Identified neuron classes in vertebrate cortical [1-4] and subcortical [5-8] areas and invertebrate peripheral [9-11] and central [12-14] brain neuropils encode specific visual features of a panorama. How downstream neurons integrate these features to control vital behaviors, like escape, is unclear [15]. In Drosophila, the timing of a single spike in the giant fiber (GF) descending neuron [16-18] determines whether a fly uses a short or long takeoff when escaping a looming predator [13]. We previously proposed that GF spike timing results from summation of two visual features whose detection is highly conserved across animals [19]: an object's subtended angular size and its angular velocity [5-8, 11, 20, 21]. We attributed velocity encoding to input from lobula columnar type 4 (LC4) visual projection neurons, but the size-encoding source remained unknown. Here, we show that lobula plate/lobula columnar, type 2 (LPLC2) visual projection neurons anatomically specialized to detect looming [22] provide the entire GF size component. We find LPLC2 neurons to be necessary for GF-mediated escape and show that LPLC2 and LC4 synapse directly onto the GF via reconstruction in a fly brain electron microscopy (EM) volume [23]. LPLC2 silencing eliminates the size component of the GF looming response in patch-clamp recordings, leaving only the velocity component. A model summing a linear function of angular velocity (provided by LC4) and a Gaussian function of angular size (provided by LPLC2) replicates GF looming response dynamics and predicts the peak response time. We thus present an identified circuit in which information from looming feature-detecting neurons is combined by a common post-synaptic target to determine behavioral output.

In the natural world, animals make decisions on an ongoing basis, continuously selecting which action to undertake next. In the lab, however, the neural bases of decision processes have mostly been studied using artificial trial structures. New experimental tools based on the genetic toolkit of model organisms now make it experimentally feasible to monitor and manipulate neural activity in small subsets of neurons during naturalistic behaviors. We thus propose a new approach to investigating decision processes, termed reverse neuroethology. In this approach, experimenters select animal models based on experimental accessibility and then utilize cutting-edge tools such as connectomes and genetically encoded reagents to analyze the flow of information through an animal's nervous system during naturalistic choice behaviors. We describe how the reverse neuroethology strategy has been applied to understand the neural underpinnings of innate, rapid decision making, with a focus on defensive behavioral choices in the vinegar fly Drosophila melanogaster.

In most animals, the brain makes behavioral decisions that are transmitted by descending neurons to the nerve cord circuitry that produces behaviors. In insects, only a few descending neurons have been associated with specific behaviors. To explore how descending neurons control an insect's movements, we developed a novel method to systematically assay the behavioral effects of activating individual neurons on freely behaving terrestrial D. melanogaster. We calculated a two-dimensional representation of the entire behavior space explored by these flies and we associated descending neurons with specific behaviors by identifying regions of this space that were visited with increased frequency during optogenetic activation. Applying this approach across a large collection of descending neurons, we found that (1) activation of most of the descending neurons drove stereotyped behaviors, (2) in many cases multiple descending neurons activated similar behaviors, and (3) optogenetically-activated behaviors were often dependent on the behavioral state prior to activation.

Natural behaviors are a coordinated symphony of motor acts which drive self-induced or reafferent sensory activation. Single sensors only signal presence and magnitude of a sensory cue; they cannot disambiguate exafferent (externally-induced) from reafferent sources. Nevertheless, animals readily differentiate between these sources of sensory signals to make appropriate decisions and initiate adaptive behavioral outcomes. This is mediated by predictive motor signaling mechanisms, which emanate from motor control pathways to sensory processing pathways, but how predictive motor signaling circuits function at the cellular and synaptic level is poorly understood. We use a variety of techniques, including connectomics from both male and female electron microscopy volumes, transcriptomics, neuroanatomical, physiological and behavioral approaches to resolve the network architecture of two pairs of ascending histaminergic neurons (AHNs), which putatively provide predictive motor signals to several sensory and motor neuropil. Both AHN pairs receive input primarily from an overlapping population of descending neurons, many of which drive wing motor output. The two AHN pairs target almost exclusively non-overlapping downstream neural networks including those that process visual, auditory and mechanosensory information as well as networks coordinating wing, haltere, and leg motor output. These results support the conclusion that the AHN pairs multi-task, integrating a large amount of common input, then tile their output in the brain, providing predictive motor signals to non-overlapping sensory networks affecting motor control both directly and indirectly.

Natural behaviors are a coordinated symphony of motor acts that drive reafferent (self-induced) sensory activation. Individual sensors cannot disambiguate exafferent (externally induced) from reafferent sources. Nevertheless, animals readily differentiate between these sources of sensory signals to carry out adaptive behaviors through corollary discharge circuits (CDCs), which provide predictive motor signals from motor pathways to sensory processing and other motor pathways. Yet, how CDCs comprehensively integrate into the nervous system remains unexplored. Here, we use connectomics, neuroanatomical, physiological, and behavioral approaches to resolve the network architecture of two pairs of ascending histaminergic neurons (AHNs) in Drosophila, which function as a predictive CDC in other insects. Both AHN pairs receive input primarily from a partially overlapping population of descending neurons, especially from DNg02, which controls wing motor output. Using Ca imaging and behavioral recordings, we show that AHN activation is correlated to flight behavior and precedes wing motion. Optogenetic activation of DNg02 is sufficient to activate AHNs, indicating that AHNs are activated by descending commands in advance of behavior and not as a consequence of sensory input. Downstream, each AHN pair targets predominantly non-overlapping networks, including those that process visual, auditory, and mechanosensory information, as well as networks controlling wing, haltere, and leg sensorimotor control. These results support the conclusion that the AHNs provide a predictive motor signal about wing motor state to mostly non-overlapping sensory and motor networks. Future work will determine how AHN signaling is driven by other descending neurons and interpreted by AHN downstream targets to maintain adaptive sensorimotor performance.

The fruit fly Drosophila melanogaster performs at least two distinct types of flight initiation. One kind is a stereotyped escape response to a visual stimulus that is mediated by the hard-wired giant fiber neural pathway, and the other is a more variable ;voluntary’ response that can be performed without giant fiber activation. Because the simpler escape take-offs are apparently successful, it is unclear why the fly has multiple pathways to coordinate flight initiation. In this study we use high-speed videography to observe flight initiation in unrestrained wild-type flies and assess the flight performance of each of the two types of take-off. Three-dimensional kinematic analysis of take-off sequences indicates that wing use during the jumping phase of flight initiation is essential for stabilizing flight. During voluntary take-offs, early wing elevation leads to a slower and more stable take-off. In contrast, during visually elicited escapes, the wings are pulled down close to the body during take-off, resulting in tumbling flights in which the fly translates faster but also rotates rapidly about all three of its body axes. Additionally, we find evidence that the power delivered by the legs is substantially greater during visually elicited escapes than during voluntary take-offs. Thus, we find that the two types of Drosophila flight initiation result in different flight performances once the fly is airborne, and that these performances are distinguished by a trade-off between speed and stability.

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.

The most fundamental choice an animal has to make when it detects a threat is whether to freeze, reducing its chances of being noticed, or to flee to safety. Here we show that Drosophila melanogaster exposed to looming stimuli in a confined arena either freeze or flee. The probability of freezing versus fleeing is modulated by the fly's walking speed at the time of threat, demonstrating that freeze/flee decisions depend on behavioral state. We describe a pair of descending neurons crucially implicated in freezing. Genetic silencing of DNp09 descending neurons disrupts freezing yet does not prevent fleeing. Optogenetic activation of both DNp09 neurons induces running and freezing in a state-dependent manner. Our findings establish walking speed as a key factor in defensive response choices and reveal a pair of descending neurons as a critical component in the circuitry mediating selection and execution of freezing or fleeing behaviors.

An approaching predator and self-motion toward an object can generate similar looming patterns on the retina, but these situations demand different rapid responses. How central circuits flexibly process visual cues to activate appropriate, fast motor pathways remains unclear. Here we identify two descending neuron (DN) types that control landing and contribute to visuomotor flexibility in Drosophila. For each, silencing impairs visually evoked landing, activation drives landing, and spike rate determines leg extension amplitude. Critically, visual responses of both DNs are severely attenuated during non-flight periods, effectively decoupling visual stimuli from the landing motor pathway when landing is inappropriate. The flight-dependence mechanism differs between DN types. Octopamine exposure mimics flight effects in one, whereas the other probably receives neuronal feedback from flight motor circuits. Thus, this sensorimotor flexibility arises from distinct mechanisms for gating action-specific descending pathways, such that sensory and motor networks are coupled or decoupled according to the behavioral state.

To survive, animals must convert sensory information into appropriate behaviours. Vision is a common sense for locating ethologically relevant stimuli and guiding motor responses. How circuitry converts object location in retinal coordinates to movement direction in body coordinates remains largely unknown. Here we show through behaviour, physiology, anatomy and connectomics in Drosophila that visuomotor transformation occurs by conversion of topographic maps formed by the dendrites of feature-detecting visual projection neurons (VPNs) into synaptic weight gradients of VPN outputs onto central brain neurons. We demonstrate how this gradient motif transforms the anteroposterior location of a visual looming stimulus into the fly's directional escape. Specifically, we discover that two neurons postsynaptic to a looming-responsive VPN type promote opposite takeoff directions. Opposite synaptic weight gradients onto these neurons from looming VPNs in different visual field regions convert localized looming threats into correctly oriented escapes. For a second looming-responsive VPN type, we demonstrate graded responses along the dorsoventral axis. We show that this synaptic gradient motif generalizes across all 20 primary VPN cell types and most often arises without VPN axon topography. Synaptic gradients may thus be a general mechanism for conveying spatial features of sensory information into directed motor outputs.