Nicotine-driven hyperactivation of larval locomotion
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eLife Assessment
The study presents useful findings on the behavioral effects of nicotine exposure, suggesting the Drosophila larva as a potential model organism for studying underlying neural circuits. However, the evidence supporting the claims of the authors is incomplete and would benefit from more rigorous analysis and explanations. The study falls short of identifying the neural mechanisms and is therefore of interest to those with an interest in pharmacology and behavior.
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Abstract
Balance between excitatory and inhibitory activity is essential for nervous system function. Neuroactive substances like nicotine can disrupt this balance and hyperactivate dopaminergic circuits, with combined effects on behavior and neural activity. We use Drosophila larvae to examine nicotine-induced, linked behavioral changes over multiple time scales by integrating high-resolution locomotor analysis with genetic and pharmacological manipulations. Acute nicotine exposure produces concentration-dependent hyperactivity. Manipulations of the dopaminergic system establish dopamine as the main mediator for motor responses, and further exploration establishes the γ lobe of the mushroom body as a key site for nicotine integration. Experiments with long-term and repetitive nicotine exposure suggest sustained circuit excitability. Finally, nicotine exposure history induces nicotine preference, highlighting experience-dependent plasticity contributing to addiction-like behaviors. These results help establish Drosophila larvae as a model organism to elucidate how neuroactive substances reconfigure neural circuits and behavior.
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Author response:
We appreciate the extremely helpful feedback from the reviewers and editors for our manuscript. We are happy that the reviewers have appreciated what we are doing here, performing the initial work that should set the stage with Drosophila larva as a model for hyperactive stimulant response. Every comment is certainly addressable within a reasonably short time period and we look forward to improving our paper in an upcoming revision.
We have some confusion about the “fundamental issue” of using nicotine, as we see the excitation as the fundamental effect we are studying, but we can continue to discuss and clarify this.
We plan to make significant edits to our introduction and background sections to better frame the goals of the work, and will clarify and expand on our methods, and more carefully make any claims about …
Author response:
We appreciate the extremely helpful feedback from the reviewers and editors for our manuscript. We are happy that the reviewers have appreciated what we are doing here, performing the initial work that should set the stage with Drosophila larva as a model for hyperactive stimulant response. Every comment is certainly addressable within a reasonably short time period and we look forward to improving our paper in an upcoming revision.
We have some confusion about the “fundamental issue” of using nicotine, as we see the excitation as the fundamental effect we are studying, but we can continue to discuss and clarify this.
We plan to make significant edits to our introduction and background sections to better frame the goals of the work, and will clarify and expand on our methods, and more carefully make any claims about neural mechanisms.
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eLife Assessment
The study presents useful findings on the behavioral effects of nicotine exposure, suggesting the Drosophila larva as a potential model organism for studying underlying neural circuits. However, the evidence supporting the claims of the authors is incomplete and would benefit from more rigorous analysis and explanations. The study falls short of identifying the neural mechanisms and is therefore of interest to those with an interest in pharmacology and behavior.
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Reviewer #1 (Public review):
Summary:
Dancausse et al. investigate behavioral responses to nicotine exposure in Drosophila larvae. They discover that high concentrations of nicotine lead to less movement and twitching, which recover slowly after several hours. Exposure to lower concentrations, however, increases locomotion and leads to hyperactive behavior. The authors also perform pharmacological and genetic manipulations to address the role of dopamine for these behavioral changes. Additionally, they test the role of MB intrinsic neurons by genetic silencing. Both Dopamine and MB manipulations affect responses to nicotine exposure. Finally, they investigate how larvae respond to repeated exposures to nicotine and find that they do not habituate. Additionally, repeated exposure to nicotine leads to a preference towards higher …
Reviewer #1 (Public review):
Summary:
Dancausse et al. investigate behavioral responses to nicotine exposure in Drosophila larvae. They discover that high concentrations of nicotine lead to less movement and twitching, which recover slowly after several hours. Exposure to lower concentrations, however, increases locomotion and leads to hyperactive behavior. The authors also perform pharmacological and genetic manipulations to address the role of dopamine for these behavioral changes. Additionally, they test the role of MB intrinsic neurons by genetic silencing. Both Dopamine and MB manipulations affect responses to nicotine exposure. Finally, they investigate how larvae respond to repeated exposures to nicotine and find that they do not habituate. Additionally, repeated exposure to nicotine leads to a preference towards higher concentrations in a gradient assay.
Strengths:
The authors use rigorous behavioral analysis and discover interesting concentration and experience-dependent effects of nicotine exposure on locomotion in fly larvae, which will be worth investigating in the future to decipher the underlying neural mechanism.
Weaknesses:
As the manuscript currently stands, the results of genetic manipulations are hard to interpret and rather inconclusive. The genetic manipulations have been performed using broadly expressing genetic driver lines, which weakens the conclusions drawn by the authors. Thus, no specific neural populations or brain regions have been discovered, and there is little insight into the underlying neural mechanism.
Based on gradient experiments, the authors suggest that fly larvae could serve as a model organism for addiction. This claim is quite strong, but no control experiments are shown for shorter exposure or a single exposure with a longer resting period before the gradient test. To compare this to addiction-like behaviors, more control experiments should be performed.
The authors should clarify better how experiments were performed in Materials and Methods. Generally, the authors perform novel behavioral analysis, which is not explained in enough detail. The nicotine concentration that has been used for most experiments is this a relevant concentration comparable to other studies? This information would be useful to put into context with other findings.
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Reviewer #2 (Public review):
Summary:
CNS function relies on a balance of excitatory and inhibitory activity. Use of addictive stimulants such as nicotine results in a chronic imbalance of these activities, and often this activity acts through dopamine pathways. To address how stimulants cause dysfunctional signaling in the DA neurotransmitter system and how this impacts neural circuit activity and behavior, the authors of this study begin to establish Drosophila larvae as a model for studying nicotine exposure.
They focus on three questions:
(1) In what ways does nicotine-driven hyperactivation modulate behavior?
(2) What roles do neural circuits play in these responses?
(3) What are the mechanisms of drug dependence and addiction-like plasticity?To this end, the authors use high-resolution behavioral, genetic, and pharmacological …
Reviewer #2 (Public review):
Summary:
CNS function relies on a balance of excitatory and inhibitory activity. Use of addictive stimulants such as nicotine results in a chronic imbalance of these activities, and often this activity acts through dopamine pathways. To address how stimulants cause dysfunctional signaling in the DA neurotransmitter system and how this impacts neural circuit activity and behavior, the authors of this study begin to establish Drosophila larvae as a model for studying nicotine exposure.
They focus on three questions:
(1) In what ways does nicotine-driven hyperactivation modulate behavior?
(2) What roles do neural circuits play in these responses?
(3) What are the mechanisms of drug dependence and addiction-like plasticity?To this end, the authors use high-resolution behavioral, genetic, and pharmacological methods.
The authors show that exposure to nicotine alters the behavioral repertoire of larval Drosophila, with effects that are long-lasting (hours) and dose-dependent. Most of the study uses a 5-minute exposure to "moderate" levels of nicotine because this dosage produces the greatest potentiation of larval crawling speed. Concomitant with increases in crawling speed, they find alterations in other behavioral parameters-crawl "efficiency" and turn rate are reduced; whereas head swings are faster and more likely to be accepted. They find that reducing the activity of dopaminergic neurons reverses the valence of behavioral change upon exposure to nicotine. For example, crawling speed is decreased upon nicotine exposure in a Ple>Kir2.1 manipulation in comparison to controls. Moreover, they demonstrate that the effect of nicotine on the quantified set of behaviors depends on dopamine signaling. Beyond implicating dopamine signaling, they implicate the mushroom body, and particularly the gamma-neurons, in mediating exposure to nicotine.
The authors further probe how nicotine exposure alters larval behavior. First, they determine what happens to crawling speed with multiple exposures, finding sustained higher crawling speeds relative to controls. Second, as a model for addition-like behavior, they examine larval behavior on a nicotine gradient after repeated nicotine exposure. The data in Figure 7D are particularly compelling, showing that after nicotine exposure, larvae prefer high concentrations of nicotine.
Strengths:
In a concise set of experiments, the authors demonstrate a nicotine-induced behavioral change, its interaction with a neurotransmitter system, and a locus of action within the CNS. Thus, the authors set the stage for the use of Drosophila larvae as a model to better understand addiction-related behaviors.
Weaknesses:
This is a clear advance for the field of larval neurogenetics, but the extent to which it changes the way we think about nicotine exposure more generally is less clear. Nonetheless, the authors clearly achieved the goal they set out to attain.
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Reviewer #3 (Public review):
Summary:
Dancausse et al. examine behavioral responses to nicotine administration in larvae. The study first distinguishes between spasms and extreme hyperexcitability elicited at high doses from a hyperactivity state triggered at lower (~1 mM feeding) doses. They then focus on the hyperactivity state and examine if dopaminergic neuron function is involved (via transgenic and pharmacological manipulations). Next, the role of the Mushroom body, a site of integration in the larval brain, is interrogated. In these studies, the authors use multiple approaches to draw complementary conclusions. The last section examines the effect of repeated nicotine exposure and of nicotine preference following repeated exposure. The findings are foundational for future studies looking to use Drosophila larvae as a system to …
Reviewer #3 (Public review):
Summary:
Dancausse et al. examine behavioral responses to nicotine administration in larvae. The study first distinguishes between spasms and extreme hyperexcitability elicited at high doses from a hyperactivity state triggered at lower (~1 mM feeding) doses. They then focus on the hyperactivity state and examine if dopaminergic neuron function is involved (via transgenic and pharmacological manipulations). Next, the role of the Mushroom body, a site of integration in the larval brain, is interrogated. In these studies, the authors use multiple approaches to draw complementary conclusions. The last section examines the effect of repeated nicotine exposure and of nicotine preference following repeated exposure. The findings are foundational for future studies looking to use Drosophila larvae as a system to study nicotine addiction.
Strengths:
Overall, I think the study is of broad importance. The neurogenetics community gets valuable insight into how ACh excitation interplays with DA signaling to regulate movement. For the addiction community, the work describes a valuable system to further interrogate genetic and environmental factors potentially driving addiction under well-controlled conditions. The quantitative analysis is generally well done, and the use of multiple experimental strategies to buttress conclusions is commendable.
Weaknesses:
(1) Conceptual point. Insects use ACh as the primary excitatory neurotransmitter, with nAChRs broadly expressed, while vertebrates use Glutamate in this role. (Arguably, nicotine expression in tobacco plants evolved as an insecticide, broadly disrupting the central excitatory neurotransmitter.) In vertebrates, central ACh neurons are relatively sparse - primarily originating from the basal forebrain.
Based on these distinctions, it is important to consider/contrast nicotine-driven hyperexcitation from other methods to produce broad hyperexcitation (e.g., inhibition of GABA, high K+, elevated temperature, etc). Many of these methods to induce hyperexcitability would also modulate DA circuitry.
A discussion of the role of ACh in insect vs. vertebrate brains is necessary to interpret the experimental design and findings with regard to addiction. These points should be addressed in the intro and discussion.
(2) (Figure 1) Relatedly, how do the behaviors elicited in Figure 1B (30 or 60 mM) compare to the convulsions described following electroshock stimulation to induce a seizure? My suspicion is that you're essentially triggering a seizure (or seizures) in these larvae.
(3) (Figure 4) Is a statistical analysis between the CS, Ple>Kir, Ple, and Kir locomotion at baseline done? Presumably, these manipulations would alter the intrinsic activity levels of the larvae?
(4) (General quantitative question) How do the parameters co-vary across individuals following nicotine admin? Crawl speed and peristalsis frequency are analyzed. Turning doesn't seem to be considered. Do individuals that show large increases in velocity also show the largest reductions in turn rate? Are these relations preserved following the DA metabolism and MB function interventions?
(5) (Discussion / general question) Beyond DA, other monoamines are involved in regulating larval locomotion - OA and TA are a clear example from Fox et al. (2006). Could the authors comment on whether they would expect similar findings in other neurotransmitter systems or if these neurotransmitter systems are involved in the ACh -> DA interplay studied here?
(6) (Discussion) Following the establishment of nicotine preference, do larvae exhibit signs of 'withdrawal' or changes in baseline behavior when deprived of nicotine? For example, in Figure 6, does the speed following nic administration ever 'go below' the H2O line?
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