Global change in brain state during spontaneous and forced walk in Drosophila is composed of combined activity patterns of different neuron classes
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eLife assessment
This paper expands on prior work by using whole-brain calcium imaging in Drosophila to examine how spontaneous and forced walking and turning affect neural activity in the brain. The measurements presented will serve as a valuable resource for the fly systems neuroscience community and suggest many testable hypotheses that may serve as the basis for future studies. Analyses of the data are solid, but conclusions drawn should be presented with more caveats. This article will be of interest to neuroscientists engaged with the central problem of how behavior modulates neural activity.
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Abstract
Movement-correlated brain activity has been found across species and brain regions. Here, we used fast whole brain lightfield imaging in adult Drosophila to investigate the relationship between walk and brain-wide neuronal activity. We observed a global change in activity that tightly correlated with spontaneous bouts of walk. While imaging specific sets of excitatory, inhibitory, and neuromodulatory neurons highlighted their joint contribution, spatial heterogeneity in walk- and turning-induced activity allowed parsing unique responses from subregions and sometimes individual candidate neurons. For example, previously uncharacterized serotonergic neurons were inhibited during walk. While activity onset in some areas preceded walk onset exclusively in spontaneously walking animals, spontaneous and forced walk elicited similar activity in most brain regions. These data suggest a major contribution of walk and walk-related sensory or proprioceptive information to global activity of all major neuronal classes.
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Author Response
Reviewer #1 (Public Review):
This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate …
Author Response
Reviewer #1 (Public Review):
This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate asymmetrically in concert with spontaneous or forced turning.
Strengths: Overall, the strength of this paper is in its careful descriptions and analyses of whole brain activation patterns that correlate with spontaneous and forced behaviors. Showing how the pattern of activity relates to broad classes of cells is also useful for understanding brain activation. Especially in brain regions identified as preceding spontaneous walking and in being asymmetrically involved in spontaneous and forced turning, it provides a wealth of potential hypotheses for new experiments. Overall, it contributes to a coarse-grained understanding of broad changes in brain activity during behavior.
Weaknesses: The primary weakness of this paper is that it presents some speculative interpretations and conclusions too strongly. Most importantly, average activity in a neuropil can represent the calcium activity of hundreds or thousands of neurons, and it is hard to know what fraction is active, for instance, or how expression pattern differences might play into calcium signals. Calcium signals also do not reliably indicate hyperpolarization, so a net increase in the average Ca++ indicator signal does not necessarily reflect that the average neuron is becoming more active, just that some labeled neurons are becoming more active, while others may be inactive or hyperpolarized. The conclusions about regions triggering walk (rather than just preceding it) are too strong for the manipulations in this paper, as are some of the links with individual neuron types. Thus, more presenting substantial caveats is required for the conclusions being drawn from the data presented here.
We thank the reviewer for their assessment and the positive comments on our manuscript. We have made these caveats clear throughout the manuscript by adding text and removing overly strong conclusions and speculations.
Reviewer #2 (Public Review):
Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future studies. Finally, the authors show that brain-wide activity is similar between spontaneous and forced walk and that severing the connection between the ventral nerve cord and central brain abolishes walk-related increases in brain activity. These results suggest that increases in brain-wide activity during walking may be largely attributed to sensory and proprioceptive feedback ascending to the central brain from the ventral nerve cord rather than to top-down executive and motor control programs. The observations presented in this study suggest hypotheses that may be tested in future studies.
Strengths: This paper presents a rich imaging dataset that is well-analyzed and cataloged, which will be valuable for researchers who use this paper for future hypothesis generation. The comparison of many different reagents, imaging speeds, and behavioral conditions suggests that the observed increases in brain-wide activity during walking are quite robust to imaging methods in adult fruit flies.
Weaknesses: This study is largely observational, and the few experimental manipulations presented are insufficient to support the author's broad claims about the generation of brain-wide neural activity.
We thank the reviewer for their assessment and have toned down claims throughout the paper accordingly.
Notably, the authors suggest that their image analysis can reveal individual cell types that are important for walking by matching their morphologies to registered components from whole-brain imaging experiments. While these predictions are a useful starting point for future experiments, they have not convincingly shown that their method can identify individual cell types in genetic reagents with more restricted expression patterns. Adding further validation to show that genetically subtracting the candidate neurons from the overall expression pattern of the calcium indicator abolishes that component from the response would strengthen this claim. Furthermore, imaging the matched candidate neuronal cell type to show that it recapitulates the activity dynamics of the proposed component would add additional evidence.
We agree that the correspondence to specific neuron types is often very speculative. We have clarified this throughout the manuscript. There are a few exceptions where the neurons we discuss are the only known neurons in a specific GAL4 expression pattern in a given region, and where we find the exact anatomical pattern matching these neurons’ anatomy. Together, this makes us quite confident that the activity results indeed from these neurons. However, the experiments proposed by the reviewer would be interesting complementary approaches. We believe, however, that abolishing activity in one neuron will be difficult to interpret regarding the neuron type as it would affect the activity of other neurons in the network (which is, in our opinion, an interesting point and research direction). Nevertheless, we plan to perform such experiments and experiments looking at the activity in more restricted drivers in the future.
In addition, increases in neural activity prior to walk onset in specific brain regions are intriguing but insufficient to demonstrate the neurons in these regions trigger walking. This claim should await further studies that employ targeted and acute manipulation of neural activity, as noted by the authors. Furthermore, that activity in these brain regions is significantly increased prior to walk onset awaits more rigorous statistical testing, as do the authors' claims that spontaneous versus forced walking alters these dynamics. The suggestion that walking increases brain-wide activity via feedback from the ventral nerve cord is an interesting possibility and would also benefit from additional experimental validation. Activating and silencing neurons that provide proprioceptive feedback from the legs and determining the effect of this manipulation on brain-wide neural activity would be a good starting point.
We have removed claims of causality in the result section. We have also added a statistical test for activation before walk onset. Activating and silencing proprioceptive neurons from the legs would be interesting follow up experiments although it is likely to affect walking. Nevertheless, we are planning to carry out such experiments in the future. We have added this point in the discussion.
Reviewer #3 (Public Review):
Aimon and colleagues investigated brain activity in flies during spontaneous and forced walking. They used light-field microscopy to image calcium activity in the brain at high temporal resolution as the animal walked on a ball and they used the statistical inference methods PCA and ICA to tease out subregions of the brain that had distinct patterns of activity. They then sought to relate those patterns to walking. Most interesting are the experiments they performed comparing forced walking to spontaneous walking because this provides a framework to generate hypotheses about which aspects of neural activity are reporting the animal's movements versus generating those movements. The authors identify subregions and neuron types that may be involved in generating vs reporting walking. Their analysis is reasonable but could be further strengthened with a more powerful statistical framework that explicitly considered the multiple hypotheses being tested. More broadly, the work serves as a starting point to investigate the role of different regions in the brain and should spur follow-up investigations that involve more perturbative approaches in addition to the correlative approaches presented here.
We thank the reviewer for their overall positive assessment of our work and fully agree with the conclusion of its current limitations.
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eLife assessment
This paper expands on prior work by using whole-brain calcium imaging in Drosophila to examine how spontaneous and forced walking and turning affect neural activity in the brain. The measurements presented will serve as a valuable resource for the fly systems neuroscience community and suggest many testable hypotheses that may serve as the basis for future studies. Analyses of the data are solid, but conclusions drawn should be presented with more caveats. This article will be of interest to neuroscientists engaged with the central problem of how behavior modulates neural activity.
-
Reviewer #1 (Public Review):
This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate asymmetrically in concert with …
Reviewer #1 (Public Review):
This paper uses light field microscopy to measure calcium signals across the fly brain while it is walking and turning, and also while the fly is externally driven to walk and turn, using a treadmill. The authors drive calcium indicator expression using pan-neuronal drivers, as well as drivers specific to individual neurotransmitters and neuromodulators. From their experiments, the authors show that inhibitory and excitatory neurons in the brain are activated in similar patterns by walking and that neurons expressing machinery for different neuromodulatory amines tend to show differentially strong calcium signals during walking. By examining spontaneous and forced walking and turning, the authors identify brain regions that activate before spontaneous turning and that activate asymmetrically in concert with spontaneous or forced turning.
Strengths: Overall, the strength of this paper is in its careful descriptions and analyses of whole brain activation patterns that correlate with spontaneous and forced behaviors. Showing how the pattern of activity relates to broad classes of cells is also useful for understanding brain activation. Especially in brain regions identified as preceding spontaneous walking and in being asymmetrically involved in spontaneous and forced turning, it provides a wealth of potential hypotheses for new experiments. Overall, it contributes to a coarse-grained understanding of broad changes in brain activity during behavior.
Weaknesses: The primary weakness of this paper is that it presents some speculative interpretations and conclusions too strongly. Most importantly, average activity in a neuropil can represent the calcium activity of hundreds or thousands of neurons, and it is hard to know what fraction is active, for instance, or how expression pattern differences might play into calcium signals. Calcium signals also do not reliably indicate hyperpolarization, so a net increase in the average Ca++ indicator signal does not necessarily reflect that the average neuron is becoming more active, just that some labeled neurons are becoming more active, while others may be inactive or hyperpolarized. The conclusions about regions triggering walk (rather than just preceding it) are too strong for the manipulations in this paper, as are some of the links with individual neuron types. Thus, more presenting substantial caveats is required for the conclusions being drawn from the data presented here.
-
Reviewer #2 (Public Review):
Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future …
Reviewer #2 (Public Review):
Aimon et al. used fast whole-brain imaging to investigate the relationship between walking and neural activity in adult fruit flies. They find that increases in brain-wide activity are tightly correlated with walking behavior, and not with grooming or flailing, and are independent of visual input. They reveal that excitatory, inhibitory, and neuromodulatory neurons all contribute to brain-wide increases in neural activity during walk. Aimon et al. extend their observations of brain-wide activity to reveal that activity in some inferior brain regions is more correlated with walk than in other brain regions. The authors further analyzed their imaging dataset to identify candidate brain regions and cell types that may be important for walking behavior, which will be useful in hypothesis generation in future studies. Finally, the authors show that brain-wide activity is similar between spontaneous and forced walk and that severing the connection between the ventral nerve cord and central brain abolishes walk-related increases in brain activity. These results suggest that increases in brain-wide activity during walking may be largely attributed to sensory and proprioceptive feedback ascending to the central brain from the ventral nerve cord rather than to top-down executive and motor control programs. The observations presented in this study suggest hypotheses that may be tested in future studies.
Strengths: This paper presents a rich imaging dataset that is well-analyzed and cataloged, which will be valuable for researchers who use this paper for future hypothesis generation. The comparison of many different reagents, imaging speeds, and behavioral conditions suggests that the observed increases in brain-wide activity during walking are quite robust to imaging methods in adult fruit flies.
Weaknesses: This study is largely observational, and the few experimental manipulations presented are insufficient to support the author's broad claims about the generation of brain-wide neural activity.
Notably, the authors suggest that their image analysis can reveal individual cell types that are important for walking by matching their morphologies to registered components from whole-brain imaging experiments. While these predictions are a useful starting point for future experiments, they have not convincingly shown that their method can identify individual cell types in genetic reagents with more restricted expression patterns. Adding further validation to show that genetically subtracting the candidate neurons from the overall expression pattern of the calcium indicator abolishes that component from the response would strengthen this claim. Furthermore, imaging the matched candidate neuronal cell type to show that it recapitulates the activity dynamics of the proposed component would add additional evidence.
In addition, increases in neural activity prior to walk onset in specific brain regions are intriguing but insufficient to demonstrate the neurons in these regions trigger walking. This claim should await further studies that employ targeted and acute manipulation of neural activity, as noted by the authors. Furthermore, that activity in these brain regions is significantly increased prior to walk onset awaits more rigorous statistical testing, as do the authors' claims that spontaneous versus forced walking alters these dynamics. The suggestion that walking increases brain-wide activity via feedback from the ventral nerve cord is an interesting possibility and would also benefit from additional experimental validation. Activating and silencing neurons that provide proprioceptive feedback from the legs and determining the effect of this manipulation on brain-wide neural activity would be a good starting point.
-
Reviewer #3 (Public Review):
Aimon and colleagues investigated brain activity in flies during spontaneous and forced walking. They used light-field microscopy to image calcium activity in the brain at high temporal resolution as the animal walked on a ball and they used the statistical inference methods PCA and ICA to tease out subregions of the brain that had distinct patterns of activity. They then sought to relate those patterns to walking. Most interesting are the experiments they performed comparing forced walking to spontaneous walking because this provides a framework to generate hypotheses about which aspects of neural activity are reporting the animal's movements versus generating those movements. The authors identify subregions and neuron types that may be involved in generating vs reporting walking. Their analysis is …
Reviewer #3 (Public Review):
Aimon and colleagues investigated brain activity in flies during spontaneous and forced walking. They used light-field microscopy to image calcium activity in the brain at high temporal resolution as the animal walked on a ball and they used the statistical inference methods PCA and ICA to tease out subregions of the brain that had distinct patterns of activity. They then sought to relate those patterns to walking. Most interesting are the experiments they performed comparing forced walking to spontaneous walking because this provides a framework to generate hypotheses about which aspects of neural activity are reporting the animal's movements versus generating those movements. The authors identify subregions and neuron types that may be involved in generating vs reporting walking. Their analysis is reasonable but could be further strengthened with a more powerful statistical framework that explicitly considered the multiple hypotheses being tested. More broadly, the work serves as a starting point to investigate the role of different regions in the brain and should spur follow-up investigations that involve more perturbative approaches in addition to the correlative approaches presented here.
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