Multiple neuronal networks coordinate Hydra mechanosensory behavior
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Summary: Specifically, all of the reviewers agreed that the emerging Hydra system holds great promise for neuroscience discoveries. Moreover, some of the findings presented here have the potential to be of use to other scientists who work in this system. However, we felt that the findings here were too preliminary and underdeveloped. In particular reviewers felt that 1) multiple locations across the Hydra's body should be stimulated coupled with mapping the behavioral and neuronal correlates of such stimulation, 2) the pan-neuronal nature of the bulk calcium measurements made it challenging to fully appreciate which neuronal circuits might be driving the sensorimotor responses, 3) uniform proxies for measuring/plotting the behavior would be useful, 4) the ablation studies lacked cellular resolution, similar to the calcium imaging experiments.
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Abstract
Hydra vulgaris is an emerging model organism for neuroscience due to its small size, transparency, genetic tractability, and regenerative nervous system; however, fundamental properties of its sensorimotor behaviors remain unknown. Here, we use microfluidic devices combined with fluorescent calcium imaging and surgical resectioning to study how the diffuse nervous system coordinates Hydra 's mechanosensory response. Mechanical stimuli cause animals to contract, and we find this response relies on at least two distinct networks of neurons in the oral and aboral regions of the animal. Different activity patterns arise in these networks depending on whether the animal is contracting spontaneously or contracting in response to mechanical stimulation. Together, these findings improve our understanding of how Hydra ’s diffuse nervous system coordinates sensorimotor behaviors. These insights help reveal how sensory information is processed in an animal with a diffuse, radially symmetric neural architecture unlike the dense, bilaterally symmetric nervous systems found in most model organisms.
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Reviewer #3:
The authors probe mechanosensory processing in Hydra by measuring calcium activity in neurons and muscles in response to precise mechanosensory stimulation in whole and resected animals. The authors' claims are well supported by the evidence. The development of a mechanosensory delivery system for Hydra is also a significant methodological advancement. Taken together, the work advances our understanding of the Hydra nervous system and is a needed step towards developing Hydra as a powerful model for systems neuroscience.
Substantive concerns:
One weakness is that different measures of "mechanosensory response" are used at different places in the manuscript. In some contexts, a response is defined as calcium activity in neurons (Fig 2), and elsewhere as calcium activity in muscles (Fig 3 and 4). And in Fig2 SuppFig2 muscle …
Reviewer #3:
The authors probe mechanosensory processing in Hydra by measuring calcium activity in neurons and muscles in response to precise mechanosensory stimulation in whole and resected animals. The authors' claims are well supported by the evidence. The development of a mechanosensory delivery system for Hydra is also a significant methodological advancement. Taken together, the work advances our understanding of the Hydra nervous system and is a needed step towards developing Hydra as a powerful model for systems neuroscience.
Substantive concerns:
One weakness is that different measures of "mechanosensory response" are used at different places in the manuscript. In some contexts, a response is defined as calcium activity in neurons (Fig 2), and elsewhere as calcium activity in muscles (Fig 3 and 4). And in Fig2 SuppFig2 muscle contractions are also measured using MeKs. The relation between neural activity, muscle activity and body movement is of course of high interest, and the paper explores this. But, if technically possible, it would be helpful to report a single metric of behavior that could be used in all experiments. For example, it might be possible to use video of the animal's pose or body length to measure contractions in all experiments. At a minimum the reasoning behind choice of measurement of response for each experiment could be discussed explicitly.
Related: Without a consistent measure of behavior, it will be important to further clarify figures so that a reader can tell at-a-glance how contraction probability is being measured.
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Reviewer #2:
The Hydra, in the phylum cnidaria, is a near microscopic freshwater animal that has recently resurfaced as an attractive model organism in neuroscience due to its optically accessible transparent body, sparsely distributed neural network, and simple behaviors. In this manuscript, Badhiwala and colleagues use calcium imaging of the Hydra neural network, combined with surgical resection and microfluidics pressure stimulation to identify body regions indispensable for mechanosensory activity. They report that while resection of the aboral region did not abolish the mechanical response, resection of the oral region attenuated this response, while combined resection of oral and aboral regions showed the greatest effect. They also find a correlation between reduced stimulated activity and spontaneous activity, suggesting a common …
Reviewer #2:
The Hydra, in the phylum cnidaria, is a near microscopic freshwater animal that has recently resurfaced as an attractive model organism in neuroscience due to its optically accessible transparent body, sparsely distributed neural network, and simple behaviors. In this manuscript, Badhiwala and colleagues use calcium imaging of the Hydra neural network, combined with surgical resection and microfluidics pressure stimulation to identify body regions indispensable for mechanosensory activity. They report that while resection of the aboral region did not abolish the mechanical response, resection of the oral region attenuated this response, while combined resection of oral and aboral regions showed the greatest effect. They also find a correlation between reduced stimulated activity and spontaneous activity, suggesting a common mechanism that gives rise to both activities. While this study takes on an innovative approach by using a microfluidics device to mechanically stimulate the hydra under optical recording there are a number of conceptual and technical limitations. Perhaps my biggest reservation is that despite real potential, the data are rather low resolution (body transections and bulk calcium responses) and as such the conclusions that can be reasonably drawn do not extend what is known in a significant way.
Major comments:
The authors have designed a microfluidic device that allows them to simultaneously mechanically stimulate, monitor movement and functionally image a hydra. The highly quantifiable nature of the microfluidic device is a great asset, although this potential is not deeply explored. While I can see how the microfluidic stimulation could offer benefits over fluid jet or blunt probe, more in-depth characterization is needed.
What is the spatial distribution of the pressure pulse stimulus on the Hydra body? How far does the mechanical force spread from the region directly touching the pressure valve?
The use of the microfluidic device was limited. Have the authors attempted to map mechanical sensitivity across the Hydra body by stimulating different sites?
The authors have not attempted to record calcium responses from single neurons, but rather spatially average a population response from a large region of interest. This should be specifically stated in the results section. More importantly, to provide insight into network function much smaller ROIs over multiple sites are needed instead of the bulk activity of the entire peduncle. This seems like a real lost opportunity as the lure of the optically clear and small hyda is that neural representation and coding can be tracked over large portions of the network at cellular resolution.
It is unclear where the recorded signals are coming from and if movement is creating artifacts. Have authors made any attempts to correct for movement? The supplemental movies show a stationary region of interest and moving animal, in some cases parts of animal moving in and out. Furthermore, is background subtracted and how? There is a large fluorescent signal coming from the entire body/ middle columnar part of the body and spontaneous firing that makes interpretation of the data difficult.
Contraction is a behavioral response of the animal; however, the authors use 'contraction' do describe calcium imaging responses throughout the figures and text. This should be avoided.
I am unsure if the title of the paper is accurate. I do not think this work has demonstrated "multiple nerve rings" are important for coordinating mechanosensory behavior.
Furthermore, the claim that the observed "linear relationship" between the spontaneous contraction probability and resection type is evidence for shared neural pathways is a stretch. These data are fairly coarse resolution and include only 3 animals in each group with highly variable responses (Figure 4C). Additionally, they do not provide evidence to distinguish the motor circuits they hypothesized these neural nets converge upon.
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Reviewer #1:
The manuscript by Badhiwala et al. is an interesting study using the emerging model system Hydra, which has many advantages for studying the entire nervous system of an animal during simple behavior. Some of the foundational neuroscience papers in this field have only come out in the past few years, and new studies such as the one here, might have the potential to contribute to an important early literature. Despite clear reasons for enthusiasm, the many shortcomings in this work greatly diminished my enthusiasm and support for this study. Although I appreciate building the microfluidic devise with simultaneous pan-neuronal imaging, the nature of the new biological insights provided here seems quite limited and easily predicted based on prior studies in hydra and other model systems. Moreover, the crude nature of some …
Reviewer #1:
The manuscript by Badhiwala et al. is an interesting study using the emerging model system Hydra, which has many advantages for studying the entire nervous system of an animal during simple behavior. Some of the foundational neuroscience papers in this field have only come out in the past few years, and new studies such as the one here, might have the potential to contribute to an important early literature. Despite clear reasons for enthusiasm, the many shortcomings in this work greatly diminished my enthusiasm and support for this study. Although I appreciate building the microfluidic devise with simultaneous pan-neuronal imaging, the nature of the new biological insights provided here seems quite limited and easily predicted based on prior studies in hydra and other model systems. Moreover, the crude nature of some experiments inhibits my ability to make fair judgement of potential findings.
Major concerns:
The pressurized stimulation of the hydra appeared to be specific to the center of the body. The authors don't mention why this region was chosen, which seems critical to this study. Relatedly, why didn't they test multiple areas across the hydra with this system? Might we expect to see different sensorimotor behaviors, and thus different neural outputs?
The authors reference a recent single cell study characterizing multiple neuronal cell types in hydra. This work would greatly benefit by using some cell-type resolution studies to determine the functional nature of the neurons being activated as opposed to solely using pan-neuronal GCAMP imaging. If they can put GCAMP in all neurons, why not put it in specific subsets of neurons based on cellular identity? This point becomes more salient because a major take-home from this paper is that the spontaneous behavior and firing patterns is nearly identical to the stimulus evoked patterns, except for an apparent increase in firing rate. The true nature of the mechanosensory response might be revealed with cell-type specific experiments.
Although the authors reference whole animal imaging, they focus imaging analysis on peduncle and hypostomal nerve rings, despite the videos showing calcium activity in other areas throughout the body. Moreover, are the authors certain their pan-neuronal genetic strategy equally samples neurons throughout the body? In other words, is the apparent increase in activity in the nerve ring over other areas driven by a technical artifact of these neurons being labeled better?
While I appreciate the resection studies to get at "loss-of-function" experiments, this approach seems rather crude, and potentially confounding to clear interpretation. Exactly which neurons are killed and to what extent, and how many, if any began to regenerate throughout this process? My alarm here is raised especially in light of the author's surprising finding that "footless" animals show that the aboral nerve ring is not required for spontaneous or mechanosensory responses. What if residual activity from neurons not ablated is driving this response?
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Summary: Specifically, all of the reviewers agreed that the emerging Hydra system holds great promise for neuroscience discoveries. Moreover, some of the findings presented here have the potential to be of use to other scientists who work in this system. However, we felt that the findings here were too preliminary and underdeveloped. In particular reviewers felt that 1) multiple locations across the Hydra's body should be stimulated coupled with mapping the behavioral and neuronal correlates of such stimulation, 2) the pan-neuronal nature of the bulk calcium measurements made it challenging to fully appreciate which neuronal circuits might be driving the sensorimotor responses, 3) uniform proxies for measuring/plotting the behavior would be useful, 4) the ablation studies lacked cellular resolution, similar to the calcium imaging …
Summary: Specifically, all of the reviewers agreed that the emerging Hydra system holds great promise for neuroscience discoveries. Moreover, some of the findings presented here have the potential to be of use to other scientists who work in this system. However, we felt that the findings here were too preliminary and underdeveloped. In particular reviewers felt that 1) multiple locations across the Hydra's body should be stimulated coupled with mapping the behavioral and neuronal correlates of such stimulation, 2) the pan-neuronal nature of the bulk calcium measurements made it challenging to fully appreciate which neuronal circuits might be driving the sensorimotor responses, 3) uniform proxies for measuring/plotting the behavior would be useful, 4) the ablation studies lacked cellular resolution, similar to the calcium imaging experiments.
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