Synchronous multi-segmental activity between metachronal waves controls locomotion speed in Drosophila larvae

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    Exploiting the power of the Drosophila larva as a model, Liu et al.'s important study sheds light on the neuronal mechanisms of speed regulation during locomotion. The data obtained using a combination of functional and structural approaches are mostly rigorous and convincing, but there are concerns about the small number of animals analysed in some of the behavioural experiments.

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The ability to adjust the speed of locomotion is essential for survival. In limbed animals, the frequency of locomotion is modulated primarily by changing the duration of the stance phase. The underlying neural mechanisms of this selective modulation remain an open question. Here, we report a neural circuit controlling a similarly selective adjustment of locomotion frequency in Drosophila larvae. Drosophila larvae crawl using peristaltic waves of muscle contractions. We find that larvae adjust the frequency of locomotion mostly by varying the time between consecutive contraction waves, reminiscent of limbed locomotion. A specific set of muscles, the lateral transverse (LT) muscles, co-contract in all segments during this phase, the duration of which sets the duration of the interwave phase. We identify two types of GABAergic interneurons in the LT neural network, premotor neuron A26f and its presynaptic partner A31c, which exhibit segmentally synchronized activity and control locomotor frequency by setting the amplitude and duration of LT muscle contractions. Altogether, our results reveal an inhibitory central circuit that sets the frequency of locomotion by controlling the duration of the period in between peristaltic waves. Further analysis of the descending inputs onto this circuit will help understand the higher control of this selective modulation.

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  1. eLife assessment

    Exploiting the power of the Drosophila larva as a model, Liu et al.'s important study sheds light on the neuronal mechanisms of speed regulation during locomotion. The data obtained using a combination of functional and structural approaches are mostly rigorous and convincing, but there are concerns about the small number of animals analysed in some of the behavioural experiments.

  2. Reviewer #1 (Public Review):

    Liu et al, use a combination of Calcium imaging, behavioral analysis, optogenetics, and synaptic connectivity analysis to investigate the neuronal mechanisms underlying the regulation of speed during crawling in Drosophila larvae. They find that the larva, although a crawling animal uses a similar strategy as limb animals to control locomotion speed, by differentially varying the different phases of the locomotion cycle. Similar to limbed animals, in which the stance phase is varied, while the swing phase remains mostly constant, in Drosophila larva where crawling is generated through waves of propagation along the body, the duration of the inter-wave phase is varied. The precise techniques in the larva allowed the authors to tackle the mechanisms underlying this type of speed regulation. In a behavioral screen, they identified one type of GABA-ergic neuron (A31c) that is activated during the inter-wave phase, synchronously across multiple segments. They found this neuron is GABA-ergic. Using EM connectomics they identified one of its post-synaptic partners: another GABA-ergic neuron: A26f that strongly innervates the transverse motor neurons that contract perpendicular to the crawling direction. A26f also shows synchronized activity across multiple segments. Activating and silencing A26f using optogenetics they determine that A26f is required and sufficient to modulate the amplitude and duration of the contraction of LT muscles. This type of implementation of speed regulation based on inhibitory neural circuit motifs could be a general neural circuit mechanism shared across species.

    The paper uses a combination of cutting-edge techniques to investigate the mechanisms of speed regulation in Drosophila larvae and makes parallels in the mechanics of the Drosophila larva crawling and limbed locomotion. While the evidence is compelling and the analysis rigorous, at times the presentation and writing could be clearer.

  3. Reviewer #2 (Public Review):

    In general, the authors do a thorough exploration of Drosophila larval crawling, specifically looking at where within the crawl cycle changes in speed are manifest. They find a strong correlation between changes in speed and with contraction of transverse muscles. The authors then characterize a neuron (A31c) known to be activated during the time in the crawl cycle where speed is altered. Unlike many neurons that have a wave of activity across the segments of the body during crawling, these neurons are active synchronously in the segments along the anterior-posterior axis. Also, the A31c neurons have synaptic output onto pre-motor neurons (A26f) whose output ultimately controls transverse muscles.

    Experimentally, the authors use all the tools at hand to make a compelling case for their conclusions. They look at crawling in freely crawling animals. Although the data on muscle length is obtained in restrained or semi-dissected animals, it is convincing. It would be more convincing if done in intact freely moving animals, but that is a technical reach. To look at neuron morphology they use immunofluorescence and connectomic data in an effective manner. They are able to manipulate the activity of their neurons of interest in order to show that they have control over transverse muscle length and crawling speed.

    Finally, the authors claim that the neurons they examine are sufficient and required to alter crawling speed. They appear to be sufficient to alter transverse muscle length and crawling speed, but the data do not support the requirement for these neurons for these processes. One way to show this would be to inhibit or ablate the neurons and find that the larvae were unable to alter the speed.

  4. Reviewer #3 (Public Review):

    The paper from Liu et al. investigates the mechanism of speed control in the fruit fly where movement is generated by the coordination of contraction across several segments (peristaltic wave). They show very convincing behavioural data demonstrating that the interwave phase is regulated to control speed and that one of the Lateral transverse muscles (LT2) is constantly contracted during this period. They describe two presynaptic inhibitory neurons to LT2 motorneuron (A31c and A26f) that have patterns of activity that suggest they could be involved in the process. When the neuronal activity has manipulated the contraction of LT2, the interwave time and the speed of locomotion seems to be modified. The data regarding the pattern of activity of A31c and A26f neurons in the isolated nervous system is not completely convincing due to the clear overlap of the neuronal activity with the contraction of abdominal segments. Also, the n number of certain behavioural experiments is very low. Overall, it is a very interesting paper that describes a new mechanism of speed control, but several points need to be solved.

    The main strength of the paper is that it describes a new mechanism of speed control. The behaviour data demonstrating that the time of the interwave is modified to control speed during crawling is very convincing.

    Then, they analysed the contraction of LT2 muscles and found that they only contract during the interwave phase. The behavioural setup and the tool to evaluate muscle contraction (tendon driver) are different from the ones that have been used in the past, but the striking difference in the pattern of contraction should have been explored in more detail. For example, repeating the experiment with a muscle reporter, or analysing more precisely the movies from Figures 5, 6, or 7. More clarity regarding the difference between their data and the data showing how LT muscles contract during the wave (Heckscher et al 2012; Zwart et al. 2016; Zarin et al. 2019) is needed.

    Liu et al. also discover two inhibitory neurons that are connected with the MN21-22 and could potentially control speed. They perform behavioural optogenetic experiments inhibiting or increasing neuronal activity and found interesting data that strongly suggest that the neurons are indeed controlling the interwave time. However, doubts are raised by the low n number of animals analysed (n often = 5 or 7). In order to compensate for a low n number the authors decided to use bootstrapping, but working with Drosophila they should have increased the number of animals analysed.
    These behavioural experiments are the strongest evidence the authors have of the mechanism of control of speed, they should be immutable.

    Another weakness is that the phase of activity of A31c and A26f neurons overlaps with part of the peristaltic wave, not matching the suggested pattern of activity during the interwave phase. When observing waves in fictive crawling (for example the long recordings from Pulver et al. 2015), it seems that there is no interwave time and that waves happen one after the other in bouts. It is possible that the sensory feedback is essential to set the interwave time and that the slightly un-phased activity is due to this lack. The authors do not give us any explanation. Is the activity of the Bar-H1+ motor neurons inhibited when A31c activity is high? What happens when A26f neurons are active? Or is it that the role of these neurons is somewhat different from what is stated? What is the actual phase relationship between A26f and A31c? Figure 4F shows us two different segments the A31c presynaptic that has an anteriorly projecting connection in a4 and the postsynaptic in a5. We should see the pattern of expression of all the segments where A26f is expressed.

    Overall, the paper is very interesting data but more rigour in the description and interpretation of the data is required. Also, a few replicates are needed to confirm what, at the moment, are very suggestive data.