Pupal behavior emerges from unstructured muscle activity in response to neuromodulation in Drosophila
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Curated by eLife
Evaluation Summary:
This paper will be of interest of neuroscience and ethology, as it bridges the gap between traditional and computational ethology by performing high-resolution imaging of muscle activity as an indicator of motor neuron activity, enabling the identification and analysis of behavior with computer algorithms. It also revealed the role of hormones in shaping and modifying the nervous system and animal behavior. The authors' major claims are supported by the data collected from thoughtfully designed animal experiments and computational analysis, although a few results can be interpreted better.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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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.
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Author Response:
Reviewer #1 (Public Review):
This paper examines muscle activity at single muscle level during Drosophila ecdysis (adult hatching) behavior. The premise is that quantifying behavior or motor neuron activity is insufficient to understand how the CNS generates behavior - it is also critical to quantify muscle activity. They show that abdominal body wall muscles generate stereotyped patterns of activity during four developmental stages; (phase 0, stochastic activity; phase 1-3, each with different patterns of activity. Co-active groups of muscles form "syllables" which are used in different combinations to generate the stereotyped activity seen in phases 1-3. This analysis was facilitated by use of a convoluted neural network. Interestingly, they found examples where muscle contraction did not match muscle activity …
Author Response:
Reviewer #1 (Public Review):
This paper examines muscle activity at single muscle level during Drosophila ecdysis (adult hatching) behavior. The premise is that quantifying behavior or motor neuron activity is insufficient to understand how the CNS generates behavior - it is also critical to quantify muscle activity. They show that abdominal body wall muscles generate stereotyped patterns of activity during four developmental stages; (phase 0, stochastic activity; phase 1-3, each with different patterns of activity. Co-active groups of muscles form "syllables" which are used in different combinations to generate the stereotyped activity seen in phases 1-3. This analysis was facilitated by use of a convoluted neural network. Interestingly, they found examples where muscle contraction did not match muscle activity (GCaMP elevation), showing the importance of measuring both attributes.
In addition to mapping the stereotyped muscle activity at single muscle resolution in the generation of ecdysis behavior, they find that phase 1 and 3 are quite variable, and speculate that other constraints on the CNS output (e.g. during larval locomotion) may prevent a sharpening up of muscle patterns. They show that the hormone ETH is required for initiating phase 1, and the neuromodulators bursicon and CCAP are required for initiating phase 2. Failure to initiate either phase is lethal. Lastly, they show that in addition to initiating phase 1 or 2, the hormone/neuromodulators result in more coherent muscle activity.
Overall this study sets the stage for a detailed analysis of motor neuron function in driving muscle activity patterns, and then further into the CNS to understand the role of premotor neurons. Ecdysis behavior has the potential to be a powerful system for understanding how the CNS generates behavior at the single muscle /single motor neuron level, as well as for understanding how neuromodulators act to regulate muscle/motor neuron activity.
The figures are almost all too small to see the salient information, and the color scheme is often difficult to resolve. Please enlarge the key aspects of the figures; and try to use more distinctive colors where critical comparisons need to be made. Some examples: left/right colored lines in 1G; panel 3D; lines in 3E; all data in 5G (this is the worst for tiny data); 6C,D,J; all of 7.
Thank you for your thoughtful review and your suggestions on how to improve the manuscript. Some figure panels (e.g. 5G) have been completely replaced. The others mentioned have been divided into multiple figures or panels, which allowed us to enlarge the material in each. Fig. 7 was deleted from the revised manuscript because it was generally found unhelpful. We also felt that the other revisions rendered this figure unnecessary. The revised manuscript now has 11 main figures and 9 figure supplements with more generous layouts for individual panels so that details are more easily resolved. In addition, we attempted to improve the color scheme to facilitate clarity, using the color palette recommended for the color-blind. Other specific changes are referenced in our responses to individual concerns below.
Reviewer #2 (Public Review):
The manuscript by Diao et al. is an important extension of their eLife paper of 2017. Their development of new tools that allow them to follow Ca2+ transients in single muscle fibers over the whole animal through the behavioral sequence and also to independently monitor the Ca2+ transients in the endplates of the motor neurons that innervate these muscles. Their goal is to break down the movements that control the ecdysis sequence into elemental "syllables" and then to defined the role of these syllables in constructing progressively complex behavioral programs and as targets of neuropeptide modulation.
A crucial behavior that occurs during P1 in higher flies is the movement of the gas bubble but this event is largely ignored in the paper. Prior to pupal ecdysis, gas is expelled into the posterior puparial space and then actively translocated, via muscular contractions of the body wall, to the anterior end of the puparium during the latter portion of P1 (shown nicely in the author's 2017 Video). A detailed study by C.G. Chadfield & J.C.Sparrow (1985. Dev. Genetics 5: 103) of pupal ecdysis in Drosophila emphasized the importance of this translocation for head eversion. When they simply removed the operculum at the start of bubble movement, then the gas bubble could not push the animal backwards in the puparial case and head eversion could not occur. However, they saw normal pupation and head eversion if the removed operculum was immediately replaced and sealed down with petroleum jelly.
During translocation, the bubble moves in a fragmented fashion between the pupal cuticle and the puparium. Ignoring this movement leads to statements like on line 378 "Because pupal ecdysis is independent of environmental factors and executed in the absence of competing physiological needs, it is likely that its variability is intrinsic to the ecdysis network." For the pupating animal, its "environment" is the inside of the puparial case and the moving bubble is an unpredictable variable in this environment. The trajectory and route of bubble movement is not fixed, and it is likely that variation in sensory feed-back from the gas movement explains the motor variability and reduced stereotypy during P1. The role for proprioception during this phase is likely to inform the CNS of the progression of the bubble fragments. The author's finding that the blockage of proprioceptors suppresses the behavior progression could mean that this sensory information is needed to signal that an anterior space has been produced, and without this signal, the behavior does not progress to its next phase. This should be addressed in the text if not experimentally.
We very much appreciate the reviewer’s point that the environment within the puparium may affect the pupa’s motor performance. We have now amended our comment on environmental influences to include this point (ll. 479-481 [515-517]), and we elaborate in the Discussion on conditions within the puparium that may influence movement and sensory processing (ll. 457-477 [493-513]). Following the reviewer’s advice, we note that the gas bubble and its dispersion during P1 must be considered a possible determinant of pupal movement. In addition, we mention other possible determinants that we did not previously discuss, namely substrate and surface tension interactions between the body wall, puparium, and residual molting fluid. In line with the Reviewer’s point that understanding the environment of the puparium is critical, we stress the need to account for all external forces acting on the pupal body to achieve a complete understanding of the pupal motor output. In the Discussion, we also now mention the Reviewers’ interesting hypothesis that creation of the anterior space at the end of P1 may provide sensory information necessary for progression of the behavioral sequence (ll. 534-535 [601-602])
Another aspect of the background that is missing is considering earlier studies on the ontogeny of behaviors leading up to ecdysis/hatching. Notable are studies of the progressive construction of the flight motor program during metamorphosis in moths (Kammer & Rheuben 1976 J. Exp. Biol. 65:65.) and a similar feature of assembly of motor programs prior to hatching in Drosophila (Crisp et al., 2008 Development 135:3707). In the moth studies, complex motor programs were gradually assembled during ontogeny with motor neurons firing but without muscle contraction (as the authors see in prepupae during P0 - Fig 2C). A lack of excitation-contraction coupling in the moth prevents muscle movement through most of development. This suppression of contraction is essential because prior to production of adult cuticle, muscle contraction would rip the developing animal apart. The same requirement to suppress muscle contraction would be seen in fly prepupa until sufficient pupal cuticle has been secreted to prevent rupture from actual muscle contractions! This should be addressed in the text.
We thank the reviewer for his comments and for the references on motor program assembly. We agree that this is topic deserved more attention than it was originally given. We have now amended our discussion of P0 to contextualize our observations, pointing to the previous literature on both suppressed muscle activity and latent motor programs observed in other developing animals (ll. 487-500 [523-536]).
Besides not being explicit about how the syllables combine to build the eight basic movements, it is not clear how these basic movements then combine to support the major behaviors of each phase. This is seen in P1, where we see that swing and brace movements can co-occur (e.g., Fig 3D) but is a swing on one side always associated with a brace on the other? What are their phase relationships? Does their temporal association remain stable as the bouts progress? Another example is in Phase 3. There appear to be 5 basic behaviors associated with bouts in Phase 3. The example in Fig 1H shows double peak bouts in phase 3, and the bulk Ca data show a preponderance of double peaks. The different shapes suggest that there are different movements during the two peaks. Their discussion of P3 movements (around line 273), though, does not address this feature of the double peaks. The example in Fig 7A suggests that some movements, like the PostSwing occur at half the frequency of other movements such as the PostCon and AntComp. Is this the basis of the double peaks and how is that reflected in the movements that are finally produced? This should be addressed in the text.
We regret the confusion on these points. As described there, we have made numerous changes to the manuscript to clarify how elements of behavior at one level (e.g. movements) derive from lower-level elements (e.g. syllables) and are used to build higher-level elements (e.g. phases). We describe the phase relationships at all levels for P1 and P2 and summarize the more variable constituents of P3 movements in the text (Figs. Fig. 7D, E and ll. 247-275 [274-302]). The specific questions raised by the reviewer are also now answered in the text. In brief, early P2 bouts (roughly those prior to head eversion) differ from later bouts in containing only a Swing. Later bouts contain in addition to the Swing a Brace performed concomitantly on the contralateral side of the body (l. 182-183 [197-199]). The movements contributing to the peak-double peak motif common to P3 are now more carefully described at ll. 351-360 [383-393])
One approach that I did not find useful was dividing the analysis into compartments - anterior versus posterior and dorsal-lateral-ventral. This may provide a way of generating some statistical analysis, but it did not illuminate anything about the behavior. The line between anterior and posterior segments seems to be arbitrary. Of course, it is important to know if there is directionality of movement [waves going anteriorly versus posteriorly], but beyond that, I am not sure what it adds. [Indeed, it made Fig 7 very confusing!] Also, I could not see a rationale for considering separate dorsal-lateral-ventral compartments. This should be addressed in the text.
We thank the reviewer for this question, which we now address in a revised section of the Discussion on the topic of neuromodulation and compartmentalization (ll. 539-588 [606-655]). To briefly expand upon our explanation there, we think that compartmental activity allows a useful coarse-grained description of the sequential body wall contractions that give rise to movement as indicated by the SequenceMatcher similarity scores (Fig. 6E in the revised manuscript). Second, and more important, we think that how activity flows across compartments provides clues about both the central organization and the neuromodulatory control of ecdysis behavior. Both ETHRB and CCAP neuron suppression exert selective effects on A-P compartments. ETHRB neuron suppression blocks the Lift, a movement of the posterior compartment, while suppressing CCAP neurons prematurely terminates the first (and only) swing-like movement by blocking its progression into the anterior compartment. Additionally, the distribution of CCAP-R appears to reflect mechanisms for selectively regulating distinct D-V compartments. Myotopic maps of larval motor neuron dendrites show that MNs innervating dorsal and ventral muscles are spatially segregated from those innervating lateral muscles and have distinct inputs. This suggests distinct regulation of activity in D-V and L compartments and likely distinct functions. Importantly, CCAP-R is expressed only in motor neurons of the D and V compartments, but in the L compartment it is expressed in muscles. As we suggest, this may allow the different regulatory mechanisms of compartmental regulation to synergize during P2. Finally, our subdivision of the A-P axis at the boundary between segments 5 and 6 has both anatomical and functional importance. At the pupal stage, selective muscle loss imposes differences in muscle composition of segments anterior and posterior to this boundary. Most importantly, anterior segments contain M12, which is a major contributor to behavior only after P1 and is targeted by neuromodulatory Type III terminals containing CCAP and Bursicon. In addition, the A-P boundary also conforms to the functionally and neuroanatomically defined “hinge” region of Tastekin et al. (2018, eLife,), which regulates the switch from forward to backward movement in the larva. Because the compartmental subdivisions we define conform with neuroanatomical differences and appear to underlie functional differences, our working hypothesis is that they will be important landmarks for mapping behaviorally relevant CNS activity as we begin to image it in the next phase of our work.
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Reviewer #3 (Public Review):
Neurobiologists direct a fundamental level of questions to ask how the brain controls sequential patterns of muscle activation and thus organizes coordinated movements. This paper addresses that problem in an insect model system and it focuses on the behavioral sequence by which the larval fruit fly transforms to a largely inert, intermediate life stage, called the pupa. Insect development is an exceptionally useful context in which to study the sequence of events that define specific ontological epochs. Other systems share many of the same developmental sequalae, but in insects the developmental progression of events proceeds at a largely invariant pace, and so it assures a highly-predictable timetable which helps to make observations and propose correlations. The work by Elliott and colleagues takes …
Reviewer #3 (Public Review):
Neurobiologists direct a fundamental level of questions to ask how the brain controls sequential patterns of muscle activation and thus organizes coordinated movements. This paper addresses that problem in an insect model system and it focuses on the behavioral sequence by which the larval fruit fly transforms to a largely inert, intermediate life stage, called the pupa. Insect development is an exceptionally useful context in which to study the sequence of events that define specific ontological epochs. Other systems share many of the same developmental sequalae, but in insects the developmental progression of events proceeds at a largely invariant pace, and so it assures a highly-predictable timetable which helps to make observations and propose correlations. The work by Elliott and colleagues takes advantage of the invariant insect development sequence to ask a neuroethological question - how does the coordination of muscle activation arise that underlies pupal ecdysis (emergence) in the fruit fly Drosophila? The authors use an innovative combination of calcium imaging, multi-viewpoint, whole animal muscle recording, genetic manipulations and convolutional analysis. They present a comprehensive overview of the individual motor elements and the emergence of regulatory coordination in neuromuscular physiology that lead invariably to the ecdysial behavioral events. This paper is successful in presenting a model whereby complex and coordinate behaviors arise during an invariant developmental sequence from what is termed seemingly uncorrelated patterns of muscle activation. Further they investigate the molecular basis for the emerging regulatory coordination and underscore the influence of specific regulatory peptides and peptide hormones. The authors do an excellent job making a detailed, data-rich and highly specific set of observations accessible to a general audience. That accessibility, and the novel approach of analyzing behavior at the resolution of single muscle cells, should promote consideration of similar developmental sequences in other model systems.
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Reviewer #2 (Public Review):
The manuscript by Diao et al. is an important extension of their eLife paper of 2017. Their development of new tools that allow them to follow Ca2+ transients in single muscle fibers over the whole animal through the behavioral sequence and also to independently monitor the Ca2+ transients in the endplates of the motor neurons that innervate these muscles. Their goal is to break down the movements that control the ecdysis sequence into elemental "syllables" and then to defined the role of these syllables in constructing progressively complex behavioral programs and as targets of neuropeptide modulation.
A crucial behavior that occurs during P1 in higher flies is the movement of the gas bubble but this event is largely ignored in the paper. Prior to pupal ecdysis, gas is expelled into the posterior puparial …
Reviewer #2 (Public Review):
The manuscript by Diao et al. is an important extension of their eLife paper of 2017. Their development of new tools that allow them to follow Ca2+ transients in single muscle fibers over the whole animal through the behavioral sequence and also to independently monitor the Ca2+ transients in the endplates of the motor neurons that innervate these muscles. Their goal is to break down the movements that control the ecdysis sequence into elemental "syllables" and then to defined the role of these syllables in constructing progressively complex behavioral programs and as targets of neuropeptide modulation.
A crucial behavior that occurs during P1 in higher flies is the movement of the gas bubble but this event is largely ignored in the paper. Prior to pupal ecdysis, gas is expelled into the posterior puparial space and then actively translocated, via muscular contractions of the body wall, to the anterior end of the puparium during the latter portion of P1 (shown nicely in the author's 2017 Video). A detailed study by C.G. Chadfield & J.C.Sparrow (1985. Dev. Genetics 5: 103) of pupal ecdysis in Drosophila emphasized the importance of this translocation for head eversion. When they simply removed the operculum at the start of bubble movement, then the gas bubble could not push the animal backwards in the puparial case and head eversion could not occur. However, they saw normal pupation and head eversion if the removed operculum was immediately replaced and sealed down with petroleum jelly.
During translocation, the bubble moves in a fragmented fashion between the pupal cuticle and the puparium. Ignoring this movement leads to statements like on line 378 "Because pupal ecdysis is independent of environmental factors and executed in the absence of competing physiological needs, it is likely that its variability is intrinsic to the ecdysis network." For the pupating animal, its "environment" is the inside of the puparial case and the moving bubble is an unpredictable variable in this environment. The trajectory and route of bubble movement is not fixed, and it is likely that variation in sensory feed-back from the gas movement explains the motor variability and reduced stereotypy during P1. The role for proprioception during this phase is likely to inform the CNS of the progression of the bubble fragments. The author's finding that the blockage of proprioceptors suppresses the behavior progression could mean that this sensory information is needed to signal that an anterior space has been produced, and without this signal, the behavior does not progress to its next phase. This should be addressed in the text if not experimentally.
Another aspect of the background that is missing is considering earlier studies on the ontogeny of behaviors leading up to ecdysis/hatching. Notable are studies of the progressive construction of the flight motor program during metamorphosis in moths (Kammer & Rheuben 1976 J. Exp. Biol. 65:65.) and a similar feature of assembly of motor programs prior to hatching in Drosophila (Crisp et al., 2008 Development 135:3707). In the moth studies, complex motor programs were gradually assembled during ontogeny with motor neurons firing but without muscle contraction (as the authors see in prepupae during P0 - Fig 2C). A lack of excitation-contraction coupling in the moth prevents muscle movement through most of development. This suppression of contraction is essential because prior to production of adult cuticle, muscle contraction would rip the developing animal apart. The same requirement to suppress muscle contraction would be seen in fly prepupa until sufficient pupal cuticle has been secreted to prevent rupture from actual muscle contractions! This should be addressed in the text.
Besides not being explicit about how the syllables combine to build the eight basic movements, it is not clear how these basic movements then combine to support the major behaviors of each phase. This is seen in P1, where we see that swing and brace movements can co-occur (e.g., Fig 3D) but is a swing on one side always associated with a brace on the other? What are their phase relationships? Does their temporal association remain stable as the bouts progress? Another example is in Phase 3. There appear to be 5 basic behaviors associated with bouts in Phase 3. The example in Fig 1H shows double peak bouts in phase 3, and the bulk Ca data show a preponderance of double peaks. The different shapes suggest that there are different movements during the two peaks. Their discussion of P3 movements (around line 273), though, does not address this feature of the double peaks. The example in Fig 7A suggests that some movements, like the PostSwing occur at half the frequency of other movements such as the PostCon and AntComp. Is this the basis of the double peaks and how is that reflected in the movements that are finally produced? This should be addressed in the text.
One approach that I did not find useful was dividing the analysis into compartments - anterior versus posterior and dorsal-lateral-ventral. This may provide a way of generating some statistical analysis, but it did not illuminate anything about the behavior. The line between anterior and posterior segments seems to be arbitrary. Of course, it is important to know if there is directionality of movement [waves going anteriorly versus posteriorly], but beyond that, I am not sure what it adds. [Indeed, it made Fig 7 very confusing!] Also, I could not see a rationale for considering separate dorsal-lateral-ventral compartments. This should be addressed in the text.
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Reviewer #1 (Public Review):
This paper examines muscle activity at single muscle level during Drosophila ecdysis (adult hatching) behavior. The premise is that quantifying behavior or motor neuron activity is insufficient to understand how the CNS generates behavior - it is also critical to quantify muscle activity. They show that abdominal body wall muscles generate stereotyped patterns of activity during four developmental stages; (phase 0, stochastic activity; phase 1-3, each with different patterns of activity. Co-active groups of muscles form "syllables" which are used in different combinations to generate the stereotyped activity seen in phases 1-3. This analysis was facilitated by use of a convoluted neural network. Interestingly, they found examples where muscle contraction did not match muscle activity (GCaMP elevation), …
Reviewer #1 (Public Review):
This paper examines muscle activity at single muscle level during Drosophila ecdysis (adult hatching) behavior. The premise is that quantifying behavior or motor neuron activity is insufficient to understand how the CNS generates behavior - it is also critical to quantify muscle activity. They show that abdominal body wall muscles generate stereotyped patterns of activity during four developmental stages; (phase 0, stochastic activity; phase 1-3, each with different patterns of activity. Co-active groups of muscles form "syllables" which are used in different combinations to generate the stereotyped activity seen in phases 1-3. This analysis was facilitated by use of a convoluted neural network. Interestingly, they found examples where muscle contraction did not match muscle activity (GCaMP elevation), showing the importance of measuring both attributes.
In addition to mapping the stereotyped muscle activity at single muscle resolution in the generation of ecdysis behavior, they find that phase 1 and 3 are quite variable, and speculate that other constraints on the CNS output (e.g. during larval locomotion) may prevent a sharpening up of muscle patterns. They show that the hormone ETH is required for initiating phase 1, and the neuromodulators bursicon and CCAP are required for initiating phase 2. Failure to initiate either phase is lethal. Lastly, they show that in addition to initiating phase 1 or 2, the hormone/neuromodulators result in more coherent muscle activity.
Overall this study sets the stage for a detailed analysis of motor neuron function in driving muscle activity patterns, and then further into the CNS to understand the role of premotor neurons. Ecdysis behavior has the potential to be a powerful system for understanding how the CNS generates behavior at the single muscle /single motor neuron level, as well as for understanding how neuromodulators act to regulate muscle/motor neuron activity.
The figures are almost all too small to see the salient information, and the color scheme is often difficult to resolve. Please enlarge the key aspects of the figures; and try to use more distinctive colors where critical comparisons need to be made. Some examples: left/right colored lines in 1G; panel 3D; lines in 3E; all data in 5G (this is the worst for tiny data); 6C,D,J; all of 7.
-
Evaluation Summary:
This paper will be of interest of neuroscience and ethology, as it bridges the gap between traditional and computational ethology by performing high-resolution imaging of muscle activity as an indicator of motor neuron activity, enabling the identification and analysis of behavior with computer algorithms. It also revealed the role of hormones in shaping and modifying the nervous system and animal behavior. The authors' major claims are supported by the data collected from thoughtfully designed animal experiments and computational analysis, although a few results can be interpreted better.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed …
Evaluation Summary:
This paper will be of interest of neuroscience and ethology, as it bridges the gap between traditional and computational ethology by performing high-resolution imaging of muscle activity as an indicator of motor neuron activity, enabling the identification and analysis of behavior with computer algorithms. It also revealed the role of hormones in shaping and modifying the nervous system and animal behavior. The authors' major claims are supported by the data collected from thoughtfully designed animal experiments and computational analysis, although a few results can be interpreted better.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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