Animals move by adaptively coordinating the sequential activation of muscles. The circuit mechanisms underlying coordinated locomotion are poorly understood. Here, we report on a novel circuit for propagation of waves of muscle contraction, using the peristaltic locomotion of Drosophila larvae as a model system. We found an intersegmental chain of synaptically connected neurons, alternating excitatory and inhibitory, necessary for wave propagation and active in phase with the wave. The excitatory neurons (A27h) are premotor and necessary only for forward locomotion, and are modulated by stretch receptors and descending inputs. The inhibitory neurons (GDL) are necessary for both forward and backward locomotion, suggestive of different yet coupled central pattern generators, and its inhibition is necessary for wave propagation. The circuit structure and functional imaging indicated that the commands to contract one segment promote the relaxation of the next segment, revealing a mechanism for wave propagation in peristaltic locomotion.

Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. Here we show that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, we reconstructed the multisensory circuit supporting the synergy, spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, we identified functionally connected circuit nodes that trigger the fastest locomotor mode, and others that facilitate it, and we provide evidence that multiple levels of multimodal integration contribute to escape mode selection. We propose that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input–output functions and selective tuning to ecologically relevant combinations of cues.

The cellular circuits through which the internal state of an organism is sensed and relayed to the brain, and through which they bring about appropriate physiological and behavioral responses, remain largely unelucidated. Using whole-animal electron microscopy reconstruction of a Drosophila larva, we previously identified an Aorta sensory organ whose somata lie on the periphery of the brain, with dendritic arborizations in the wall of the anterior aorta, and whose axons project to the protocerebrum and synapse onto the insulin-producing cells (IPCs). Here we characterize the full anatomy and connectivity of this sensory organ. We show that the aorta sensory neurons make their strongest direct synaptic contacts onto IPCs and the neurosecretory cells producing Dromyosuppressin (DMS), with only weaker direct contacts to diuretic hormone 44 (DH44). They also make direct contacts to the enteric serotonergic neurons modulating swallowing (Se0ens), and to two single mushroom body neurons, DAN-j1 and MBON-d3, of the learning and memory center of Drosophila. Calcium imaging shows that the neurons are activated by fructose, and optogenetic manipulation reveals that their activity influences peptide content in IPCs, DH44 and DMS cells. Polysynaptic paths to the brain run through two morphologically distinct interneuron families. One, formed by the Hugin protocerebral neurons, targets the neuroendocrine system and is associated with bitter taste and pathogen induced feeding suppression. The other, formed by the BAmas projection neurons, targets dopaminergic input neurons and output neurons of the mushroom body. The Aorta sensory pathway therefore couples internal hemolymph state to two parallel central systems — endocrine state control and mushroom body-associated value updating — and provides a candidate substrate for a metabolic memory through which post-ingestive state is fed back into future feeding choice.Competing Interest StatementThe authors have declared no competing interest.

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic pre-motor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via Eaat1, and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory post-synaptic currents in motor neurons. The neuronal synapses were always located within a micron of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. This article is protected by copyright. All rights reserved.

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis.

The mechanisms by which synaptic partners recognize each other and establish appropriate numbers of connections during embryonic development to form functional neural circuits are poorly understood. We combined electron microscopy reconstruction, functional imaging of neural activity, and behavioral experiments to elucidate the roles of (1) partner identity, (2) location, and (3) activity in circuit assembly in the embryonic nerve cord of Drosophila. We found that postsynaptic partners are able to find and connect to their presynaptic partners even when these have been shifted to ectopic locations or silenced. However, orderly positioning of axon terminals by positional cues and synaptic activity is required for appropriate numbers of connections between specific partners, for appropriate balance between excitatory and inhibitory connections, and for appropriate functional connectivity and behavior. Our study reveals with unprecedented resolution the fine connectivity effects of multiple factors that work together to control the assembly of neural circuits.

Even a simple sensory stimulus can elicit distinct innate behaviors and sequences. During sensorimotor decisions, competitive interactions among neurons that promote distinct behaviors must ensure the selection and maintenance of one behavior, while suppressing others. The circuit implementation of these competitive interactions is still an open question. By combining comprehensive electron microscopy reconstruction of inhibitory interneuron networks, modeling, electrophysiology, and behavioral studies, we determined the circuit mechanisms that contribute to the Drosophila larval sensorimotor decision to startle, explore, or perform a sequence of the two in response to a mechanosensory stimulus. Together, these studies reveal that, early in sensory processing, (1) reciprocally connected feedforward inhibitory interneurons implement behavioral choice, (2) local feedback disinhibition provides positive feedback that consolidates and maintains the chosen behavior, and (3) lateral disinhibition promotes sequence transitions. The combination of these interconnected circuit motifs can implement both behavior selection and the serial organization of behaviors into a sequence.

Postural control, the capacity of an animal to detect and correct an inappropriate body orientation, is a widespread and fundamental neurobiological function, yet the neural circuits that implement it remain poorly resolved in most species. The larva of Drosophila melanogaster performs a posture control stereotyped self-righting (SR) manoeuvre when turned upside-down, a behaviour previously shown to depend on a pair of segmentally repeated lateral transverse motor neurons (LT1/2-MNs) and on the normal expression of several Hox-targeting microRNAs. Here, we use a connectomics and a functional behavioural approach to trace and test the sensory, interneuronal and motor architecture of the SR circuit along the antero-posterior axis. Starting from the LT1/2-MNs, we identify a set of pre-motor interneurons, their principal upstream partners, and a population of class IV multidendritic sensory neurons as core components of the circuit, and show, through neuron-specific thermogenetic silencing, that inhibiting the great majority of these elements significantly impairs SR performance. We further find substantial overlap between the SR circuit and the previously described nociceptive rolling and touch crawling circuits, converging on shared interneurons including DnB, A02o (Wave-1) and TePn05. Network and axial connectivity analyses reveal a nested, hub-like organisation, a small number of integrator neurons bridging sensory and motor sub-networks, and a consistent decline in synapse number and density towards posterior segments. Together, these data yield the first axial wiring diagram for a postural control circuit in any animal and offer a set of structural principles including: hub organisation, shared sensory-motor structure, and antero-posterior connectivity gradients, which may extend to other segmentally organised nervous systems.

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.

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.