Linking neural circuits to the mechanics of animal behavior in Drosophila larval locomotion.

The motions that make up animal behavior arise from the interplay between neural circuits and the mechanical parts of the body. Therefore, in order to comprehend the operational mechanisms governing behavior, it is essential to examine not only the underlying neural network but also the mechanical characteristics of the animal's body. The locomotor system of fly larvae serves as an ideal model for pursuing this integrative approach. By virtue of diverse investigation methods encompassing connectomics analysis and quantification of locomotion kinematics, research on larval locomotion has shed light on the underlying mechanisms of animal behavior. These studies have elucidated the roles of interneurons in coordinating muscle activities within and between segments, as well as the neural circuits responsible for exploration. This review aims to provide an overview of recent research on the neuromechanics of animal locomotion in fly larvae. We also briefly review interspecific diversity in fly larval locomotion and explore the latest advancements in soft robots inspired by larval locomotion. The integrative analysis of animal behavior using fly larvae could establish a practical framework for scrutinizing the behavior of other animal species.

Synchronous multi-segmental activity between metachronal waves controls locomotion speed in Drosophila larvae.

The ability to adjust the speed of locomotion is essential for survival. In limbed animals, the frequency of locomotion is modulated primarily by changing the duration of the stance phase. The underlying neural mechanisms of this selective modulation remain an open question. Here, we report a neural circuit controlling a similarly selective adjustment of locomotion frequency in Drosophila larvae. Drosophila larvae crawl using peristaltic waves of muscle contractions. We find that larvae adjust the frequency of locomotion mostly by varying the time between consecutive contraction waves, reminiscent of limbed locomotion. A specific set of muscles, the lateral transverse (LT) muscles, co-contract in all segments during this phase, the duration of which sets the duration of the interwave phase. We identify two types of GABAergic interneurons in the LT neural network, premotor neuron A26f and its presynaptic partner A31c, which exhibit segmentally synchronized activity and control locomotor frequency by setting the amplitude and duration of LT muscle contractions. Altogether, our results reveal an inhibitory central circuit that sets the frequency of locomotion by controlling the duration of the period in between peristaltic waves. Further analysis of the descending inputs onto this circuit will help understand the higher control of this selective modulation.

A vacuum-actuated soft robot inspired by Drosophila larvae to study kinetics of crawling behaviour.

Peristalsis, a motion generated by the propagation of muscular contraction along the body axis, is one of the most common locomotion patterns in limbless animals. While the kinematics of peristalsis has been examined intensively, its kinetics remains unclear, partially due to the lack of suitable physical models to simulate the locomotion patterns and inner drive in soft-bodied animals. Inspired by a soft-bodied animal, Drosophila larvae, we propose a vacuum-actuated soft robot mimicking its crawling behaviour. The soft structure, made of hyperelastic silicone rubber, was designed to imitate the larval segmental hydrostatic structure. Referring to a numerical simulation by the finite element method, the dynamical change in the vacuum pressure in each segment was controlled accordingly, and the soft robots could exhibit peristaltic locomotion. The soft robots successfully reproduced two previous experimental phenomena on fly larvae: 1. Crawling speed in backward crawling is slower than in forward crawling. 2. Elongation of either the segmental contraction duration or intersegmental phase delay makes peristaltic crawling slow. Furthermore, our experimental results provided a novel prediction for the role of the contraction force in controlling the speed of peristaltic locomotion. These observations indicate that soft robots could serve to examine the kinetics of crawling behaviour in soft-bodied animals.

Extraction of bouton-like structures from neuropil calcium imaging data.

The neuropil, the plexus of axons and dendrites, plays a critical role in operating the circuit processing of the nervous system. Revealing the spatiotemporal activity pattern within the neuropil would clarify how the information flows throughout the nervous system. However, calcium imaging to examine the circuit dynamics has mainly focused on the soma population due to their discrete distribution. The development of a methodology to analyze the calcium imaging data of a densely packed neuropil would provide us with new insights into the circuit dynamics. Here, we propose a new method to decompose calcium imaging data of the neuropil into populations of bouton-like synaptic structures with a standard desktop computer. To extract bouton-like structures from calcium imaging data, we introduced a new type of modularity, a widely used quality measure in graph theory, and optimized the clustering configuration by a simulated annealing algorithm, which is established in statistical physics. To assess this method's performance, we conducted calcium imaging of the neuropil of Drosophila larvae. Based on the obtained data, we established artificial neuropil imaging datasets. We applied the decomposition procedure to the artificial and experimental calcium imaging data and extracted individual bouton-like structures successfully. Based on the extracted spatiotemporal data, we analyzed the network structure of the central nervous system of fly larvae and found it was scale-free. These results demonstrate that neuropil calcium imaging and its decomposition could provide new insight into our understanding of neural processing.

Electrophysiological validation of monosynaptic connectivity between premotor interneurons and the aCC motoneuron in the Drosophila larval CNS.

The Drosophila connectome project aims to map the synaptic connectivity of entire larval and adult fly neural networks, which is essential for understanding nervous system development and function. So far, the project has produced an impressive amount of electron microscopy data that has facilitated reconstructions of specific synapses, including many in the larval locomotor circuit. While this breakthrough represents a technical tour-de-force, the data remain under-utilised, partly due to a lack of functional validation of reconstructions. Attempts to validate connectivity posited by the connectome project, have mostly relied on behavioural assays and/or GRASP or GCaMP imaging. While these techniques are useful, they have limited spatial or temporal resolution. Electrophysiological assays of synaptic connectivity overcome these limitations. Here, we combine patch clamp recordings with optogenetic stimulation in male and female larvae, to test synaptic connectivity proposed by connectome reconstructions. Specifically, we use multiple driver lines to confirm that several connections between premotor interneurons and the anterior corner cell (aCC) motoneuron are, as the connectome project suggests, monosynaptic. In contrast, our results also show that conclusions based on GRASP imaging may provide false positive results regarding connectivity between cells. We also present a novel imaging tool, based on the same technology as our electrophysiology, as a favourable alternative to GRASP. Finally, of eight Gal4 lines tested, five are reliably expressed in the premotors they are targeted to. Thus, our work highlights the need to confirm functional synaptic connectivity, driver line specificity, and use of appropriate genetic tools to support connectome projects.SIGNIFICANCE STATEMENTThe Drosophila connectome project aims to provide a complete description of connectivity between neurons in an organism that presents experimental advantages over other models. It has reconstructed over 80 percent of the fly larva's synaptic connections by manual identification of anatomical landmarks present in serial section transmission electron microscopy (ssTEM) volumes of the larval CNS. We use a highly reliable electrophysiological approach to verify these connections, so provide useful insight into the accuracy of work based on ssTEM. We also present a novel imaging tool for validating excitatory monosynaptic connections between cells, and show that several genetic driver lines designed to target neurons of the larval connectome exhibit non-specific and/or unreliable expression.

A neuromechanical model for Drosophila larval crawling based on physical measurements.

BACKGROUND: Animal locomotion requires dynamic interactions between neural circuits, the body (typically muscles), and surrounding environments. While the neural circuitry of movement has been intensively studied, how these outputs are integrated with body mechanics (neuromechanics) is less clear, in part due to the lack of understanding of the biomechanical properties of animal bodies. Here, we propose an integrated neuromechanical model of movement based on physical measurements by taking Drosophila larvae as a model of soft-bodied animals. RESULTS: We first characterized the kinematics of forward crawling in Drosophila larvae at a segmental and whole-body level. We then characterized the biomechanical parameters of fly larvae, namely the contraction forces generated by neural activity, and passive elastic and viscosity of the larval body using a stress-relaxation test. We established a mathematical neuromechanical model based on the physical measurements described above, obtaining seven kinematic values characterizing crawling locomotion. By optimizing the parameters in the neural circuit, our neuromechanical model succeeded in quantitatively reproducing the kinematics of larval locomotion that were obtained experimentally. This model could reproduce the observation of optogenetic studies reported previously. The model predicted that peristaltic locomotion could be exhibited in a low-friction condition. Analysis of floating larvae provided results consistent with this prediction. Furthermore, the model predicted a significant contribution of intersegmental connections in the central nervous system, which contrasts with a previous study. This hypothesis allowed us to make a testable prediction for the variability in intersegmental connection in sister species of the genus Drosophila. CONCLUSIONS: We generated a neurochemical model based on physical measurement to provide a new foundation to study locomotion in soft-bodied animals and soft robot engineering.

Interspecies variation of larval locomotion kinematics in the genus Drosophila and its relation to habitat temperature.

BACKGROUND: Speed and trajectory of locomotion are the characteristic traits of individual species. Locomotion kinematics may have been shaped during evolution towards increased survival in the habitats of each species. Although kinematics of locomotion is thought to be influenced by habitats, the quantitative relation between the kinematics and environmental factors has not been fully revealed. Here, we performed comparative analyses of larval locomotion in 11 Drosophila species. RESULTS: We found that larval locomotion kinematics are divergent among the species. The diversity is not correlated to the body length but is correlated instead to the habitat temperature of the species. Phylogenetic analyses using Bayesian inference suggest that the evolutionary rate of the kinematics is diverse among phylogenetic tree branches. CONCLUSIONS: The results of this study imply that the kinematics of larval locomotion has diverged in the evolutionary history of the genus Drosophila and evolved under the effects of the ambient temperature of habitats.

Regulation of coordinated muscular relaxation in Drosophila larvae by a pattern-regulating intersegmental circuit.

Typical patterned movements in animals are achieved through combinations of contraction and delayed relaxation of groups of muscles. However, how intersegmentally coordinated patterns of muscular relaxation are regulated by the neural circuits remains poorly understood. Here, we identify Canon, a class of higher-order premotor interneurons, that regulates muscular relaxation during backward locomotion of Drosophila larvae. Canon neurons are cholinergic interneurons present in each abdominal neuromere and show wave-like activity during fictive backward locomotion. Optogenetic activation of Canon neurons induces relaxation of body wall muscles, whereas inhibition of these neurons disrupts timely muscle relaxation. Canon neurons provide excitatory outputs to inhibitory premotor interneurons. Canon neurons also connect with each other to form an intersegmental circuit and regulate their own wave-like activities. Thus, our results demonstrate how coordinated muscle relaxation can be realized by an intersegmental circuit that regulates its own patterned activity and sequentially terminates motor activities along the anterior-posterior axis.

Optogenetics in Drosophila.

The fruit fly Drosophila melanogaster, an insect 4 mm long, has served as the experimental subject in a wide range of biological research, including neuroscience. In this chapter, we briefly introduce optogenetic applications in Drosophila neuroscience research. First, we describe the development of Drosophila from egg to adult. In fly neuroscience, temperature-controlled perturbation of neural activity, sometimes called "thermogenetics," has been an invaluable tool that predates the advent of optogenetics. After briefly introducing this perturbation technique, we describe the process of generating transgenic flies that express optogenetic probes in a specific group of cells. Transgenic techniques are crucial in the application of optogenetics in Drosophila neuroscience; here we introduce the transposon P-elements, ϕC31 integrase, and CRISPR-Cas9 methods. As for cell-specific gene expression techniques, the binary expression systems utilizing Gal4-UAS, LexA-lexAop, and Q-system are described. We also present a short and basic optogenetic experiment with Drosophila larvae as a practical example. Finally, we review a few recent studies in Drosophila neuroscience that made use of optogenetics. In this overview of fly development, transgenic methods, and applications of optogenetics, we present an introductory background to optogenetics in Drosophila.

Regulation of forward and backward locomotion through intersegmental feedback circuits in Drosophila larvae.

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system.

System level analysis of motor-related neural activities in larval Drosophila.

The way in which the central nervous system (CNS) governs animal movement is complex and difficult to solve solely by the analyses of muscle movement patterns. We tackle this problem by observing the activity of a large population of neurons in the CNS of larval Drosophila. We focused on two major behaviors of the larvae - forward and backward locomotion - and analyzed the neuronal activity related to these behaviors during the fictive locomotion that occurs spontaneously in the isolated CNS. We expressed a genetically-encoded calcium indicator, GCaMP and a nuclear marker in all neurons and then used digitally scanned light-sheet microscopy to record (at a fast frame rate) neural activities in the entire ventral nerve cord (VNC). We developed image processing tools that automatically detected the cell position based on the nuclear staining and allocate the activity signals to each detected cell. We also applied a machine learning-based method that we recently developed to assign motor status in each time frame. Our experimental procedures and computational pipeline enabled systematic identification of neurons that showed characteristic motor activities in larval Drosophila. We found cells whose activity was biased toward forward locomotion and others biased toward backward locomotion. In particular, we identified neurons near the boundary of the subesophageal zone (SEZ) and thoracic neuromeres, which were strongly active during an early phase of backward but not forward fictive locomotion.

Data-driven analysis of motor activity implicates 5-HT2A neurons in backward locomotion of larval Drosophila.

Rhythmic animal behaviors are regulated in part by neural circuits called the central pattern generators (CPGs). Classifying neural population activities correlated with body movements and identifying the associated component neurons are critical steps in understanding CPGs. Previous methods that classify neural dynamics obtained by dimension reduction algorithms often require manual optimization which could be laborious and preparation-specific. Here, we present a simpler and more flexible method that is based on the pre-trained convolutional neural network model VGG-16 and unsupervised learning, and successfully classifies the fictive motor patterns in Drosophila larvae under various imaging conditions. We also used voxel-wise correlation mapping to identify neurons associated with motor patterns. By applying these methods to neurons targeted by 5-HT2A-GAL4, which we generated by the CRISPR/Cas9-system, we identified two classes of interneurons, termed Seta and Leta, which are specifically active during backward but not forward fictive locomotion. Optogenetic activation of Seta and Leta neurons increased backward locomotion. Conversely, thermogenetic inhibition of 5-HT2A-GAL4 neurons or application of a 5-HT2 antagonist decreased backward locomotion induced by noxious light stimuli. This study establishes an accelerated pipeline for activity profiling and cell identification in larval Drosophila and implicates the serotonergic system in the modulation of backward locomotion.

Divergent Connectivity of Homologous Command-like Neurons Mediates Segment-Specific Touch Responses in Drosophila.

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.

Gap Junction-Mediated Signaling from Motor Neurons Regulates Motor Generation in the Central Circuits of Larval Drosophila.

In this study, we used the peristaltic crawling of Drosophila larvae as a model to study how motor patterns are regulated by central circuits. We built an experimental system that allows simultaneous application of optogenetics and calcium imaging to the isolated ventral nerve cord (VNC). We then investigated the effects of manipulating local activity of motor neurons (MNs) on fictive locomotion observed as waves of MN activity propagating along neuromeres. Optical inhibition of MNs with halorhodopsin3 in a middle segment (A4, A5, or A6), but not other segments, dramatically decreased the frequency of the motor waves. Conversely, local activation of MNs with channelrhodopsin2 in a posterior segment (A6 or A7) increased the frequency of the motor waves. Since peripheral nerves mediating sensory feedback were severed in the VNC preparation, these results indicate that MNs send signals to the central circuits to regulate motor pattern generation. Our results also indicate segmental specificity in the roles of MNs in motor control. The effects of the local MN activity manipulation were lost in shaking-B2 (shakB2 ) or ogre2 , gap-junction mutations in Drosophila, or upon acute application of the gap junction blocker carbenoxolone, implicating electrical synapses in the signaling from MNs. Cell-type-specific RNAi suggested shakB and ogre function in MNs and interneurons, respectively, during the signaling. Our results not only reveal an unexpected role for MNs in motor pattern regulation, but also introduce a powerful experimental system that enables examination of the input-output relationship among the component neurons in this system.SIGNIFICANCE STATEMENT Motor neurons are generally considered passive players in motor pattern generation, simply relaying information from upstream interneuronal circuits to the target muscles. This study shows instead that MNs play active roles in the control of motor generation by conveying information via gap junctions to the central pattern-generating circuits in larval Drosophila, providing novel insights into motor circuit control. The experimental system introduced in this study also presents a new approach for studying intersegmentally coordinated locomotion. Unlike traditional electrophysiology methods, this system enables the simultaneous recording and manipulation of populations of neurons that are genetically specified and span multiple segments.

Neural Circuits Underlying Fly Larval Locomotion.

Locomotion is a complex motor behavior that may be expressed in different ways using a variety of strategies depending upon species and pathological or environmental conditions. Quadrupedal or bipedal walking, running, swimming, flying and gliding constitute some of the locomotor modes enabling the body, in all cases, to move from one place to another. Despite these apparent differences in modes of locomotion, both vertebrate and invertebrate species share, at least in part, comparable neural control mechanisms for locomotor rhythm and pattern generation and modulation. Significant advances have been made in recent years in studies of the genetic aspects of these control systems. Findings made specifically using Drosophila (fruit fly) models and preparations have contributed to further understanding of the key role of genes in locomotion. This review focuses on some of the main findings made in larval fruit flies while briefly summarizing the basic advantages of using this powerful animal model for studying the neural locomotor system.

A circuit mechanism for the propagation of waves of muscle contraction in Drosophila.

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 the 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.

Functional dissociation in sweet taste receptor neurons between and within taste organs of Drosophila.

Finding food sources is essential for survival. Insects detect nutrients with external taste receptor neurons. Drosophila possesses multiple taste organs that are distributed throughout its body. However, the role of different taste organs in feeding remains poorly understood. By blocking subsets of sweet taste receptor neurons, we show that receptor neurons in the legs are required for immediate sugar choice. Furthermore, we identify two anatomically distinct classes of sweet taste receptor neurons in the leg. The axonal projections of one class terminate in the thoracic ganglia, whereas the other projects directly to the brain. These two classes are functionally distinct: the brain-projecting neurons are involved in feeding initiation, whereas the thoracic ganglia-projecting neurons play a role in sugar-dependent suppression of locomotion. Distinct receptor neurons for the same taste quality may coordinate early appetitive responses, taking advantage of the legs as the first appendages to contact food.

Identification of Inhibitory Premotor Interneurons Activated at a Late Phase in a Motor Cycle during Drosophila Larval Locomotion.

Rhythmic motor patterns underlying many types of locomotion are thought to be produced by central pattern generators (CPGs). Our knowledge of how CPG networks generate motor patterns in complex nervous systems remains incomplete, despite decades of work in a variety of model organisms. Substrate borne locomotion in Drosophila larvae is driven by waves of muscular contraction that propagate through multiple body segments. We use the motor circuitry underlying crawling in larval Drosophila as a model to try to understand how segmentally coordinated rhythmic motor patterns are generated. Whereas muscles, motoneurons and sensory neurons have been well investigated in this system, far less is known about the identities and function of interneurons. Our recent study identified a class of glutamatergic premotor interneurons, PMSIs (period-positive median segmental interneurons), that regulate the speed of locomotion. Here, we report on the identification of a distinct class of glutamatergic premotor interneurons called Glutamatergic Ventro-Lateral Interneurons (GVLIs). We used calcium imaging to search for interneurons that show rhythmic activity and identified GVLIs as interneurons showing wave-like activity during peristalsis. Paired GVLIs were present in each abdominal segment A1-A7 and locally extended an axon towards a dorsal neuropile region, where they formed GRASP-positive putative synaptic contacts with motoneurons. The interneurons expressed vesicular glutamate transporter (vGluT) and thus likely secrete glutamate, a neurotransmitter known to inhibit motoneurons. These anatomical results suggest that GVLIs are premotor interneurons that locally inhibit motoneurons in the same segment. Consistent with this, optogenetic activation of GVLIs with the red-shifted channelrhodopsin, CsChrimson ceased ongoing peristalsis in crawling larvae. Simultaneous calcium imaging of the activity of GVLIs and motoneurons showed that GVLIs' wave-like activity lagged behind that of motoneurons by several segments. Thus, GVLIs are activated when the front of a forward motor wave reaches the second or third anterior segment. We propose that GVLIs are part of the feedback inhibition system that terminates motor activity once the front of the motor wave proceeds to anterior segments.

A group of segmental premotor interneurons regulates the speed of axial locomotion in Drosophila larvae.

BACKGROUND: Animals control the speed of motion to meet behavioral demands. Yet, the underlying neuronal mechanisms remain poorly understood. Here we show that a class of segmentally arrayed local interneurons (period-positive median segmental interneurons, or PMSIs) regulates the speed of peristaltic locomotion in Drosophila larvae. RESULTS: PMSIs formed glutamatergic synapses on motor neurons and, when optogenetically activated, inhibited motor activity, indicating that they are inhibitory premotor interneurons. Calcium imaging showed that PMSIs are rhythmically active during peristalsis with a short time delay in relation to motor neurons. Optogenetic silencing of these neurons elongated the duration of motor bursting and greatly reduced the speed of larval locomotion. CONCLUSIONS: Our results suggest that PMSIs control the speed of axial locomotion by limiting, via inhibition, the duration of motor outputs in each segment. Similar mechanisms are found in the regulation of mammalian limb locomotion, suggesting that common strategies may be used to control the speed of animal movements in a diversity of species.

Serotonin and downstream leucokinin neurons modulate larval turning behavior in Drosophila.

Serotonin (5-HT) is known to modulate motor outputs in a variety of animal behaviors. However, the downstream neural pathways of 5-HT remain poorly understood. We studied the role of 5-HT in directional change, or turning, behavior of fruit fly (Drosophila melanogaster) larvae. We analyzed light- and touch-induced turning and found that turning is a combination of three components: bending, retreating, and rearing. Serotonin transmission suppresses rearing; when we inhibited 5-HT neurons with Shibire or Kir2.1, rearing increased without affecting the occurrence of bending or retreating. Increased rearing in the absence of 5-HT transmission often results in slower or failed turning, indicating that suppression of rearing by 5-HT is critical for successful turning. We identified a class of abdominal neurons called the abdominal LK neurons (ABLKs), which express the 5-HT1B receptor and the neuropeptide leucokinin, as downstream targets of 5-HT that are involved in the control of turning. Increased rearing was observed when neural transmission or leucokinin synthesis was inhibited in these cells. Forced activation of ABLKs also increased rearing, suggesting that an appropriate level of ABLK activity is critical for the control of turning. Calcium imaging revealed that ABLKs show periodic activation with an interval of ∼15 s. The activity level of ABLKs increased and decreased in response to a 5-HT agonist and antagonist, respectively. Our results suggest that 5-HT modulates larval turning by regulating the activity level of downstream ABLK neurons and secretion of the neuropeptide leucokinin.