Animals use sensory information to move toward more favorable conditions. Drosophila larvae can move up or down gradients of odors (chemotax), light (phototax), and temperature (thermotax) by modulating the probability, direction, and size of turns based on sensory input. Whether larvae can anemotax in gradients of mechanosensory cues is unknown. Further, although many of the sensory neurons that mediate taxis have been described, the central circuits are not well understood. Here, we used high-throughput, quantitative behavioral assays to demonstrate Drosophila larvae anemotax in gradients of wind speeds and to characterize the behavioral strategies involved. We found that larvae modulate the probability, direction, and size of turns to move away from higher wind speeds. This suggests that similar central decision-making mechanisms underlie taxis in somatosensory and other sensory modalities. By silencing the activity of single or very few neuron types in a behavioral screen, we found two sensory (chordotonal and multidendritic class III) and six nerve cord neuron types involved in anemotaxis. We reconstructed the identified neurons in an electron microscopy volume that spans the entire larval nervous system and found they received direct input from the mechanosensory neurons or from each other. In this way, we identified local interneurons and first- and second-order subesophageal zone (SEZ) and brain projection neurons. Finally, silencing a dopaminergic brain neuron type impairs anemotaxis. These findings suggest that anemotaxis involves both nerve cord and brain circuits. The candidate neurons and circuitry identified in our study provide a basis for future detailed mechanistic understanding of the circuit principles of anemotaxis.

Serial electron microscopic analysis shows that the Drosophila brain at hatching possesses a large fraction of developmentally arrested neurons with a small soma, heterochromatin-rich nucleus, and unbranched axon lacking synapses. We digitally reconstructed all 802 "small undifferentiated" (SU) neurons and assigned them to the known brain lineages. By establishing the coordinates and reconstructing trajectories of the SU neuron tracts, we provide a framework of landmarks for the ongoing analyses of the L1 brain circuitry. To address the later fate of SU neurons, we focused on the 54 SU neurons belonging to the DM1-DM4 lineages, which generate all columnar neurons of the central complex. Regarding their topologically ordered projection pattern, these neurons form an embryonic nucleus of the fan-shaped body ("FB pioneers"). Fan-shaped body pioneers survive into the adult stage, where they develop into a specific class of bi-columnar elements, the pontine neurons. Later born, unicolumnar DM1-DM4 neurons fasciculate with the fan-shaped body pioneers. Selective ablation of the fan-shaped body pioneers altered the architecture of the larval fan-shaped body primordium but did not result in gross abnormalities of the trajectories of unicolumnar neurons, indicating that axonal pathfinding of the two systems may be controlled independently. Our comprehensive spatial and developmental analysis of the SU neurons adds to our understanding of the establishment of neuronal circuitry.

We reconstructed, from a whole CNS EM volume, the synaptic map of input and output neurons that underlie food intake behavior of larvae. Input neurons originate from enteric, pharyngeal and external sensory organs and converge onto seven distinct sensory synaptic compartments within the CNS. Output neurons consist of feeding motor, serotonergic modulatory and neuroendocrine neurons. Monosynaptic connections from a set of sensory synaptic compartments cover the motor, modulatory and neuroendocrine targets in overlapping domains. Polysynaptic routes are superimposed on top of monosynaptic connections, resulting in divergent sensory paths that converge on common outputs. A completely different set of sensory compartments is connected to the mushroom body calyx. The mushroom body output neurons are connected to interneurons that directly target the feeding output neurons. Our results illustrate a circuit architecture in which monosynaptic and multisynaptic connections from sensory inputs traverse onto output neurons via a series of converging paths.

Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. In the present work, we identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, we reconstructed the main pathway that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. Our results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation.

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming (we define 'command-like neuron' as a neuron whose activation elicits or 'commands' a specific behavior). In most animals it remains unknown how neural circuits switch between antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of larval 'mooncrawler descending neurons' (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by stimulating a backward-active premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct monosynaptic reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals.

The brain adaptively integrates present sensory input, past experience, and options for future action. The insect mushroom body exemplifies how a central brain structure brings about such integration. Here we use a combination of systematic single-cell labeling, connectomics, transgenic silencing, and activation experiments to study the mushroom body at single-cell resolution, focusing on the behavioral architecture of its input and output neurons (MBINs and MBONs), and of the mushroom body intrinsic APL neuron. Our results reveal the identity and morphology of almost all of these 44 neurons in stage 3 Drosophila larvae. Upon an initial screen, functional analyses focusing on the mushroom body medial lobe uncover sparse and specific functions of its dopaminergic MBINs, its MBONs, and of the GABAergic APL neuron across three behavioral tasks, namely odor preference, taste preference, and associative learning between odor and taste. Our results thus provide a cellular-resolution study case of how brains organize behavior.

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. Yet, how noxious stimuli are transformed to coordinated escape behaviors remains poorly understood. Inlarvae, noxious stimuli trigger sequential body bending and corkscrew-like rolling behavior. We identified a population of interneurons in the nerve cord of, termed Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Electron microscopic circuit reconstruction shows that DnBs are targets of nociceptive and mechanosensory neurons, are directly presynaptic to pre-motor circuits, and link indirectly to Goro rolling command-like neurons. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior.

Neuroscientists are now able to acquire data at staggering rates across spatiotemporal scales. However, our ability to capitalize on existing datasets, tools, and intellectual capacities is hampered by technical challenges. The key barriers to accelerating scientific discovery correspond to the FAIR data principles: findability, global access to data, software interoperability, and reproducibility/re-usability. We conducted a hackathon dedicated to making strides in those steps. This manuscript is a technical report summarizing these achievements, and we hope serves as an example of the effectiveness of focused, deliberate hackathons towards the advancement of our quickly-evolving field.

Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, we identify premotor circuits including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.

During postembryonic development, the nervous system must adapt to a growing body. How changes in neuronal structure and connectivity contribute to the maintenance of appropriate circuit function remains unclear. In a previous paper (Schneider-Mizell et al., 2016), we measured the cellular neuroanatomy underlying synaptic connectivity in Drosophila. Here, we examined how neuronal morphology and connectivity change between 1st instar and 3rd instar larval stages using serial section electron microscopy. We reconstructed nociceptive circuits in a larva of each stage and found consistent topographically arranged connectivity between identified neurons. Five-fold increases in each size, number of terminal dendritic branches, and total number of synaptic inputs were accompanied by cell-type specific connectivity changes that preserved the fraction of total synaptic input associated with each presynaptic partner. We propose that precise patterns of structural growth act to conserve the computational function of a circuit, for example determining the location of a dangerous stimulus.