Decision letter: Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single-cell resolution, we here describe a multiphasic behavioral sequence in Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, with sequential increases in stereotypy dependent upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution. eLife digest How do we find out how the brain works? One way is to use imaging techniques to visualise an animal’s brain in action as it performs simple behaviours: as the animal moves, parts of its brain light up under the microscope. For laboratory animals like fruit flies, which have relatively small brains, this lets us observe their brain activity right down to the level of individual brain cells. The brain directs movements via collective activity of the body’s muscles. Our ability to track the activity of individual muscles is, however, more limited than our ability to observe single brain cells: even modern imaging technology still cannot monitor the activity of all the muscle cells in an animal’s body as it moves about. Yet this is precisely the information that scientists need to fully understand how the brain generates behaviour. Fruit flies perform specific behaviours at certain stages of their life cycle. When the fly pupa begins to metamorphose into an adult insect, it performs a fixed sequence of movements involving a set number of muscles, which is called the pupal ecdysis sequence. This initial movement sequence and the rest of metamorphosis both occur within the confines of the pupal case, which is a small, hardened shell surrounding the whole animal. Elliott et al. set out to determine if the fruit fly pupa’s ecdysis sequence could be used as a kind of model, to describe a simple behaviour at the level of individual muscles. Imaging experiments used fly pupae that were genetically engineered to produce an activity-dependent fluorescent protein in their muscle cells. Pupal cases were treated with a chemical to make them transparent, allowing easy observation of their visually ‘labelled’ muscles. This yielded a near-complete record of muscle activity during metamorphosis. Initially, individual muscles became active in small groups. The groups then synchronised with each other over the different regions of the pupa’s body to form distinct movements, much as syllables join to form words. This synchronisation was key to progression through metamorphosis and was co-ordinated at each step by specialised nerve cells that produce or respond to specific hormones. These results reveal how the brain might direct muscle activity to produce movement patterns. In the future, Elliott et al. hope to compare data on muscle activity with comprehensive records of brain cell activity, to shed new light on how the brain, muscles, and other factors work together to control behaviour. Introduction A major goal of neuroscience is explaining how nervous systems generate and organize behavior. This requires describing behavior in terms that can be correlated with neural activity. The dynamics of brain activity can be observed in whole brains at single-cell resolution (Ahrens et al., 2013; Ardiel et al., 2017; Cong et al., 2017; Lemon et al., 2015; Nguyen et al., 2016; Pulver et al., 2015), but behavioral dynamics has not been captured at a similar level of detail (Datta et al., 2019). While progress in fine-mapping natural behavior, or ‘computational ethology’ (Anderson and Perona, 2014), has benefited from recent advances in visual tracking (Johnson et al., 2020), 3D imaging (Hong et al., 2015), machine vision (Dankert et al., 2009), machine learning (Kabra et al., 2013; Machado et al., 2015), and image feature extraction (Berman et al., 2014; Mathis et al., 2018; Wiltschko et al., 2015), the primary focus of these efforts has been kinematic, seeking to define anatomical movements at higher resolution. While movements are the essential components of behavior, they are complex products of motor neuron activity, which must balance the contractions of agonist and antagonist muscles and must also promote anatomical rigidity that supports movement. Here, we bridge the gap between motor neuron activity and movement by describing muscle activity at the single-cell level. Comprehensively monitoring muscle activity in behaving animals is achievable with genetically encoded Ca++ indicators and has been demonstrated at single-cell resolution in hydra (Szymanski and Yuste, 2019), roundworms (Ardiel et al., 2017), and larval fruit flies (Heckscher et al., 2012; Zarin et al., 2019). However, the application of muscle Ca++ imaging to characterize more complex sequences has been constrained by the challenge of tracking behavior in freely moving animals. This problem is resolved in pupal fruit flies where behavior is restricted to the puparium (Kim et al., 2006), which can be clarified for optical access to the confined animal. The pupa maintains a fixed position and orientation during movement, and all behavior is executed within a delimited field of view. Pupal Ca++ activity imaging of muscles has been previously demonstrated using GCaMP6s by Diao et al., 2017, who showed that the bulk Ca++ signal collected over ventral muscles exhibits temporal patterns that conform well to known body wall movements. The behavioral hallmark of pupal development is the Drosophila pupal ecdysis sequence, which is one of a general class of behavioral sequences used by insects to molt (Truman, 2005; Zitnan and Adams, 2012). These sequences typically divide into three principal phases during which the animal first loosens and then sheds its old exoskeleton before expanding its newly secreted one. Ecdysis sequences are strongly dependent on the action of hormones for their initiation and progression, and together with vertebrate reproductive behaviors, they have long served as a useful model of hormone-behavior interactions. Neural control of ecdysis behaviors is typically exercised by a conserved complement of hormones including eclosion hormone, ecdysis triggering hormone (ETH), crustacean cardioactive peptide (CCAP), and Bursicon (Ewer and Reynolds, 2002; White and Ewer, 2014). The sites of action of these hormones within the nervous system have been principally studied in Drosophila, but detailed neuroethological studies have been undertaken in larger insects, such as the locust, Schistocerca gregaria (Hughes, 1980a; Hughes, 1980b; Hughes, 1980c), and cricket, Teleogryllus oceanicus (Carlson, 1977; Carlson and Bentley, 1977). Motor program organization of the adult ecdysis sequences in these insects is quite stereotyped, suggesting central control of motor execution, but sensory feedback can also modulate behavioral performance. While characterization of central nervous system (CNS) activity underlying ecdysis sequences has remained limited in larger insects, progress has been made in Drosophila where a fictive ecdysis sequence can be elicited in an excised pupal brain by exposure to ETH (Diao et al., 2017; Kim et al., 2015; Kim et al., 2006; Mena et al., 2016). Fictive activity imaged using Ca++ indicators grossly correlates with the motor patterns executed during pupal ecdysis, but comprehensively interpreting CNS activity remains a challenge. The Drosophila pupal ecdysis sequence occurs at the onset of metamorphosis (Figure 1A) and has been adapted to initiate transformation of the body plan (Kim et al., 2006). Although it occurs in the context of the pupal molt and has three principal phases characterized by distinct motor programs, its function in molting is limited to casting off the cuticular linings of the gut and trachea. Its primary function is, instead, to create internal pressure at points along the body to evert adult parts such as the head, legs, and wings (Denlinger and Zdarek, 1994). ETH initiates the pupal ecdysis sequence after a long period of behavioral quiescence and targets neurons that express its two receptor isoforms, ETHRA and ETHRB (Kim et al., 2006). Neurons expressing ETHRB are largely dispensable during larval life, but are essential for pupal ecdysis (Diao et al., 2016). Neurons expressing ETHRA include the neuroendocrine cells that secrete CCAP and Bursicon. These cells are likewise essential at pupal, but not larval, ecdysis (Clark et al., 2008; Park et al., 2003). They become active approximately 10 min after the onset of pupal ecdysis, and their activation mediates the transition to the next behavioral phase. Understanding how the nervous system transforms such hormonal signals into temporally ordered behavioral sequences will require a more complete description of the motor neuron activity that dictates the behavioral sequences themselves. Figure 1 Download asset Open asset Pupal ecdysis behavior and muscle anatomy. (A) Timeline of metamorphosis from puparium formation (PF at 0 hr) to eclosion (~100 hr) with the time of pupal ecdysis (arrow) indicated. Other significant events in pupal development include two major periods of tissue histolysis prior to and following pupal ecdysis as well as the times of larval-to-pupal and pupal-to-adult apolysis when separation from the cuticle of the previous developmental stage occurs. L3: third larval instar; APF: after puparium formation. (B) Timeline of pupal ecdysis with phases indicated. Arrows indicate times of hormone release. T = 0, onset of P1. (C) Schematics show puparium (brown) and pupa (beige) from dorsal view. Arrows indicate directionality of body wall movements for each phase. (D) Pupal muscles of hemisegment (HS) A3 stained with phalloidin (gray) and for postsynaptic marker DLG1 (cyan). Scale bar, 25 μm. (E) Pupal and larval musculature stained with phalloidin (gray) showing left HSA1–A7 (A3, boxed). Scale bar, 250 μm. (F) Larval muscles in a representative HS (e.g., dotted box in (E), right; based on six fillets). Outlined muscles degrade before pupal ecdysis. Muscles are labeled and coded blue (ventral), green (lateral), and fuchsia (dorsal). (G) Pupal muscles in a representative HS (e.g., dotted box in E, left; based on 17 fillets). Dotted lines indicate those present only in anterior segments. Colors as in (F). See also Video 1. Here, we use body-wide fluorescence imaging from the dorsal, lateral, and ventral views to characterize the pupal ecdysis sequence at single-cell resolution. Using improved imaging methods—including a new pan-muscle LexA driver that permits dual imaging of muscle and neuron activity—we identify novel elements of the pupal ecdysis sequence, including previously undescribed movements and a phase of stochastic muscle activity preceding ecdysis. We find that muscle activity exhibits a high degree of variability, with individual muscles recruited stochastically into repeating small ensembles, which we call syllables. Syllable activity is synchronized over anatomical compartments to form movements, which are sufficiently stereotyped to be learned by a convolutional neural network (CNN; for code see https://github.com/BenjaminHWhite/muscle_activity; copy archived at swh:1:rev:66456f6ff61e8faa9fe4b442b91ef3fce3b178f9, White, 2021). We can prevent synchronization at specific motor program transitions by suppressing neuromodulatory neurons, which is lethal. The suppression of proprioceptive neurons, which blocks initiation of pupal ecdysis, is also unexpectedly lethal. Overall, our analysis at single-cell resolution reveals a dynamical system in which movements are not rigidly specified but form from variable components subject to neuromodulatory reorganization. Results Pupal behavior described at cellular resolution Previous characterization of pupal ecdysis has distinguished three principal phases (Diao et al., 2017; Kim et al., 2006; Figure 1A–C, Video 1). The first phase (P1) consists of sustained longitudinal compression (‘lifting’) of posterior abdominal segments accompanied by unilateral, anteriorly propagating, ‘rolling contractions’ of the dorsal body wall that alternate left-to-right. The second (P2) features left-to-right alternating lateral ‘swinging’ movements formed by unilateral, anteriorly directed contractions, while the third (P3) consists of alternating left-right posteriorly directed contractions that change into bilaterally symmetric, backward peristaltic contractions. These phases always proceed in the same order. The movements increase internal pressure to evert the head (i.e., force it out of the body cavity) and push the developing legs and wings to the body surface and elongate them (Zdarek and Friedman, 1986). Phalloidin labeling demonstrates that approximately half of larval muscles persist until pupal ecdysis, and all retain innervation by Ib synapses (Prokop, 2006; Figure 1D, E). The most prominent loss of muscles occurs in the ventral and posterior compartments. Only 5 of 13 larval ventral muscles survive (Figure 1F vs. G), and one of these (M12) is absent in posterior segments (Supplementary file 1). Also missing from posterior segments are muscles M4 and M5. All five dorsal longitudinal muscles, except M4, are present in all segments, as are five of the six lateral transverse muscles. This was consistent across 17 animals, indicating that pupal ecdysis is executed by a standard set of persistent larval muscles. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Pupal ecdysis behavior (lateral view). Bright-field video taken from the lateral side showing a pupa performing the pupal ecdysis sequence. Phases 1–3 are labeled. Sped up to 50 fps. Scale bars, 250 μm. Time, seconds. Although Drosophila behavior has been recorded at single-cell resolution, the drivers used to make such recordings express weakly at the pupal stage and cannot be used with Gal4 drivers targeting other cells such as neurons. We identified a striated muscle-specific gene, l(2)01289 (aka CG9432), that expresses robustly from early larval stages through adulthood, which we call hulk (hlk). We generated a Trojan exon insertion in the hlk gene that co-expresses LexA:QF and combined it with the Ca++ biosensor LexAop-GCaMP6s to report muscle activity. With this hlk-LexA>LexAop-GCaMP6s line (i.e., hlk>GCaMP6s), we imaged muscle Ca++ activity through the clarified puparium for approximately 90 min to capture pupal ecdysis behavior, in vivo (see Materials and methods; Figure 2, Figure 2—figure supplements 1 and 2). Figure 2 with 2 supplements see all Download asset Open asset Pupal ecdysis muscle activity. (A) Muscle activity in a P1 bout (dorsal view) from a pupa expressing hlk>GCaMP6s. Schematic on left shows dorsal muscle anatomy. Trace above images shows bulk Ca++ activity signal with movements (see Table 1). Times in min:s format. Scale bar, 250 μm. T = 0, bout onset. (B) Time traces of bulk Ca++ activity on the left (black) and right (brown dotted) sides of a pupa executing the pupal ecdysis sequence and imaged from the dorsal side, as in (A). Alternating activity is evident. Dotted lines: onset of ecdysis phases. T = 0, imaging onset. (C) Time trace of bulk Ca++ activity from a pupa imaged from the lateral side. Dotted lines, onset of ecdysis phases. Arrowheads, peak-double peak bouts of late P3. T = 0, imaging onset. (D) Muscle activity in a P1 bout (lateral view) from a pupa expressing hlk>GCaMP6s. Times in min:s format. Schematic on left shows lateral muscle anatomy. Trace above images shows bulk Ca++ activity signal with movements (see Table 1). Scale bar, 250 μm. T = 0, bout onset. (E) Bouts in each phase (%) with P-to-A activity, such as shown in (D). N = 16 pupae. *p<0.05; **p<0.01. See also Figure 2—figure supplement 1, Table 1, and Videos 2 and 3 Data presented in this and other figures are available on Figshare DOI: https://doi.org/10.6084/m9.figshare.c.5489637.v1. The muscle activity patterns of animals imaged from the dorsal side match known behaviors, such as the bilateral posterior ‘Lifts’ and left-right alternating ‘rolling contraction’ (RollCon) movements of P1 (Figure 2A, Video 2). Temporal patterns of bulk Ca++ activity differentiated phases P1, P2, and P3 (Figure 2B). Traces show phase-specific oscillations of varying amplitude and frequency, with individual oscillations conforming to bouts of movement (see Materials and methods). Consistent with Diao et al., 2017, the alternating left-right oscillations of P1 persisted through P2 and P3, with coordinated bilateral activity becoming dominant only in P3. Bulk Ca++ activity imaged from the lateral side showed the same three phases (Figure 2C), and oscillations reflected bouts that included identifiable movements (Figure 2D). The Lift is performed by most muscles across the dorsal-ventral in the posterior segments, while typically activity in the ventral longitudinal muscles and of activity in P1 after head in P2 (Figure as previously shown (Kim et al., 2006). These data and previous and that the line the ecdysis sequence Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg P1 muscle activity. of a P1 bout with Lift and movements alternating bilaterally in a data were at 2 and up to 10 fps. Scale bars, 250 μm. Time, seconds. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg P1 muscle activity. of a P1 bout with Lift and movements in a data were at 2 and up to 10 fps. Scale bars, 250 μm. Time, seconds. Muscle imaging reveals an initial phase of activity Ca++ imaging a phase of muscle activity prior to the onset of pupal ecdysis (Figure which we call 0 begins approximately 3 before P1 and into distinct bouts of muscle activation (Figure Video muscle during are small to Figure supplement 1A) and with small body wall than movements. are also observed during motor development and are but become et al., 2008; et al., determine if muscle activation is or we a fly line with of the fluorescent Ca++ biosensor in the muscle and a protein in motor neurons, where it to the Figure In vivo imaging Ca++ activity at the min prior to the first muscle Ca++ response (Figure and muscle activity increases until the onset of P1, when all muscles and their are active (Figure except which remains to until P2 (Figure supplement muscle may as a for the ecdysis sequence and is by proprioceptive neurons, and as during larval et al., and present at the pupal stage at through ecdysis (Figure bulk Ca++ activity in sensory neurons is correlated with muscle activity during (Figure and sensory neurons show correlated Ca++ activity with muscles (Figure When class neurons were with Ca++ became sustained and during before (Figure supplement 2A, all animals before P1 = Figure 3 with 2 supplements see all Download asset Open asset muscle activity the ecdysis sequence. (A) of muscle Ca++ activity (lateral view) in five Muscle activity is distinct for each as image were to Scale bar, 250 μm. (B) Muscle and activity in body wall (HS) active Scale bar, μm. (C) generated from in and activity for the muscles and their P1 onset. T = 0, imaging onset. (D) for and activity in multiple during and late temporal to P1 onset. N = N = late N = (E) HS A3 from pupa expressing in class neurons using the driver et al., 2019). muscles Scale bar, 50 μm. (F) for bulk Ca++ activity in muscles labeled with and sensory neurons labeled with N = 10 pupae. (G) compare Ca++ activity of the muscle with those in an sensory neuron during T = 0, imaging onset. See also Figure supplements 1 and 2 and Video Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg muscle activity. of in a data were at 2 and up to 10 fps. Scale bars, 250 μm. Time, seconds. Muscle activity patterns identify movements The transition from to P1 is accompanied by the first movements, with bouts typically of a Lift by a The transition from P1 to P2 is by a behavioral in which a P1 bout is by bouts only a We used muscle activity patterns with body wall to define five movements, all executed in anatomical compartments (Figure Video Table 1). We also define a onset for P3, which previously been difficult (e.g., see Diao et al., 2017; Kim et al., 2006). Figure with 1 supplement see all Download asset Open asset Muscle activity patterns identify movements. (A) of muscle activity (lateral view) executed during labeled movements of the phases. Scale bars, 250 μm. (B) Muscle activity a from (A) by time showing the P-to-A of coordinated activation across the Times in min:s format. Schematic on left muscle anatomy. Scale bars, 250 μm. (C) Ca++ traces from a single bout for dorsal ventral and lateral muscles in hemisegment (HS) show activity across compartments. (D) and lateral P2 muscle Ca++ activity during the active lateral muscles, with movement on the side of the animal (dorsal view). White, dorsal Times in min:s format. Scale bars, 250 μm. T = 0, movement onset. See also Figure supplement 1 and Video Table 1 and function of pupal ecdysis movements. head, shed Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Muscle activity of the movements (lateral view) in a data were at 2 and up to 10 except the posterior which is 5 fps. Scale bars, 250 μm. Time, seconds. Muscle activity in a is coordinated across the as it anteriorly (Figure While initial are they after head (Kim et al., and are then accompanied by a movement we call the (Figure The is performed by of lateral transverse muscles and in anterior by of these same muscles in posterior (Figure lateral view). The begins the of activity from P-to-A in P1 to in P3. of P3 is by a movement we call the (Figure The first the P2 bout after a relatively long and ventral contractions in the posterior with dorsal contractions in the anterior The and complex movements that the include we call the and The first two have been (Kim et al., 2006). All of these movements are to P3. of the movements in Figure and Video 5 is with the activity of specific muscles, and we a to and the movements (Figure supplement Using the to movement (Figure supplement together with of bout and phase (see Materials and we characterized the variability of pupal ecdysis behavior at the level of and movements. stereotypy increases with spatiotemporal level of description The stereotypy of the pupal ecdysis sequence can be from bulk Ca++ traces (Figure but in the bout and of all phases (Supplementary file 2, Figure and in the bout number of P1 and P2 (Figure of were for P2 for all phase and bout (Supplementary file 2). This is consistent with the developmental of P2 and that its is the most of all the phases. also showed variability, with (Supplementary file 2). However, the in which movements were executed as by the (see Materials and considerable for movements were for all bouts within each animal by phase and across animals. The of the sequences for P1, P2, and P3 were all above a for (Figure with of P3 the and P2 the Figure 5 Download asset Open asset Temporal variability in muscle activity. (A) Muscle Ca++ traces for five representative pupae imaged from the lateral side. bulk moving Phases as indicated. T = 0, imaging all traces to P2 (B) and for pupae = *p<0.05; (C) of P1 and P2 activity bouts for pupae in (D) for movement sequences of P1, P2, and P3. See also 1 and the stereotypy of the muscle activation patterns used to generate individual movements, we also used the The in which muscles were in bouts of P1 yielded an of indicating However, this from that of