolateral protocerebrum, an integrative sensory hub, as well as discrete actions to the gnathal ganglia, a locus for action selection. Additionally, AN projection patterns within the motor system are predictive of their encoding. Thus, ascending populations are well poised to inform distinct brain hubs of self-motion and ongoing behaviors and may provide an important substrate for computations that are required for adaptive behavior.