Synaptic architecture of leg and wing premotor control networks in Drosophila.

Animal movement is controlled by motor neurons (MNs), which project out of the central nervous system to activate muscles1. MN activity is coordinated by complex premotor networks that facilitate the contribution of individual muscles to many different behaviours2-6. Here we use connectomics7 to analyse the wiring logic of premotor circuits controlling the Drosophila leg and wing. We find that both premotor networks cluster into modules that link MNs innervating muscles with related functions. Within most leg motor modules, the synaptic weights of each premotor neuron are proportional to the size of their target MNs, establishing a circuit basis for hierarchical MN recruitment. By contrast, wing premotor networks lack proportional synaptic connectivity, which may enable more flexible recruitment of wing steering muscles. Through comparison of the architecture of distinct motor control systems within the same animal, we identify common principles of premotor network organization and specializations that reflect the unique biomechanical constraints and evolutionary origins of leg and wing motor control.

Connectomic reconstruction of a female Drosophila ventral nerve cord.

A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.

Bridging two insect flight modes in evolution, physiology and robophysics.

Since taking flight, insects have undergone repeated evolutionary transitions between two seemingly distinct flight modes1-3. Some insects neurally activate their muscles synchronously with each wingstroke. However, many insects have achieved wingbeat frequencies beyond the speed limit of typical neuromuscular systems by evolving flight muscles that are asynchronous with neural activation and activate in response to mechanical stretch2-8. These modes reflect the two fundamental ways of generating rhythmic movement: time-periodic forcing versus emergent oscillations from self-excitation8-10. How repeated evolutionary transitions have occurred and what governs the switching between these distinct modes remain unknown. Here we find that, despite widespread asynchronous actuation in insects across the phylogeny3,6, asynchrony probably evolved only once at the order level, with many reversions to the ancestral, synchronous mode. A synchronous moth species, evolved from an asynchronous ancestor, still preserves the stretch-activated muscle physiology. Numerical and robophysical analyses of a unified biophysical framework reveal that rather than a dichotomy, these two modes are two regimes of the same dynamics. Insects can transition between flight modes across a bridge in physiological parameter space. Finally, we integrate these two actuation modes into an insect-scale robot11-13 that enables transitions between modes and unlocks a new self-excited wingstroke strategy for engineered flight. Together, this framework accounts for repeated transitions in insect flight evolution and shows how flight modes can flip with changes in physiological parameters.

Wing structure and neural encoding jointly determine sensing strategies in insect flight.

Animals rely on sensory feedback to generate accurate, reliable movements. In many flying insects, strain-sensitive neurons on the wings provide rapid feedback that is critical for stable flight control. While the impacts of wing structure on aerodynamic performance have been widely studied, the impacts of wing structure on sensing are largely unexplored. In this paper, we show how the structural properties of the wing and encoding by mechanosensory neurons interact to jointly determine optimal sensing strategies and performance. Specifically, we examine how neural sensors can be placed effectively on a flapping wing to detect body rotation about different axes, using a computational wing model with varying flexural stiffness. A small set of mechanosensors, conveying strain information at key locations with a single action potential per wingbeat, enable accurate detection of body rotation. Optimal sensor locations are concentrated at either the wing base or the wing tip, and they transition sharply as a function of both wing stiffness and neural threshold. Moreover, the sensing strategy and performance is robust to both external disturbances and sensor loss. Typically, only five sensors are needed to achieve near-peak accuracy, with a single sensor often providing accuracy well above chance. Our results show that small-amplitude, dynamic signals can be extracted efficiently with spatially and temporally sparse sensors in the context of flight. The demonstrated interaction of wing structure and neural encoding properties points to the importance of understanding each in the context of their joint evolution.

Transcription factor Pebbled/RREB1 regulates injury-induced axon degeneration.

Genetic studies of Wallerian degeneration have led to the identification of signaling molecules (e.g., dSarm/Sarm1, Axundead, and Highwire) that function locally in axons to drive degeneration. Here we identify a role for the Drosophila C2H2 zinc finger transcription factor Pebbled [Peb, Ras-responsive element binding protein 1 (RREB1) in mammals] in axon death. Loss of Peb in Drosophila glutamatergic sensory neurons results in either complete preservation of severed axons, or an axon death phenotype where axons fragment into large, continuous segments, rather than completely disintegrate. Peb is expressed in developing and mature sensory neurons, suggesting it is required to establish or maintain their competence to undergo axon death. peb mutant phenotypes can be rescued by human RREB1, and they exhibit dominant genetic interactions with dsarm mutants, linking peb/RREB1 to the axon death signaling cascade. Surprisingly, Peb is only able to fully block axon death signaling in glutamatergic, but not cholinergic sensory neurons, arguing for genetic diversity in axon death signaling programs in different neuronal subtypes. Our findings identify a transcription factor that regulates axon death signaling, and peb mutant phenotypes of partial fragmentation reveal a genetically accessible step in axon death signaling.

Single mechanosensory neurons encode lateral displacements using precise spike timing and thresholds.

During locomotion, animals rely on multiple sensory modalities to maintain stability. External cues may guide behaviour, but they must be interpreted in the context of the animal's own body movements. Mechanosensory cues that can resolve dynamic internal and environmental conditions, like those from vertebrate vestibular systems or other proprioceptors, are essential for guided movement. How do afferent proprioceptor neurons transform movement into a neural code? In flies, modified hindwings known as halteres detect forces produced by body rotations and are essential for flight. However, the mechanisms by which haltere neurons transform forces resulting from three-dimensional body rotations into patterns of neural spikes are unknown. We use intracellular electrodes to record from haltere primary afferent neurons during a range of haltere motions. We find that spike timing activity of individual neurons changes with displacement and propose a mechanism by which single neurons can encode three-dimensional haltere movements during flight.

MOTOR NERVE CONDUCTION VELOCITIES OF THE MEDIAN AND SCIATIC-TIBIAL NERVES IN EIGHT NORMAL LARGE FLYING FOXES ( PTEROPUS VAMPYRUS).

Electrodiagnostic testing is an integral part of the evaluation of the motor unit in many neurologic conditions. Literature about the peripheral nervous system of flying foxes ( Pteropus spp) is sparse, and reference range values for motor nerve conduction velocities in vivo have not been established in Chiropterans. The goals of this study were to determine reference range conduction velocities in flying fox for the thoracic and pelvic limb nerve. Eight Pteropus vampyrus, large flying foxes, of varying ages and gender underwent nerve conduction studies of the median nerve and sciatic-tibial nerve. Mean (SD) conduction velocity values were 49.8 (12.7) m/sec for the median nerve and 42.1 (10.2) m/sec for the sciatic-tibial nerve. Median nerve conduction velocities were not significantly faster than sciatic-tibial nerve conduction velocities, although a trend was seen. Differences by sex or age class were not statistically significant. It was also noted that flying foxes rapidly lose body heat under general anesthesia.

A Defensive Kicking Behavior in Response to Mechanical Stimuli Mediated by Drosophila Wing Margin Bristles.

Mechanosensation, one of the fastest sensory modalities, mediates diverse behaviors including those pertinent for survival. It is important to understand how mechanical stimuli trigger defensive behaviors. Here, we report that Drosophila melanogaster adult flies exhibit a kicking response against invading parasitic mites over their wing margin with ultrafast speed and high spatial precision. Mechanical stimuli that mimic the mites' movement evoke a similar kicking behavior. Further, we identified a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle that forms an array along the wing margin as being essential sensory components for this behavior. Our electrophysiological recordings demonstrated that the mechanosensitivity of recurved bristles requires Nanchung and Nanchung-expressing neurons. Together, our results reveal a novel neural mechanism for innate defensive behavior through mechanosensation. SIGNIFICANCE STATEMENT: We discovered a previously unknown function for recurved bristles on the Drosophila melanogaster wing. We found that when a mite (a parasitic pest for Drosophila) touches the wing margin, the fly initiates a swift and accurate kick to remove the mite. The fly head is dispensable for this behavior. Furthermore, we found that a TRPV channel, Nanchung, and a specific Nanchung-expressing neuron under each recurved bristle are essential for its mechanosensitivity and the kicking behavior. In addition, touching different regions of the wing margin elicits kicking directed precisely at the stimulated region. Our experiments suggest that recurved bristles allow the fly to sense the presence of objects by touch to initiate a defensive behavior (perhaps analogous to touch-evoked scratching; Akiyama et al., 2012).

Neural evidence supports a dual sensory-motor role for insect wings.

Flying insects use feedback from various sensory modalities including vision and mechanosensation to navigate through their environment. The rapid speed of mechanosensory information acquisition and processing compensates for the slower processing times associated with vision, particularly under low light conditions. While halteres in dipteran species are well known to provide such information for flight control, less is understood about the mechanosensory roles of their evolutionary antecedent, wings. The features that wing mechanosensory neurons (campaniform sensilla) encode remains relatively unexplored. We hypothesized that the wing campaniform sensilla of the hawkmoth, Manduca sexta, rapidly and selectively extract mechanical stimulus features in a manner similar to halteres. We used electrophysiological and computational techniques to characterize the encoding properties of wing campaniform sensilla. To accomplish this, we developed a novel technique for localizing receptive fields using a focused IR laser that elicits changes in the neural activity of mechanoreceptors. We found that (i) most wing mechanosensors encoded mechanical stimulus features rapidly and precisely, (ii) they are selective for specific stimulus features, and (iii) there is diversity in the encoding properties of wing campaniform sensilla. We found that the encoding properties of wing campaniform sensilla are similar to those for haltere neurons. Therefore, it appears that the neural architecture that underlies the haltere sensory function is present in wings, which lends credence to the notion that wings themselves may serve a similar sensory function. Thus, wings may not only function as the primary actuator of the organism but also as sensors of the inertial dynamics of the animal.

Dendrites are dispensable for basic motoneuron function but essential for fine tuning of behavior.

Dendrites are highly complex 3D structures that define neuronal morphology and connectivity and are the predominant sites for synaptic input. Defects in dendritic structure are highly consistent correlates of brain diseases. However, the precise consequences of dendritic structure defects for neuronal function and behavioral performance remain unknown. Here we probe dendritic function by using genetic tools to selectively abolish dendrites in identified Drosophila wing motoneurons without affecting other neuronal properties. We find that these motoneuron dendrites are unexpectedly dispensable for synaptic targeting, qualitatively normal neuronal activity patterns during behavior, and basic behavioral performance. However, significant performance deficits in sophisticated motor behaviors, such as flight altitude control and switching between discrete courtship song elements, scale with the degree of dendritic defect. To our knowledge, our observations provide the first direct evidence that complex dendrite architecture is critically required for fine-tuning and adaptability within robust, evolutionarily constrained behavioral programs that are vital for mating success and survival. We speculate that the observed scaling of performance deficits with the degree of structural defect is consistent with gradual increases in intellectual disability during continuously advancing structural deficiencies in progressive neurological disorders.

PINK1-mediated phosphorylation of Parkin boosts Parkin activity in Drosophila.

Two genes linked to early onset Parkinson's disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.

Antennal mechanosensory neurons mediate wing motor reflexes in flying Drosophila.

Although many behavioral studies have shown the importance of antennal mechanosensation in various aspects of insect flight control, the identities of the mechanosensory neurons responsible for these functions are still unknown. One candidate is the Johnston's organ (JO) neurons that are located in the second antennal segment and detect phasic and tonic rotations of the third antennal segment relative to the second segment. To investigate how different classes of JO neurons respond to different types of antennal movement during flight, we combined 2-photon calcium imaging with a machine vision system to simultaneously record JO neuron activity and the antennal movement from tethered flying fruit flies (Drosophila melanogaster). We found that most classes of JO neurons respond strongly to antennal oscillation at the wing beat frequency, but not to the tonic deflections of the antennae. To study how flies use input from the JO neurons during flight, we genetically ablated specific classes of JO neurons and examined their effect on the wing motion. Tethered flies flying in the dark require JO neurons to generate slow antiphasic oscillation of the left and right wing stroke amplitudes. However, JO neurons are not necessary for this antiphasic oscillation when visual feedback is available, indicating that there are multiple pathways for generating antiphasic movement of the wings. Collectively, our results are consistent with a model in which flying flies use JO neurons to detect increases in the wing-induced airflow and that JO neurons are involved in a response that decreases contralateral wing stoke amplitude.

Bat wing sensors support flight control.

Bats are the only mammals capable of powered flight, and they perform impressive aerial maneuvers like tight turns, hovering, and perching upside down. The bat wing contains five digits, and its specialized membrane is covered with stiff, microscopically small, domed hairs. We provide here unique empirical evidence that the tactile receptors associated with these hairs are involved in sensorimotor flight control by providing aerodynamic feedback. We found that neurons in bat primary somatosensory cortex respond with directional sensitivity to stimulation of the wing hairs with low-speed airflow. Wing hairs mostly preferred reversed airflow, which occurs under flight conditions when the airflow separates and vortices form. This finding suggests that the hairs act as an array of sensors to monitor flight speed and/or airflow conditions that indicate stall. Depilation of different functional regions of the bats' wing membrane altered the flight behavior in obstacle avoidance tasks by reducing aerial maneuverability, as indicated by decreased turning angles and increased flight speed.

Sensors and sensory processing for airborne vibrations in silk moths and honeybees.

Insects use airborne vibrations caused by their own movements to control their behaviors and produce airborne vibrations to communicate with conspecific mates. In this review, I use two examples to introduce how insects use airborne vibrations to accurately control behavior or for communication. The first example is vibration-sensitive sensilla along the wing margin that stabilize wingbeat frequency. There are two specialized sensors along the wing margin for detecting the airborne vibration caused by wingbeats. The response properties of these sensors suggest that each sensor plays a different role in the control of wingbeats. The second example is Johnston's organ that contributes to regulating flying speed and perceiving vector information about food sources to hive-mates. There are parallel vibration processing pathways in the central nervous system related with these behaviors, flight and communication. Both examples indicate that the frequency of airborne vibration are filtered on the sensory level and that on the central nervous system level, the extracted vibration signals are integrated with other sensory signals for executing quick adaptive motor response.

Homeostatic interactions at the front of migration control the integrity and the efficiency of a migratory glial chain.

In metazoans, cell migration often occurs in a collective manner: the cells move while physically and functionally connected to their neighbors. The coordinated and timely movement of the cells eventually ensures the proper organization of tissues, and deregulation in such a process contributes to the development of severe diseases. Thus, understanding the cellular mechanisms underlying coordinated cell movement is of great interest in basic and medical science. The developing Drosophila wing provides an excellent model to follow the chain migration of glial cells in vivo. Cells at the tip of the glial collective have been shown to control the timely movement of the chain. In the present study, we show that while pioneers trigger chain migration, they cannot move as single cells. We also show that isolating cell clusters at the chain tip restores the formation of smaller migratory communities. Interestingly, the migratory efficiency of these de novo formed communities depends on the number of cells and progressively improves as the size of the cluster increases. Thus, homeostatic events at the migratory front control community integrity, efficiency, and coordination, emphasizing the importance of interactions and cell counting in fine-tuning collective processes.

Temperature compensation of aerobic capacity and performance in the Antarctic pteropod, Clione antarctica, compared with its northern congener, C. limacina.

In ectotherms living in cold waters, locomotory performance is constrained by a slower generation of the ATP that is needed to fuel muscle contraction. Both polar and temperate pteropods of the genus Clione, however, are able to swim continuously by flapping their parapodia (wings) at comparable frequencies at their respective habitat temperatures. Therefore, we expected polar species to have increased aerobic capacities in their wing muscles when measured at common temperatures. We investigated muscle and mitochondrial ultrastructure of Clione antarctica from the Southern Ocean (-1.8°C) and populations of a sister species, Clione limacina, from the Arctic (-0.5 to 3°C) and from the North Atlantic (10°C). We also measured oxygen consumption and the activity of the mitochondrial enzyme citrate synthase (CS) in isolated wings of the two species. The Antarctic species showed a substantial up-regulation of the density of oxidative muscle fibers, but at the expense of fast-twitch muscle fibers. Mitochondrial capacity was also substantially increased in the Antarctic species, with the cristae surface density (58.2±1.3µm(2)µm(-3)) more than twice that found in temperate species (34.3±0.8µm(2)µm(-3)). Arctic C. limacina was intermediate between these two populations (43.7±0.5µm(2)µm(-3)). The values for cold-adapted populations are on par with those found in high-performance vertebrates. As a result of oxidative muscle proliferation, CS activity was 4-fold greater in C. antarctica wings than in temperate C. limacina when measured at a common temperature (20°C). Oxygen consumption of isolated wing preparations was comparable in the two species when measured at their respective habitat temperatures. These findings indicate complete compensation of ATP generation in wing muscles across a 10°C temperature range, which supports similar wing-beat frequencies during locomotion at each species' respective temperature. The elevated capacity in the wing muscles is reflected in the partial compensation of whole-animal oxygen consumption and feeding rates.

Organization of the primary somatosensory cortex and wing representation in the Big Brown Bat, Eptesicus fuscus.

Bats are the only mammals capable of true powered flight. The bat wing exhibits specializations, allowing these animals to perform complicated flight maneuvers like landing upside-down, and hovering. The wing membrane contains various tactile receptors, including hair-associated Merkel receptors that might be involved in stabilizing bat flight. Here, we studied the neuronal representation of the wing membrane in the primary somatosensory cortex (S1) of the anesthetized Big Brown Bat, Eptesicus fuscus, using tactile stimulation with calibrated monofilaments (von Frey hairs) while recording from multi-neuron clusters. We also measured cortical response thresholds to tactile stimulation of the wings.The body surface is mapped topographically across the surface of S1, with the head, foot, and wing being overrepresented. The orientation of the wing representation is rotated compared to the hand representaion of terrestrial mammals, confirming results from other bat species. Although different wing membrane parts derive embryologically from different body parts, including the flank (plagiopatagium), the tactile sensitivity of the entire flight membrane (0.2-1.2 mN) is remarkably close or even higher (dactylopatagium) than the average tactile sensitivity of the human fingertip.

Postembryonic development of centrally generated flight motor patterns in the hawkmoth, Manduca sexta.

This study analyses the maturation of centrally generated flight motor patterns during metamorphosis of Manduca sexta. Bath application of the octopamine agonist chlordimeform to the isolated central nervous system of adult moths reliably induces fictive flight patterns in wing depressor and elevator motoneurons. Pattern maturation is investigated by chlordimeform application at different developmental stages. Chlordimeform also induces motor patterns in larval ganglia, which differ from fictive flight, indicating that in larvae and adults, octopamine affects different networks. First changes in motoneuron activity occur at the pupal stage P10. Rhythmic motor output is induced in depressor, but not in elevator motoneurons at P12. Adult-like fictive flight activity in motoneurons is observed at P16 and increases in speed and precision until emergence 2 days later. Pharmacological block of chloride channels with picrotoxin also induces fictive flight in adults, suggesting that the pattern-generating network can be activated by the removal of inhibition, and that proper network function does not rely on GABA(A) receptors. Our results suggest that the flight pattern-generating network becomes gradually established between P12 and P16, and is further refined until adulthood. These findings are discussed in the context of known physiological and structural CNS development during Manduca metamorphosis.

Innervation of the wing membrane in the bent-winged bat Miniopterus fuliginosus.

The nerves that innervate the fingertips and wing membrane from the upper arm of the bent-winged bat Miniopterus fuliginosus were examined under a stereomicroscope. The radial, median, ulnar and musculocutaneous nerves were formed by the brachial plexus, which ran to the wing membrane. The two suspected axillary nerves ran to the wing membrane. The radial nerve ran to the end of the first digit, while the median nerve ran along the forearm and subsequently branched-off to run along the second to fifth digits up to the end of the phalanges. The ulnar nerve ran to the plagiopatagium on the extensor side of the elbow joint. Finally, the musculocutaneous nerve passed through the ventral side of the humerus and branched out at the elbow joint to run radially to the propatagium area. In this study, the visible nerves that were distributed from the upper arm to the fingertips of Miniopterus fuliginosus were formed by C6-T1.

A neural circuit encoding mating states tunes defensive behavior in Drosophila.

Social context can dampen or amplify the perception of touch, and touch in turn conveys nuanced social information. However, the neural mechanism behind social regulation of mechanosensation is largely elusive. Here we report that fruit flies exhibit a strong defensive response to mechanical stimuli to their wings. In contrast, virgin female flies being courted by a male show a compromised defensive response to the stimuli, but following mating the response is enhanced. This state-dependent switch is mediated by a functional reconfiguration of a neural circuit labelled with the Tmc-L gene in the ventral nerve cord. The circuit receives excitatory inputs from peripheral mechanoreceptors and coordinates the defensive response. While male cues suppress it via a doublesex (dsx) neuronal pathway, mating sensitizes it by stimulating a group of uterine neurons and consequently activating a leucokinin-dependent pathway. Such a modulation is crucial for the balance between defense against body contacts and sexual receptivity.