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• W2952067902 abstract "•High-speed, 3D SCAPE microscopy is used to image freely moving Drosophila larvae•Calcium activity in proprioceptive neurons is coincident with dendrite deformation•Proprioceptor subtypes show sequential activity patterns during forward crawling•Sensors activate in different combinations during complex turns and retractions Proprioceptors provide feedback about body position that is essential for coordinated movement. Proprioceptive sensing of the position of rigid joints has been described in detail in several systems; however, it is not known how animals with a flexible skeleton encode their body positions. Understanding how diverse larval body positions are dynamically encoded requires knowledge of proprioceptor activity patterns in vivo during natural movement. Here we used high-speed volumetric swept confocally aligned planar excitation (SCAPE) microscopy in crawling Drosophila larvae to simultaneously track the position, deformation, and intracellular calcium activity of their multidendritic proprioceptors. Most proprioceptive neurons were found to activate during segment contraction, although one subtype was activated by extension. During cycles of segment contraction and extension, different proprioceptor types exhibited sequential activity, providing a continuum of position encoding during all phases of crawling. This sequential activity was related to the dynamics of each neuron’s terminal processes, and could endow each proprioceptor with a specific role in monitoring different aspects of body-wall deformation. We demonstrate this deformation encoding both during progression of contraction waves during locomotion as well as during less stereotyped, asymmetric exploration behavior. Our results provide powerful new insights into the body-wide neuronal dynamics of the proprioceptive system in crawling Drosophila, and demonstrate the utility of our SCAPE microscopy approach for characterization of neural encoding throughout the nervous system of a freely behaving animal. Proprioceptors provide feedback about body position that is essential for coordinated movement. Proprioceptive sensing of the position of rigid joints has been described in detail in several systems; however, it is not known how animals with a flexible skeleton encode their body positions. Understanding how diverse larval body positions are dynamically encoded requires knowledge of proprioceptor activity patterns in vivo during natural movement. Here we used high-speed volumetric swept confocally aligned planar excitation (SCAPE) microscopy in crawling Drosophila larvae to simultaneously track the position, deformation, and intracellular calcium activity of their multidendritic proprioceptors. Most proprioceptive neurons were found to activate during segment contraction, although one subtype was activated by extension. During cycles of segment contraction and extension, different proprioceptor types exhibited sequential activity, providing a continuum of position encoding during all phases of crawling. This sequential activity was related to the dynamics of each neuron’s terminal processes, and could endow each proprioceptor with a specific role in monitoring different aspects of body-wall deformation. We demonstrate this deformation encoding both during progression of contraction waves during locomotion as well as during less stereotyped, asymmetric exploration behavior. Our results provide powerful new insights into the body-wide neuronal dynamics of the proprioceptive system in crawling Drosophila, and demonstrate the utility of our SCAPE microscopy approach for characterization of neural encoding throughout the nervous system of a freely behaving animal. Monitoring neural activity in freely behaving animals is a key step toward understanding how sensory activity is transformed into action [1Calhoun A.J. Murthy M. Quantifying behavior to solve sensorimotor transformations: advances from worms and flies.Curr. Opin. Neurobiol. 2017; 46: 90-98Crossref PubMed Scopus (21) Google Scholar, 2Alivisatos A.P. Chun M. Church G.M. Greenspan R.J. Roukes M.L. Yuste R. The Brain Activity Map Project and the challenge of functional connectomics.Neuron. 2012; 74: 970-974Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 3Kerr J.N. Nimmerjahn A. Functional imaging in freely moving animals.Curr. Opin. Neurobiol. 2012; 22: 45-53Crossref PubMed Scopus (49) Google Scholar]. Small-invertebrate model systems with well-described sensory systems and complete or near-complete connectomes, such as C. elegans and Drosophila larvae, are ideal systems in which to uncover fundamental principles of sensorimotor integration. Light-sheet, confocal, and two-photon microscopy can capture neuronal calcium activity in isolated Drosophila brains or immobilized preparations [4Pulver S.R. Bayley T.G. Taylor A.L. Berni J. Bate M. Hedwig B. Imaging fictive locomotor patterns in larval Drosophila.J. Neurophysiol. 2015; 114: 2564-2577Crossref PubMed Scopus (61) Google Scholar, 5Ghannad-Rezaie M. Wang X. Mishra B. Collins C. Chronis N. Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae.PLoS ONE. 2012; 7: e29869Crossref PubMed Scopus (72) Google Scholar, 6Chhetri R.K. Amat F. Wan Y. Höckendorf B. Lemon W.C. Keller P.J. Whole-animal functional and developmental imaging with isotropic spatial resolution.Nat. Methods. 2015; 12: 1171-1178Crossref PubMed Scopus (154) Google Scholar, 7Lemon W.C. Pulver S.R. Höckendorf B. McDole K. Branson K. Freeman J. Keller P.J. Whole-central nervous system functional imaging in larval Drosophila.Nat. Commun. 2015; 6: 7924Crossref PubMed Scopus (124) Google Scholar, 8Royer L.A. Lemon W.C. Chhetri R.K. Wan Y. Coleman M. Myers E.W. Keller P.J. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms.Nat. Biotechnol. 2016; 34: 1267-1278Crossref PubMed Scopus (148) Google Scholar]. However, these methods have been unable to provide volumetric imaging at sufficient speeds, in unrestrained samples, to enable extended imaging of body-wide neural activity in behaving animals. Multispectral, high-speed, volumetric swept confocally aligned planar excitation (SCAPE) microscopy is capable of characterizing tissue and cellular dynamics in live behaving animals [9Bouchard M.B. Voleti V. Mendes C.S. Lacefield C. Grueber W.B. Mann R.S. Bruno R.M. Hillman E.M. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.Nat. Photonics. 2015; 9: 113-119Crossref PubMed Scopus (341) Google Scholar, 10Hillman E.M. Voleti V. Patel K. Li W. Yu H. Perez-Campos C. Benezra S.E. Bruno R.M. Galwaduge P.T. High-speed 3D imaging of cellular activity in the brain using axially-extended beams and light sheets.Curr. Opin. Neurobiol. 2018; 50: 190-200Crossref PubMed Scopus (23) Google Scholar]. We have applied this imaging technology to characterize the dynamics of multidendritic (md) neuron activity in crawling Drosophila larvae. Multifunctional md neurons are located just under the larval body wall and extend sensory dendrites along internal structures and the epidermis [11Finlayson L.H. Lowenstein O. The structure and function of abdominal stretch receptors in insects.Proc. R. Soc. Lond. B Biol. Sci. 1958; 148: 433-449Crossref PubMed Google Scholar, 12Schrader S. Merritt D.J. Dorsal longitudinal stretch receptor of Drosophila melanogaster larva—fine structure and maturation.Arthropod Struct. Dev. 2007; 36: 157-169Crossref PubMed Scopus (12) Google Scholar, 13Grueber W.B. Jan L.Y. Jan Y.N. Tiling of the Drosophila epidermis by multidendritic sensory neurons.Development. 2002; 129: 2867-2878Crossref PubMed Google Scholar, 14Hughes C.L. Thomas J.B. A sensory feedback circuit coordinates muscle activity in Drosophila.Mol. Cell. Neurosci. 2007; 35: 383-396Crossref PubMed Scopus (156) Google Scholar, 15Corty M.M. Tam J. Grueber W.B. Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.Development. 2016; 143: 1351-1362Crossref PubMed Scopus (21) Google Scholar, 16Bodmer R. Jan Y.N. Morphological differentiation of the embryonic peripheral neurons in Drosophila.Rouxs Arch. Dev. Biol. 1987; 196: 69-77Crossref PubMed Scopus (191) Google Scholar]. A subset of six of these md neurons (Figure 1A) extend axons to more dorsal neuropil regions important for motor control, suggesting that they are proprioceptors that provide feedback on body position [15Corty M.M. Tam J. Grueber W.B. Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.Development. 2016; 143: 1351-1362Crossref PubMed Scopus (21) Google Scholar, 17Merritt D.J. Whitington P.M. Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function.J. Neurosci. 1995; 15: 1755-1767Crossref PubMed Google Scholar, 18Schrader S. Merritt D.J. Central projections of Drosophila sensory neurons in the transition from embryo to larva.J. Comp. Neurol. 2000; 425: 34-44Crossref PubMed Scopus (40) Google Scholar, 19Grueber W.B. Ye B. Yang C.H. Younger S. Borden K. Jan L.Y. Jan Y.N. Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.Development. 2007; 134: 55-64Crossref PubMed Scopus (152) Google Scholar]. This feedback is thought to be particularly important during crawling, which involves periodic strides driven by peristaltic waves of muscle contractions along the body [20Heckscher E.S. Lockery S.R. Doe C.Q. Characterization of Drosophila larval crawling at the level of organism, segment, and somatic body wall musculature.J. Neurosci. 2012; 32: 12460-12471Crossref PubMed Scopus (117) Google Scholar]. However, studies investigating the activity of these sensors have been limited to dissected preparations: imaging of axon terminals in an isolated central nervous system (CNS) suggests that at least some of these neurons are active during muscle contraction [21Cheng L.E. Song W. Looger L.L. Jan L.Y. Jan Y.N. The role of the TRP channel NompC in Drosophila larval and adult locomotion.Neuron. 2010; 67: 373-380Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar], whereas an electrophysiology study has shown activity in one cell type in response to stretch in a dissected preparation [22Suslak T.J. Watson S. Thompson K.J. Shenton F.C. Bewick G.S. Armstrong J.D. Jarman A.P. Piezo Is Essential for Amiloride-Sensitive Stretch-Activated Mechanotransduction in Larval Drosophila Dorsal Bipolar Dendritic Sensory Neurons.PLoS ONE. 2015; 10: e0130969Crossref PubMed Scopus (20) Google Scholar]. Studies that disabled all or some of these six neurons observed significantly slowed crawling, suggesting that these cells are proprioceptors that provide a segment contraction “mission accomplished” signal that promotes progression of the peristaltic wave [14Hughes C.L. Thomas J.B. A sensory feedback circuit coordinates muscle activity in Drosophila.Mol. Cell. Neurosci. 2007; 35: 383-396Crossref PubMed Scopus (156) Google Scholar]. These behavioral studies concluded that this set of neurons have partially redundant functions during crawling, because silencing different subsets caused similar behavioral deficits, whereas silencing both subsets had a more severe effect. However, the diverse dendrite morphologies and positions of these proprioceptor neurons [13Grueber W.B. Jan L.Y. Jan Y.N. Tiling of the Drosophila epidermis by multidendritic sensory neurons.Development. 2002; 129: 2867-2878Crossref PubMed Google Scholar, 15Corty M.M. Tam J. Grueber W.B. Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.Development. 2016; 143: 1351-1362Crossref PubMed Scopus (21) Google Scholar] suggest that each is likely to have distinct sensitivities and functions. Identifying the specific roles of each cell type is not possible without measuring the system’s dynamic activity patterns during natural movements to examine the synergies and dynamic encoding properties of the larval proprioceptive circuit. Here, we characterized the spatiotemporal and functional dynamics of this set of Drosophila md proprioceptors by imaging neurons co-expressing GCaMP and tdTomato using SCAPE microscopy, with subsequent dynamic tracking and ratiometric calcium signal extraction. Characterization of the real-time dynamics of segment contraction and extension during crawling and exploratory head movements revealed that proprioceptors increased their calcium levels in synchrony with deformation of their dendrites. These cells provide a striking sequence of signaling during stereotyped forward crawling, suggesting an elegant continuum of sensing during movement. Furthermore, analysis of sensory responses during non-stereotyped exploration revealed a consistent relationship between activity patterns and more complex, asymmetric segment deformations. These activity patterns were found to be interpretable, via a simple linear combinatorial code, as separable representations of simultaneous turning and retracting movements. Our results provide valuable new input for models of how movements are controlled via feedback in the context of the larval connectome, and also demonstrate a new approach for characterization of body-wide neuronal dynamics in behaving Drosophila. To begin to characterize proprioceptor dynamics as larvae crawl, we focused on the ventral posterior dendritic arborization (vpda) class I neuron (Figure 1A). Class I neurons spread sensory dendrites along the body-wall epidermis, suggesting that these cells may detect cuticle folding. To investigate how vpda sensory terminals deform during crawling, we first characterized dendrite dynamics using high-speed volumetric SCAPE microscopy [9Bouchard M.B. Voleti V. Mendes C.S. Lacefield C. Grueber W.B. Mann R.S. Bruno R.M. Hillman E.M. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms.Nat. Photonics. 2015; 9: 113-119Crossref PubMed Scopus (341) Google Scholar, 10Hillman E.M. Voleti V. Patel K. Li W. Yu H. Perez-Campos C. Benezra S.E. Bruno R.M. Galwaduge P.T. High-speed 3D imaging of cellular activity in the brain using axially-extended beams and light sheets.Curr. Opin. Neurobiol. 2018; 50: 190-200Crossref PubMed Scopus (23) Google Scholar] at 10 volumes per s (VPS) as a larva crawled within a linear channel (Figures 1B and 1C). To achieve this imaging, we made numerous improvements to our original SCAPE microscopy system, including substantially improving spatial resolution to permit individual dendrites to be clearly resolved in 3D at high speed in the freely moving larva. We also increased the field of view to over 1 mm and made it sufficiently uniform to capture the entire crawling larva (see STAR Methods). During forward crawling, peristaltic muscle contractions move from posterior to anterior along the animal [20Heckscher E.S. Lockery S.R. Doe C.Q. Characterization of Drosophila larval crawling at the level of organism, segment, and somatic body wall musculature.J. Neurosci. 2012; 32: 12460-12471Crossref PubMed Scopus (117) Google Scholar]. vpda proprioceptors expressing GFP as a static marker showed repeated folding and extension: folding as a peristaltic wave entered a segment, and extending as the wave moved to anterior segments (Figures 1C–1C″′; Video S1). Viewed in cross-section, vpda dendrite tips flexed from distal to proximal, eventually angling at approximately 90° during each peristaltic contraction (Figure 1C″). Because class I dendrites are positioned along the basal surface of the epidermis [23Kim M.E. Shrestha B.R. Blazeski R. Mason C.A. Grueber W.B. Integrins establish dendrite-substrate relationships that promote dendritic self-avoidance and patterning in Drosophila sensory neurons.Neuron. 2012; 73: 79-91Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar], vpda dendrite dynamics most likely reflects body-wall dynamics. Therefore, our data indicate that vpda dendrites are positioned to respond to repeated contraction and extension of the body wall that occurs during crawling. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5ZDFhYjcyOTcwMDhkNzZiNGZjMDViMzZlZDY3OGU1OCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4OTY5NDMzfQ.ajJXUrbR2_cEhgy_P2KefT9QpZe1hmPj-7piXZTk2LVZznsES-quZVV0dwBt8dqg0DSFcWN3YCaFTQbB0r-xtS5IsSL9-1nBNN5mMqU-6iZsB6FIsT86FNWCXL24szr9qmxi0Dxsbpb9MuvZgS-0wXIu7N_Er98bASReEKGcpuLZiRTx4YdFMzTyAQog3-kNG5TWpqnQabULIxvBLA6tyWb0c_VQZLADWrsHPQ3guE30YX-oxae6t0xkyHf5A4sD0YIUQNo31znRP2AHxxPzpPj9EjQz5ThPClq5Kejw-lFqUQsC3WEpb6rZ6S1OCFQhdJBC-0Vg1QMys4NO0DL_EQ Download .mp4 (7.36 MB) Help with .mp4 files Video S1. Ventral Class I Dendrite Dynamics during Crawling, GFP Only, Related to Figure 1Class I dendrites (and class IV weakly) are labeled by 221-Gal4, UAS-mCD8::GFP. Top is ventral view MIP over a 95 μm depth range from a 160μm deep volume, bottom is orthogonal view of ventral class I neuron vpda. Note that dendrites of vpda fold with each peristaltic wave. SCAPE images are shown on a square root grayscale to reduce dynamic range for visualization of both cell bodies and dendrites. Following the real-time movie, the data is played at 4X slower speed. Posterior is to the left. Scale bar = 100μm. Next, we sought to reveal whether and how the activity of these neurons changes as the dendrites fold during segment contraction. If vpda neurons indeed function as proprioceptors, we should be able to detect activity in these cells during locomotion. We built a dual-expression line of larvae to label targeted proprioceptive cells with both calcium-sensitive GCaMP (green) and static tdTomato (red). To acquire SCAPE microscopy data in this model, we optimized parallel dual-color imaging and developed a tracking algorithm that localizes the cell bodies via their static red fluorescence. These tracked cells were then used as fiducials for quantification of movement and behavior, as well as to extract and ratiometrically correct simultaneously recorded GCaMP fluorescence from the same cells. We used the inter-cell distance between the measured neuron and a homologous neuron in the posterior or anterior segment as our measure of segment contraction and extension. We observed consistent rises in GCaMP fluorescence in vpda neurons during each segment contraction (Figure 2). Calcium signals subsided as the peristaltic wave progressed to adjacent anterior segments (Figures 2B and 2D; Videos S2 and S3). Dynamic calcium responses were also visible in dendritic arbors and axons (Figures 2C and 2C′, arrowheads and arrows). Note that because of variability in calcium signal amplitude across contraction events (Figure 2B), signals were normalized for the averages shown in Figure 2D (see STAR Methods). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1ZWZkMmRhNTk0NGE2ZTA1ZGU2OGJlYjllNjg1OTlkZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4OTY5NDMzfQ.FiYJt66CkNby9d3FeSQL0OxXLzK02dhfuyGdosZ-4aZqqq4cYD1vR-ljUkZqqMRxL7XolAYs65H8vlE4IPR80hvY2bLdoIl0buVVgC_drM4l1MgIfZ53lXpy_VYKYq_tj89o0BvM4Dl2U02lFOZNF_b7hYtZ6QCkLo85Ndrt_1JUhOixG5XgtkIfmlwVNLwkbr7HNcGqKxM1g5iFnGsL6sFslO508XSPeKB6y7Hbt_M0TAjdj5sNn5OPNAus9LqoLoR3M4l9GOs8hBCVOQHyYEVkYT75hovmEiSpNv2WsMI5eGFKeTirD2nxLaA5TI1dlByahD3xxL6uuEOnGwiVig Download .mp4 (5.79 MB) Help with .mp4 files Video S2. Tracking of Neurons and Quantification of GCaMP6f Fluorescence during Crawling Using SCAPE Microscopy, Related to Figure 2Class I neurons are labeled with 410-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato. Top panels show five tracked vpda neurons each on left and right of larva. Solid line indicates GCaMP fluorescence (measured as ΔR/R0) and dashed lines indicate distance between tracked cell and posterior cell (inter-cell distance). Middle panels depict dual channel SCAPE imaging of a crawling larva (ventral MIP from full 168μm deep imaging volume and an orthogonal MIP view, square root colorscale). CNS is observed at right margin of the movie. Bottom panel is the positions of 12 tracked neurons in 3D space. Posterior is to the left. Scale bar = 100μm. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI0MDE5OTI5MGRhNDg0MWZlNDUxMzQyNDczMzcxZjYzNyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4OTY5NDMzfQ.suWKbw5kJZa3_jQneYebeEDkzDXRmWiZH4ntxRJmX1_Q9JrJ-GZzRsg-J9aB5I_dFzhcNKcizqdUNPQMjkGt1TJbORh-IDLt0rvBg_Z13KcbNd10aOCF2-jxEQZNyM-XDsXckV6upOQXDcZIx0V3iplojG12yAe6ka8Tu9cVXWd9YDfnCqlY8CjnZh0CZ_Gf_hOO0CcDtj1VZQictS52lGEd8cR4rVEGlTFTddoZ2YOFLTE-r5EmAKs7_dj9kFVUlypp0_bZSr3DlEvVOAwxJZqfSjmE3NEPjmwOVWCpsmQTmdleGKuuOdtYdAgGXQVN9LNP_k5etL9s731i36ouvw Download .mp4 (4.09 MB) Help with .mp4 files Video S3. Ventral Class I GCaMP Dynamics during Crawling, Related to Figure 2Ventral class I (vpda) neurons are labeled with 410-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato. Ventral view MIP over a 85 μm depth range of a 153μm deep volume to exclude gut autofluorescence (square root colorscale). Top panel is tdTomato fluorescence, middle panel is green GCaMP6f fluorescence and the bottom panel is the channel merge. Posterior is to the left. Scale bar = 100μm. As a control, an additional larva line co-expressing GFP and tdTomato in vpda neurons was imaged using SCAPE during crawling. Applying the same tracking and ratiometric correction as for GCaMP, we observed insignificant changes in ratiometrically corrected GFP signal during crawling (Figure S1). Taken together, our data indicate that vpda neurons respond to body-wall folding during segment contraction. Having established this pipeline for cell characterization, SCAPE was then used to monitor the physical and functional dynamics of the remaining proprioceptive cell types, each of which has unique dendrite morphologies and positions (Figure 1A). Two additional class I neurons besides vpda project secondary dendrites along the dorsal side of the body wall (dorsal dendritic arborization neuron D [ddaD] anteriorly and dorsal dendritic arborization neuron E [ddaE] posteriorly; Figure 1A) [13Grueber W.B. Jan L.Y. Jan Y.N. Tiling of the Drosophila epidermis by multidendritic sensory neurons.Development. 2002; 129: 2867-2878Crossref PubMed Google Scholar]. These neurons are poised to detect cuticle folding on the dorsal side of the animal. In addition, dorsal and ventral bipolar dendrite md neurons (dbd and vbd, respectively) extend in an anterior-posterior direction, and it is known that at least dbd extends along internal connective tissue [12Schrader S. Merritt D.J. Dorsal longitudinal stretch receptor of Drosophila melanogaster larva—fine structure and maturation.Arthropod Struct. Dev. 2007; 36: 157-169Crossref PubMed Scopus (12) Google Scholar]. By contrast, neuron dorsal multidendritic neuron 1 (dmd1) extends an atypical thick dendrite from the body wall to the internal intersegmental nerve (ISN) [15Corty M.M. Tam J. Grueber W.B. Dendritic diversification through transcription factor-mediated suppression of alternative morphologies.Development. 2016; 143: 1351-1362Crossref PubMed Scopus (21) Google Scholar], which lies along the muscle layer, suggesting that this proprioceptor could be poised to detect muscle dynamics. Imaging of dorsal class I neurons revealed that ddaE and ddaD dendrites deform as the peristaltic wave enters each segment, and flatten as the wave passes (Figures 3A–3A′ ″; Video S4, first section). Although there is some variability in the degree of dendrite deformation, we consistently see folding in both cell types, with ddaE folding before ddaD (Figures 3A″, 3A″′, and 3B) in synchrony with the posterior-to-anterior progression of peristaltic waves. Like vpda, calcium dynamics revealed increases in dorsal class I activity during segment contraction (Figures 3C, 3D, and S2; Video S5, first section). When comparing paired ddaE and ddaD cells within the same segment, responses of posterior ddaE and anterior ddaD neurons occurred in succession during segment contraction, with ddaE responding just before ddaD (Figures 4A and 4C ), corresponding to the lag in dendrite folding. These data suggest that cellular calcium activity is a result of dendritic folding in all class I neurons. As a control, larvae were imaged under compression with a glass coverslip that prevented physical folding of the ddaD dendrites (Figure S3). In this case, no increases in ddaD calcium activity were seen during forward crawling, consistent with dendritic folding driving calcium activity. This same compression did not prevent dendritic folding in ddaE neurons, and accordingly this cell type continued to show activity during crawling. This result further highlights the importance of imaging freely crawling larvae for characterization of locomotion, because physical restraint itself appears to influence proprioceptive signaling.Figure 4Each Dorsal Proprioceptor Type Is Activated Sequentially during Segment ContractionShow full caption(A) Mean calcium response (± SD) of dbd, dmd1, ddaE, and ddaD during segment contraction. dmd1 and dbd data are the same as shown in Figures 3G and 3H. This plot includes a subset of ddaE and ddaD activity data shown in Figures 3C and 3D from paired cells within a segment (n = 4 animals, 5 cells, 5 events). Data are aligned using the time at maximum contraction (as measured by the distance between ddaE and homologous ddaE in the posterior segment), which is set at t = 0 s for each event. The time window and the amplitude of ΔR/R0 of each trace were normalized and interpolated across events (see STAR Methods).(B) To test the lag between dmd1 and ddaE activity, we compared the time at half-maximum calcium activity from paired cells within a segment in a GMR10D05-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato larva (n = 4 animals, n = 8 cells, 8 events). ddaE activity occurs significantly later than dmd1 activity (p = 0.0078) by single-tailed paired t test.(C) To test the lag between ddaE and ddaD, we compared the time at half-maximum calcium activity from 410-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato animals (n = 4 animals, n = 5 cells, 5 events). Data are from the same cell pairs as analyzed in (A). ddaD activity occurs significantly later than ddaE activity (p = 0.01) by single-tailed paired t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Mean calcium response (± SD) of dbd, dmd1, ddaE, and ddaD during segment contraction. dmd1 and dbd data are the same as shown in Figures 3G and 3H. This plot includes a subset of ddaE and ddaD activity data shown in Figures 3C and 3D from paired cells within a segment (n = 4 animals, 5 cells, 5 events). Data are aligned using the time at maximum contraction (as measured by the distance between ddaE and homologous ddaE in the posterior segment), which is set at t = 0 s for each event. The time window and the amplitude of ΔR/R0 of each trace were normalized and interpolated across events (see STAR Methods). (B) To test the lag between dmd1 and ddaE activity, we compared the time at half-maximum calcium activity from paired cells within a segment in a GMR10D05-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato larva (n = 4 animals, n = 8 cells, 8 events). ddaE activity occurs significantly later than dmd1 activity (p = 0.0078) by single-tailed paired t test. (C) To test the lag between ddaE and ddaD, we compared the time at half-maximum calcium activity from 410-Gal4, 20XUAS-IVS-GCaMP6f (x2), UAS-CD4-tdTomato animals (n = 4 animals, n = 5 cells, 5 events). Data are from the same cell pairs as analyzed in (A). ddaD activity occurs significantly later than ddaE activity (p = 0.01) by single-tailed paired t test. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyY2RlZDUzYWRlNTRiZTAxYjAyYTRlNTQwYjM0NTYwYiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4OTY5NDMzfQ.FJ-68O6dK8nEbV_iaY5wvMiXSU4pGDbIh53MX_DvZwceNWhsiGIPqQG4sUuXFfcmkqZsQsHp0tX7pRQiBFh0bUlHYg_wtWq2yHUNsyl0fIBDzFCRa6RLA9jxe8rNUD5k7zqbc4GLgLu3fHhNyFgWJ5hW-4IEJshnTVLeU4cYzPeRFdSOeTABXTA5iHQlLF4Mb86zCEctf91e1ya6yb8aPdrQaZswKtz81m2p5jY-rrfVttvrGWs8YoIByOd8W7ID0O9sd8aUD09pvRPKbVr9C5I2fbo5Th9sA8PX4YtgjkzWkhTfSj6Hl7ShXuIcFuPLRnOkyYEAe0X9PsTki4SPEw Download .mp4 (35.2 MB) Help with .mp4 files Video S4. Dendrite Dynamics of Dorsal Class I, dbd, dmd1, and vbd during Crawling, GFP or tdTomato Only, Related to Figure 3Three different imaging acquisitions are shown in sequence. In the first movie, dorsal class I (ddaE, ddaD) dendrites (and class IV weakly) are labeled by 221-Gal4, UAS-mCD8::GFP. In the second movie, dbd, dmd1, ddaE, and ddaD neurons are labeled by GMR10D05-Gal4, 20XUAS-mCD8::GFP. In the third movie, the previous real-time movie is played at 4X slower speed. In the fourth movie, vbd neurons are labeled by 1129-Gal4, UAS-CD4-tdTomato. For all movies, top is dorsal view MIP over a 80-95μm range from a 160-165μm deep volume to exclude gut autofluorescence and bottom is side view (square root grayscale). Posterior is to the left. Scale bar = 100μm. eyJraWQi" @default.
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• W2952067902 date "2019-03-01" @default.
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• W2952067902 title "Characterization of Proprioceptive System Dynamics in Behaving Drosophila Larvae Using High-Speed Volumetric Microscopy" @default.
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• W2952067902 doi "https://doi.org/10.1016/j.cub.2019.01.060" @default.
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