Intensity coded octopaminergic modulation of aversive crawling behavior in Drosophila melanogaster larvae
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Summary: This manuscript addresses an interesting question of how octopaminergic neurons regulate locomotor rhythms. Despite the interesting topic, the reviewers raised technical and mechanistic concerns that need to be addressed.
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Abstract
Activation and modulation of sensory-guided behaviors by biogenic amines assure appropriate adaptations to changes in an insect’s environment. Given its genetic tool kit Drosophila melanogaster represents an excellent model organism to study larger networks of neurons by optophysiological methods. Here, we studied stationary crawling movements of 3 rd instar larvae and revealed how the octopaminergic VUM neuron system reacts during crawling behavior and tactile stimulations. We conducted calcium imaging experiments on dissections of the isolated nervous system (missing all sensory input) and found spontaneous rhythmic wave pattern of neuronal activity in VUM neuron clusters over the range of thoracic and abdominal neuromeres in the VNC. In contrast, in vivo preparations (semi-intact animals, receiving sensory input) did not reveal such spontaneous rhythmic pattern. However, tactile stimulations activated different clusters of the VUM neuron system simultaneously in these preparations. The activation intensity of VUM neurons in the VNC was correlated with the location and degree of body wall stimulation. While VUM neuron cluster near the respective location of body wall stimulation were less activated more distant cluster showed stronger activation. Repeated gentle touch stimulations led to decreased response intensities, repeated harsh stimulations resulted in increasing intensities over trials. Optophysiological signals correlated highly with crawling behavior in freely moving larvae stimulated similarly. We conclude that the octopaminergic system is strongly coupled to the neuronal pattern generator of crawling movements and that it is simultaneously activated by physical stimulation, rather intensity than sequential coded. We hope that our work raises the interest in whole biogenic network activity and shows that octopamine release does not only underlie “the more the better” principle but instead has a more complex function in control and modulation of insect’s locomotion.
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Reviewer #2:
This short study highlights the complexity of the octopaminergic system in insect behavior. This aspect of neuromodulation has received little attention in comparison with the role of dopamine in learning and motivation. The main question being addressed is whether, how and where octopamine modulates the generation of rhythmic behavior (peristalsis) upon noxious sensory stimulation (touch and pain). Using a combination of functional imaging and behavioral inspections, the authors explore the role of octopamine released by the VUM neurons on the escape crawling behavior of the Drosophila larva.
The specific observations reported in the study are:
Isolated larval CNS preparations that do not receive sensory input (deafferented preps) show spontaneous rhythmic wave patterns of neuronal activity in octopaminergic VUM neuron …
Reviewer #2:
This short study highlights the complexity of the octopaminergic system in insect behavior. This aspect of neuromodulation has received little attention in comparison with the role of dopamine in learning and motivation. The main question being addressed is whether, how and where octopamine modulates the generation of rhythmic behavior (peristalsis) upon noxious sensory stimulation (touch and pain). Using a combination of functional imaging and behavioral inspections, the authors explore the role of octopamine released by the VUM neurons on the escape crawling behavior of the Drosophila larva.
The specific observations reported in the study are:
Isolated larval CNS preparations that do not receive sensory input (deafferented preps) show spontaneous rhythmic wave patterns of neuronal activity in octopaminergic VUM neuron cluster.
In vivo preps that receive sensory input did not show spontaneous rhythmic patterns in the neural activity of the VUM neuron cluster.
The VUM neurons show weaker responses in clusters that get sensory input from physically stimulated body segments and stronger responses in clusters that get input from segments further away from stimulated segments.
In functional (GCaMP) imaging experiments, repeated gentle (rod) touch stimulations led to decreased VUM response intensities. Repeated harsh (brush) stimulations resulted in increasing VUM intensities. The authors correlate these physiological observations of the VUM activity with an increase in crawling speed upon repeated harsh stimulations, and a decrease in crawling speed upon repeated gentle touch stimulations.
Based on observations (4), the authors propose that the differences in the behavior elicited by series of gentle touch and harsh stimulations are due to differences in adaptation of two classes of mechanosensory neurons. The class III da neurons responsible for detecting gentle touch would quickly adapt, whereas the class IV da neurons responsible for detecting harsh touch would integrate neural activity over time. The authors also conclude that (i) the octopaminergic system is strongly coupled to the CPG underlying peristalsis and (ii) "it is simultaneously activated by physical stimulation, rather intensity than sequential coded" (line 53). The first conclusion is supported by observations (1-2). While the involvement of octopamine in the modulation of a key CPG of the larva is a certainly interesting result, it represents the starting point of a mechanistic inspection. The problem is that the rest of the study falls short of testing or establishing any concrete mechanism.
Although the topic of this study is exciting and its results are generally promising, the work is largely inconclusive. In addition, some conclusions are phrased in a way that is cryptic. For instance, I found it difficult to decipher the meaning of "the octopaminergic system is simultaneously activated by physical stimulation, rather intensity than sequential coded" (line 53). This conclusion appears to contradict the observation that repeated gentle touch stimulations produce a gradual decrease in the overall activity of VUM neurons. In the discussion section, the authors nicely refer to published findings in stick insects, honey beers and locusts. Compared to these systems, the advantage of Drosophila is that it offers the neuro-genetic tools to shed mechanistic insights into the molecular and cellular bases of neuromodulation.
Questions and mechanisms that the authors might have wanted to address at a mechanistic level:
Re. observations (1-2): What explains the observation that sensory inputs present in in-vivo preps abolish the spontaneous rhythmic pattern in the VUM activity? How does this relate to the VUM activity elicited by the tactile stimulations presented in Fig 3?
It would be important to establish the importance of the VUM activity on peristalsis through loss of function experiments. Expression of Tdc2 could be restrictive to the VNC by using tshirt-Gal4. These experiments would support the authors' proposal that octopamine is released to facilitate motor coordination (in lines 474-478).
Technical concerns:
-How can you rule out that the mini-stage featured in the in-vivo prep (Fig 2A) does not sever nervous fibers innervating the VNC? The plate placed under the CNS is very large. It is difficult to believe that this plate can be inserted while leaving all nerves (afferent and efferent neurons) intact on both sides. The integrity of the preparation should be controlled anatomically.
-In Fig 2, a statistical analysis should be performed to establish a lack of correlation between the VUM activity and patterns of crawling. Trial 2.2 suggests the existence of some correlation. This correlative analysis would be important to back up the statement that "unstimulated larvae showed no consistent VUM neuron responses correlated to crawling movements" (lines 228-229; see also lines 235-236).
-Lines 234-236: How can "movements" be assessed in an isolated deafferented prep?
Re. observation (3): Do the mechanosensory inputs have an inhibitory effect on the VUM activity patterns? If so, how does the inhibition come about?
How do you explain that harsh stimulation at the posterior end inhibits activity of both the most abdominal and thoracic segments? Does this imply that the t1 and a8 segments are somehow coupled?
In line 400, the authors propose that "VUM neurons as one possible system to modulate either indirectly the endogenous input or directly the central pattern generating neurons as a response to external tactile stimulation of the body wall." How does this model and subsequent discussion fit with the observations of Fig 3? It would be helpful to test the validity of the two alternatives described in line 400.
Technical concerns:
-Line 292: The segments displaying highest activity upon tactile stimulations are said to be consistent across consecutive simulations. Are they consistent across preparations as well? Were the data of Fig 3 generated on more than one prep?
-Are the results of Fig 3 dependent on the strength of the tactile stimulations? More than one intensity should be tested to rule out intensity coding, as is stated in the abstract (lines 53 and 55).
Re. observation (4): One of the observations reported in Fig 3 is that posterior harsh stimulations produce an overall increase in VUM activity whereas anterior harsh stimulation produce a decrease in activity. In Fig 4, larvae undergo harsh physical stimulations. However, it is unclear whether the harsh stimulations are applied to the posterior or anterior end of the larva. Based on the physiological results of Fig 3, wouldn't the authors expect that harsh stimulations of the head/neck region should lead to a deceleration of the larva, as was observed for gentle touch? Couldn't this prediction be tested experimentally? For the same reason, stating in line 512 that the same stimulation is used to activate the VUM neurons in Fig 3 and Fig 4 is misleading.
The discussion about the adaptive nature of the class III and IV da neurons is compelling. However it ought to be supported by more direct experimental evidence that could be collected in the Drosophila larva.
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Reviewer #1:
In this work the authors measure the activity of the octopaminergic VUM neurons that arborize throughout the somatic body wall muscles in the Drosophila larva. They use three different larval preparations: isolated CNS (no sensory afferents), semi-intact (CNS exposed while maintaining sensory input), and intact. They find that isolated CNS has rhythmic waves of activity in the VUM neurons, but that semi-intact preparations do not show rhythmic VUM activity. They also show that "harsh" or "gentle" touch elicits different responses in VUM neurons.
There are several interesting findings. The ability of VUM neurons to show rhythmic activity in the isolated CNS is a novel finding. It would be even more interesting to register these waves to that of the glutamatergic body wall motor neurons that drive locomotion. It is also …
Reviewer #1:
In this work the authors measure the activity of the octopaminergic VUM neurons that arborize throughout the somatic body wall muscles in the Drosophila larva. They use three different larval preparations: isolated CNS (no sensory afferents), semi-intact (CNS exposed while maintaining sensory input), and intact. They find that isolated CNS has rhythmic waves of activity in the VUM neurons, but that semi-intact preparations do not show rhythmic VUM activity. They also show that "harsh" or "gentle" touch elicits different responses in VUM neurons.
There are several interesting findings. The ability of VUM neurons to show rhythmic activity in the isolated CNS is a novel finding. It would be even more interesting to register these waves to that of the glutamatergic body wall motor neurons that drive locomotion. It is also interesting that touch applied to an anterior segment results in elevated VUM activity in a posterior segment, and conversely posterior touch leads to elevated VUM activity in an anterior segment, suggesting that sensory input dampens VUM activity.
There are also issues that need to be addressed, which are listed below.
The function of the VUM neurons in locomotion was not tested, e.g. by silencing or activating them. These experiments would greatly strengthen the paper.
The three larval preparations are poorly described. (a) The fictive preparation is clearest but still should have a citation to Pulver 2015 at first use, as that paper provides a detailed description of the isolated CNS prep. (b) The semi-intact prep is not well described: is the CNS pulled from the body? How can this be done without ripping the nerves? How can the intactness of the nerves be validated? (c) The intact prep sounds simple, but how is VUM GCaMP3 fluorescence measured in an intact larva as shown in Figure 4? Is the "intact" prep the same as the "in vivo" prep? One name should be used throughout for clarity.
The semi-intact prep showed Ca++ signals in only 5% of the preps. This makes me worried that the prep is unhealthy, and that the data from the 5% are not physiological.
Experiment 1 shows four individuals, but population data for all larvae were not shown. Selecting only a subset of the analyzed larvae is not appropriate; data from all should be shown.
Experiment 2 shows low resolution data (left) that is not interpretable. The data highlighted in the right panel is much better but again, only three examples are presented; no population data or statistics are shown.
It is also unclear how many larvae were analyzed in Experiment 2. Line 163 says "...~5% of the in vivo preparations (n=27)..." but is that 1/27 or 27/540? In addition, are the different stimulation patterns done sequentially on the same larva, or independently on different larvae?
The prep used for Experiment 3 is not mentioned. Not in the text, not in the figure legend.
The prep for Experiment 4 appears to be the intact larva, but if so, how were GCaMP signals measured? How were movement artifacts handled?
In Experiment 4, the term "crawling frequency" is not defined. Is it frequency that locomotion is initiated?
How do the authors standardize harsh and gentle touches?
It says "in very rare cases..." on line 246. Please give actual numbers.
The figures are cited out of order (1, 3, 2, 4).
Many references are missing in the first part of the Introduction, e.g. lines 64. 65, 73, 78, and 83.
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Summary: This manuscript addresses an interesting question of how octopaminergic neurons regulate locomotor rhythms. Despite the interesting topic, the reviewers raised technical and mechanistic concerns that need to be addressed.
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