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Anatomy of Drosophila larvae (3 rd instar). (A) A lateral image of Drosophila larvae. Scale bar: 1mm. (B) Body wall muscles in larvae visualized by GFP expression. T1-T3, A1-A8 indicate thoracic segment 1-3 and abdominal segment 1-8. (C) Image series of exposure of central nervous system by dissection. Top: the head and tail are pinned down. Middle: The dorsal side of the body wall was cut and body wall was opened to fillet. Bottom: Internal tissues except for nervous system were removed. Dotted rectangle denotes the central nervous system (CNS). (D) Magnified image of the CNS in (C) showing the brain and the VNC (ventral nerve cord). The VNC corresponds to the vertebrate spinal cord. The SEZ (subesophageal zone) locates behind the brains. (E) Motor neurons visualized by GFP expression. Motor neurons in the VNC elongate axons through nerves (A2, A3, A4 and A8/9 nerves marked with arrowheads) to target muscles for forming the neuromuscular junction (NMJ). (A3 NMJ is marked with an arrowhead.)
Source publication
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 anot...
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... central nervous system (CNS) can be exposed experimen- tally by cutting the body wall and removing the internal tissues (intestines, a trachea, fat bodies and Malpighian tubes) (Fig. 1C). The CNS consists of two hemispheres (the brain lobes), the ventral nerve cord (VNC; thoracic and abdominal ganglia), which is analo- gous to the vertebrate spinal cord, and subesophageal zone (SEZ) in between the brain and VNC (Fig. 1D). There are a number of neu-ronal connections between the brain and VNC [21]. The VNC is segmented into three thoracic neuromeres and eight abdominal neuromeres. Muscles in each body wall segment are innervated by motor neurons in the corresponding neuromeres within the VNC (Fig. 1E). Motor neurons form neuromuscular junctions on the body wall muscle that are visible in the dissected larvae (Fig. 1E). Spatiotemporal activity of the motor neurons within the VNC un- derlies all larval locomotion. Accordingly, motor circuits in the VNC can be considered as a chain of segmental units. Based on this anatomical property, mathematical models are constructed to de- scribe the larval crawling locomotion ...
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... central nervous system (CNS) can be exposed experimen- tally by cutting the body wall and removing the internal tissues (intestines, a trachea, fat bodies and Malpighian tubes) (Fig. 1C). The CNS consists of two hemispheres (the brain lobes), the ventral nerve cord (VNC; thoracic and abdominal ganglia), which is analo- gous to the vertebrate spinal cord, and subesophageal zone (SEZ) in between the brain and VNC (Fig. 1D). There are a number of neu-ronal connections between the brain and VNC [21]. The VNC is segmented into three thoracic neuromeres and eight abdominal neuromeres. Muscles in each body wall segment are innervated by motor neurons in the corresponding neuromeres within the VNC (Fig. 1E). Motor neurons form neuromuscular junctions on the body wall muscle that are visible in the dissected larvae (Fig. 1E). Spatiotemporal activity of the motor neurons within the VNC un- derlies all larval locomotion. Accordingly, motor circuits in the VNC can be considered as a chain of segmental units. Based on this anatomical property, mathematical models are constructed to de- scribe the larval crawling locomotion ...
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... central nervous system (CNS) can be exposed experimen- tally by cutting the body wall and removing the internal tissues (intestines, a trachea, fat bodies and Malpighian tubes) (Fig. 1C). The CNS consists of two hemispheres (the brain lobes), the ventral nerve cord (VNC; thoracic and abdominal ganglia), which is analo- gous to the vertebrate spinal cord, and subesophageal zone (SEZ) in between the brain and VNC (Fig. 1D). There are a number of neu-ronal connections between the brain and VNC [21]. The VNC is segmented into three thoracic neuromeres and eight abdominal neuromeres. Muscles in each body wall segment are innervated by motor neurons in the corresponding neuromeres within the VNC (Fig. 1E). Motor neurons form neuromuscular junctions on the body wall muscle that are visible in the dissected larvae (Fig. 1E). Spatiotemporal activity of the motor neurons within the VNC un- derlies all larval locomotion. Accordingly, motor circuits in the VNC can be considered as a chain of segmental units. Based on this anatomical property, mathematical models are constructed to de- scribe the larval crawling locomotion ...
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... central nervous system (CNS) can be exposed experimen- tally by cutting the body wall and removing the internal tissues (intestines, a trachea, fat bodies and Malpighian tubes) (Fig. 1C). The CNS consists of two hemispheres (the brain lobes), the ventral nerve cord (VNC; thoracic and abdominal ganglia), which is analo- gous to the vertebrate spinal cord, and subesophageal zone (SEZ) in between the brain and VNC (Fig. 1D). There are a number of neu-ronal connections between the brain and VNC [21]. The VNC is segmented into three thoracic neuromeres and eight abdominal neuromeres. Muscles in each body wall segment are innervated by motor neurons in the corresponding neuromeres within the VNC (Fig. 1E). Motor neurons form neuromuscular junctions on the body wall muscle that are visible in the dissected larvae (Fig. 1E). Spatiotemporal activity of the motor neurons within the VNC un- derlies all larval locomotion. Accordingly, motor circuits in the VNC can be considered as a chain of segmental units. Based on this anatomical property, mathematical models are constructed to de- scribe the larval crawling locomotion ...
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... larvae grow in size between every molt. The length of the egg is about 0.5 mm, and after two moltings the 3rd instar larva is 5mm in length (Fig. 1A). The body wall of the larva is segmented, with three thoracic segments and eight abdominal segments (T1-3 and A1-8 in Fig. 1B). Each half (hemi)-segment contains about 30 muscles. The muscles can be classified, based on their orientation, as longitudinal, transverse or oblique. Peristaltic locomotion is gen- erated by sequential contraction of muscles from the posterior to anterior segments (in the case of forward locomotion). During the peristaltic wave, contraction of longitudinal muscles precedes that of transverse muscles [20]. The position and orientation of muscles in each segment are almost the same from A1 to A7, and therefore each segment can be regarded as a unit of motor ...
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... larvae grow in size between every molt. The length of the egg is about 0.5 mm, and after two moltings the 3rd instar larva is 5mm in length (Fig. 1A). The body wall of the larva is segmented, with three thoracic segments and eight abdominal segments (T1-3 and A1-8 in Fig. 1B). Each half (hemi)-segment contains about 30 muscles. The muscles can be classified, based on their orientation, as longitudinal, transverse or oblique. Peristaltic locomotion is gen- erated by sequential contraction of muscles from the posterior to anterior segments (in the case of forward locomotion). During the peristaltic wave, contraction of longitudinal muscles precedes that of transverse muscles [20]. The position and orientation of muscles in each segment are almost the same from A1 to A7, and therefore each segment can be regarded as a unit of motor ...
Citations
... Different segments have a highly organized pattern of muscle fibers, with distinct variations in the thoracic and some abdominal segments. Each of the A1 to A7 hemi-segment shows a repeating pattern for position and orientation of about 30 muscles (Schulman et al., 2015;Kohsaka et al., 2017). Maintenance of muscle integrity is essential during their growth, as muscle functionality is required for larval mobility and later for pupation. ...
We describe here a simple procedure to visualize the segmental body wall muscles of Drosophila larvae which can be useful to examine effects of gene mutations or other conditions that affect skeletal muscle development, their innervations, and formation and activity of neuro-muscular junctions.
(This is 2nd edition)
... Highly detailed serial section transmission electron microscopy (ssTEM) whole brain volumes with synaptic level resolution have been obtained for both Drosophila larval and adult (Zheng et al., 2018 ) brains, where neurons were reconstructed using a collaborative web-based software, CATMAID (Saalfeld et al., 2009 ;Schneider-Mizell et al., 2016 ). Concerted efforts from many laboratories have made tremendous advances in reconstructing the Drosophila larval connectome at synaptic level resolution and there has been great progress in mapping out select connectomes at the EM level Eschbach & Zlatic, 2020 ;Kohsaka et al., 2017 ). Connectomes have been further validated using behavioral and functional imaging studies for olfaction in combination with learning and memory (Berck et al., 2016 ;Eichler et al., 2017 ;Eschbach et al., 2020 ;Saumweber et al., 2018 ), feeding (Miroschnikow et al., 2018 ;Schlegel et al., 2016 ), visual processing (Larderet et al., 2017 ), locomotion Heckscher et al., 2015 ;Hiramoto et al., 2021 ;Kohsaka et al., 2019 ;Zarin et al., 2019 ;Zwart et al., 2016 ), chemotaxis (Tastekin et al., 2018 ), thermosensation (Hernandez-Nunez et al., 2021 ), mechanosensation (Jovanic et al., 2016 ;Jovanic et al., 2019 ;Masson et al., 2020 ), nociceptive modalities Gerhard et al., 2017 ;Hu et al., 2017 ;Imambocus et al., 2022 ;Kaneko et al., 2017 ;Ohyama et al., 2015 ;Takagi et al., 2017 ), among others (Andrade et al., 2019 ;Huckesfeld et al., 2021 ;Imura et al., 2020 ;Mark et al., 2021 ;Valdes-Aleman et al., 2021 ;Winding et al., 2022). ...
... Recent work in premotor and motor neuron connectomics has provided evidence for both labeled line and combinatorial connectivity between promotor and motor neurons that give rise to co-active motor neurons states leading to selective muscle group activation (Huang & Zarin, 2022 ;Zarin et al., 2019 ). Various Drosophila larval premotor neuron subtypes and/or motor pools have been implicated in locomotion, nociception, and innocuous mechanosensation Huang & Zarin, 2022 ;Jovanic et al., 2019 ;Kohsaka et al., 2017 ;Kohsaka et al., 2014 ;Kohsaka et al., 2019 ;Yoshino et al., 2017 ;Zarin et al., 2019 ). Neural reconstruction efforts in Drosophila larvae have revealed that somatosensory (CIII md and CIV md) neurons are not directly connected to motor neurons, but rather feed into polysynaptic pathways leading to motor neurons via premotor neurons (such as the DnB, mCSI, & Chair-1 neurons studied here) (Jovanic et al., 2019 ;Ohyama et al., 2015 ;Winding et al., 2022). ...
Metazoans detect and differentiate between innocuous (non-painful) and/or noxious (harmful) environmental cues using primary sensory neurons, which serve as the first node in a neural network that computes stimulus specific behaviors to either navigate away from injury-causing conditions or to perform protective behaviors that mitigate extensive injury. The ability of an animal to detect and respond to various sensory stimuli depends upon molecular diversity in the primary sensors and the underlying neural circuitry responsible for the relevant behavioral action selection. Recent studies in Drosophila larvae have revealed that somatosensory class III multidendritic (CIII md) neurons function as multimodal sensors regulating distinct behavioral responses to innocuous mechanical and nociceptive thermal stimuli. Recent advances in circuit bases of behavior have identified and functionally validated Drosophila larval somatosensory circuitry involved in innocuous (mechanical) and noxious (heat and mechanical) cues. However, central processing of cold nociceptive cues remained unexplored. We implicate multisensory integrators (Basins), premotor (Down-and-Back) and projection (A09e and TePns) neurons as neural substrates required for cold-evoked behavioral and calcium responses. Neural silencing of cell types downstream of CIII md neurons led to significant reductions in cold-evoked behaviors and neural co-activation of CIII md neurons plus additional cell types facilitated larval contraction (CT) responses. We further demonstrate that optogenetic activation of CIII md neurons evokes calcium increases in these neurons. Collectively, we demonstrate how Drosophila larvae process cold stimuli through functionally diverse somatosensory circuitry responsible for generating stimulus specific behaviors.
... Some aspects of the neural circuitry underlying locomotion (Kohsaka et al., 2017) and vibration response (Matsuo et al., 2014) have been characterized. The four behavioral sequences noted above are initialized by the activation of dendritic arborization neurons and chordotonal neuronal complexes lining the upper and lower portions of each larva segment (Grueber et al., 2007;Cheng et al., 2010;Ohyama et al., 2013). ...
How animals respond to repeatedly applied stimuli, and how animals respond to mechanical stimuli in particular, are important questions in behavioral neuroscience. We study adaptation to repeated mechanical agitation using the Drosophila larva. Vertical vibration stimuli elicit a discrete set of responses in crawling larvae: continuation, pause, turn, and reversal. Through high-throughput larva tracking, we characterize how the likelihood of each response depends on vibration intensity and on the timing of repeated vibration pulses. By examining transitions between behavioral states at the population and individual levels, we investigate how the animals habituate to the stimulus patterns. We identify time constants associated with desensitization to prolonged vibration, with re-sensitization during removal of a stimulus, and additional layers of habituation that operate in the overall response. Known memory-deficient mutants exhibit distinct behavior profiles and habituation time constants. An analogous simple electrical circuit suggests possible neural and molecular processes behind adaptive behavior.
... Turning is accomplished by unilateral body bending followed by peristaltic motions (Lahiri et al., 2011). The combination of these movements, such as forward crawling, backward crawling, and turning, enables larvae to move in a twodimensional space facilitating food foraging and the search for suitable molting sites ( Figure 2D; Green et al., 1983;Kohsaka et al., 2017;Clark et al., 2018). Kohsaka 10.3389/fncir.2023.1175899 ...
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.
... Forward crawling is the most predominant mode in fly larval locomotion [24,25]. By propagating segmental contraction from the posterior to anterior segments, larvae move forward [26][27][28], and neural circuits for crawling have been intensively examined [29][30][31][32][33][34][35][36][37]. Previous simulation studies have succeeded in building models describing the propagative nature of crawling behaviour qualitatively [10,[38][39][40]. ...
... The model in this study could describe forward crawling, the most frequent behaviour in larvae. Recent neuroscience studies have revealed numerous neural circuit modules involved in distinct aspects of behaviour, including speed control [32], bilateral coordination [35], intrasegmental coordination [36], intersegmental coordination [31], backward crawling [58,59], turning [60], escaping [61], and sensory input-guided navigation [34,62]. By integrating these circuit modules, it would be possible to establish a neuromechanical model that reproduces multiple and natural larval behaviours. ...
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.
... Particularly in Drosophila, there is a vivid interest in dissecting the motor circuit. 1,2 We have previously focused on eve-positive (eve + ) neurons, aCC and RP2, which are segmentally repeated in the ventral nerve cord (VNC). [3][4][5] Both motoneurons innervate body wall muscles (Figure 1a, dots). ...
Visualization and manipulation of defined motoneurons have provided significant insights into how motor circuits are assembled in Drosophila. A conventional approach for molecular and cellular analyses of subsets of motoneurons involves the expression of a wide range of UAS transgenes using available GAL4 drivers (eg, eve promoter-fused GAL4). However, a more powerful toolkit could be one that enables a single-cell characterization of interactions between neurites from neurons of interest. Here we show the development of a UAS > LexA > QF expression system to generate randomly selected neurons expressing one of the 2 binary expression systems. As a demonstration, we apply it to visualize dendrite-dendrite interactions by genetically labeling eve ⁺ neurons with distinct fluorescent reporters.
... How can we approach the neural circuit mechanisms underlying the interspecies divergence in larval locomotion? Circuit mechanisms in larval locomotion have been examined intensively in Drosophila melanogaster. Recent connectomics studies have identified several key neurons for larval locomotion in Drosophila melanogaster [20,30,[59][60][61][62][63][64][65][66][67]. Regarding bending, the thoracic neuromere was shown to be important in bending in chemotaxis [30]. ...
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.
... Drosophila larval locomotion is an excellent model for investigation of sensorimotor circuits at the single cell level [16][17][18] (Fig. 1a). The larva has a segmented body and normally moves by forward locomotion. ...
... The larva has a segmented body and normally moves by forward locomotion. However, when exposed to noxious stimulus to the head, it performs backward locomotion as an escape behavior 16,19,20 . Forward and backward locomotion are symmetric axial movements achieved by propagation of muscular contraction and relaxation along the body. ...
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.
... These parameters can be utilized to examine the neuromuscular performance in an array of genotypic/environmental backgrounds. Comparative analysis of such parameters can offer an opportunity to correlate genes to behavior, resolving sensory to motor output at cellular and molecular levels (Kohsaka et al., 2017). Therefore, the crawling behavior of larvae can be exploited to study effect of drugs, modulated gene expression and olfactory malfunctioning. ...
... In particular, we wanted to assess whether the loss of FEZ1 could contribute to defects in gross and fine motor skills as has been reported in JS patients. We decided to model this behavior using Drosophila, an organism that has been extensively used to study locomotion behavior (29,30). ...
FEZ1-mediated axonal transport plays important roles in central nervous system development but its involvement in the peripheral nervous system is not well-characterised. FEZ1 is deleted in Jacobsen syndrome (JS), an 11q terminal deletion developmental disorder. JS patients display impaired psychomotor skills, including gross and fine motor delay, suggesting that FEZ1 deletion may be responsible for these phenotypes, given its association with the development of motor-related circuits. Supporting this hypothesis, our data shows that FEZ1 is selectively expressed in the rat brain and spinal cord. Its levels progressively increase over the developmental course of human motor neurons derived from embryonic stem cells. Deletion of FEZ1 strongly impaired axon and dendrite development, and significantly delayed the transport of synaptic proteins into developing neurites. Concurring with these observations, Drosophila unc-76 mutants showed severe locomotion impairments, accompanied by a strong reduction of synaptic boutons at neuromuscular junctions. These abnormalities were ameliorated by pharmacological activation of UNC-51/ATG1, a FEZ1-activating kinase, with rapamycin and metformin. Collectively, the results highlight a role for FEZ1 in motor neuron development and implicate its deletion as an underlying cause of motor impairments in JS patients.
