Early-life experience reorganizes neuromodulatory regulation of stage-specific behavioral responses and individuality dimensions during development

  1. Reemy Ali Nasser
  2. Yuval Harel
  3. Shay Stern

  • Curated by eLife

    eLife logo

    eLife assessment

    Early life stress can have profound effects on animal behavior, including potential influences on individuality. In this valuable work, the authors use a rich new dataset to solidly demonstrate that the behavioral consequences of early life stress in C. elegans can be buffered by neuromodulators previously implicated in patterns of individuality. While much remains to be learned about the mechanisms by which stress might influence individuality, these studies provide an important entry point that will be of interest to neurobiologists studying interactions between behavior, neuromodulation, stress, and individuality.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Early-life experiences may promote stereotyped behavioral alterations that are dynamic across development time, but also behavioral responses that are variable among individuals, even when initially exposed to the same stimulus. Here, by utilizing longitudinal monitoring of Caenorhabditis elegans individuals throughout development we show that behavioral effects of early-life starvation are exposed during early and late developmental stages and buffered during intermediate stages of development. We further found that both dopamine and serotonin shape the discontinuous behavioral responses by opposite and temporally segregated functions across development time. While dopamine buffers behavioral responses during intermediate developmental stages, serotonin promotes behavioral sensitivity to stress during early and late stages. Interestingly, unsupervised analysis of individual biases across development uncovered multiple individuality dimensions that coexist within stressed and unstressed populations and further identified experience-dependent effects on variation within specific individuality dimensions. These results provide insight into the complex temporal regulation of behavioral plasticity across developmental timescales, structuring shared and unique individual responses to early-life experiences.

Article activity feed

  1. Version published to 10.7554/elife.84312 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In this interesting manuscript, Nasser et al explore long-term patterns of behavior and individuality in C. elegans following early-life nutritional stress. Using a rigorous, highly quantitative, high-throughput approach, they track patterns of motor behavior in many individual nematodes from L1 to young adulthood. Interestingly, they find that early-life food deprivation leads to decreased activity in young larvae and adults, but that activity between these times, during L2-L4, is largely unaffected. Further, they show that this "buffering" of stress requires dopamine signaling, as L2-L4 activity is significantly reduced by early-life starvation in cat-2 mutants. The paper also provides evidence that serotonin signaling has a role in modulating sensitivity to stress in L1 larvae and adults, but the size of these effects is modest. To evaluate patterns of individuality, the authors use principal components analysis to find that three temporal patterns of activity account for much of the variation in the data. While the paper refers to these as "individuality types," it may be more reasonable to think of these as "dimensions of individuality." Further, they provide evidence that stress may alter the strength and/or features of these dimensions. Though the circuit mechanisms underlying individuality and stress-induced changes in behavior remain unknown, this paper lays an important foundation for evaluating these questions. As the authors note, the behaviors studied here represent only a small fraction of the behavioral repertoire of this system. As such, the findings here are an interesting and very promising entry point for a deeper understanding of behavioral individuality, particularly because of the cellular/synaptic-level analysis that is possible in this system. This paper should be of interest to those studying C. elegans behavior and also more generally to those interested in behavioral plasticity and individuality.

    We thank the reviewer for finding our results interesting.

    Reviewer #2 (Public Review):

    This paper set out to understand the impact of early life stress on the behavior and individuality of animals, and how that impact might be amplified or masked by neuromodulation. To do so, the authors built on a previously established assay (Stern et al 2017) to measure the roaming fraction and speed of individuals. This technique allowed the authors to assess the effects of early life starvation on behavior across the entire developmental trajectory of the individual. By combining this with strains with mutant neuromodulatory systems, this enabled the authors to produce a rich dataset ripe for analysis to analyze the complicated interactions between behavior, starvation intensity, developmental time, individuality, and neuromodulatory systems.

    The richness of this dataset - 2 behavioral measures continuous across 5 developmental stages, 3 different neuromodulatory conditions (with the dopamine system subject to decomposition by receptor types) and 4 different levels of starvation, with ~50-500 individuals in each condition-underlies the strength of this paper. This dataset enabled the authors to convincingly demonstrate that starvation triggers a behavioral effect in L1 and adult animals that is largely masked in intermediate stages, and that this effect becomes larger with increased severity of starvation. Furthermore, they convincingly show that the masking of the effect of starvation in L2-L4 animals depends on dopaminergic systems. The richness of the dataset also allowed a careful analysis of individuality, though only neuromodulatory mutants convincingly manipulated individuality, recapitulating earlier research. Nonetheless, a few caveats exist on some of their findings and conclusions:

    We thank the reviewer for the constructive comments. In the revised manuscript we include additional analyses and textual changes as detailed below, to address the points raised.

    1. Lack of quantitative analysis for effects within developmental stages. In making the argument for buffered effects of starvation on behavior during periods of larval development, the authors make claims regarding the temporal structure of behavior within specific stages. However, no formal analysis is performed and and the traces are provided without confidence intervals, making it difficult to judge the significance of potential deviations between starvation conditions.

    In the revised manuscript, we include additional analyses of roaming fraction effects across shorter developmental-windows, showing within-stage differences in behavioral patterns following starvation (Figure 1 - figure supplement 1E; Figure 3 - figure supplement 1C). In addition, we further temper and rewrite our conclusions to clearly describe these effects (now- “…while 1 day of early starvation modified within-stage temporal behavioral structures by shifting roaming activity peaks to later time-windows during the L2 and L3 stages…” in p. 4 and “Interestingly, during the L2 intermediate stage the effects on roaming activity patterns were more pronounced during earlier time-windows of the stage…” in p. 8).

    1. Incorrect inferences from differences in significance demonstrating significant differences. The authors claim that there is an increase in PC1 inter-individual variation in tph-1 individuals, however the difference in significance is not evidence of a significant difference between conditions (see Nieuwenhuis et al. 2011). This undermines claims about an interaction of starvation, neuromodulators, and individuality.

    In the revised manuscript we provide now a direct comparison of PCs inter-individual variances between starved and unstarved populations, demonstrating significant differences in inter-individual variation in specific PC individuality dimensions following stress (Figure 6 and Figure 6 - figure supplement 1). These results include the increase in PC1 inter-individual variation in tph-1 mutants following 3 and 4 days of starvation (Figure 6A,E).

    1. Sensitivity of analysis to baseline effects and assumptions of additive/proportional effects. The neuromodulatory and stress conditions in this paper have a mixture of effects on baseline activity and differences from baseline. The authors normalize to the roaming fraction without starvation, making the reasonable assumption that the effect due to starvation is proportional to baseline, rather than an additive effect. This confound is most visible in the adult subpanel of figure 5d, where an ~2-3 fold difference in relative roaming due to starvation is clearly noted, however, this is from a baseline roaming fraction in tph-1 animals that are ~2 fold higher, suggesting that the effect could plausibly be comparable in absolute terms.

    Unavoidably, any such assumptions on the expected interaction between multiple effects will be a gross simplification in complicated nonlinear systems, and the data are largely shown with sufficient clarity to allow the reader to make their own conclusions. However, some of the interpretations in the paper lean heavily on an assumption that the data support a direct interpretation (e.g. "neuronal mechanisms actively buffer behavioral alterations at specific development times") rather than an indirect interpretation (e.g. that serotonin reduces baseline roaming fraction which makes a fixed sized effect more noticeable). Parsing the differences requires either more detailed mechanistic study or careful characterization of the effect of different baselines on the sensitivity of behavior to perturbation-barring that it's worth noting that many of these interactions may be due to differences in biological and experimental sensitivity to change under different conditions, rather than a direct interaction of stress and neuromodulatory processes or evidence of differing neuromodulatory activity at different stages of development.

    In the revised manuscript we added a discussion of the potential complicated interactions between neuromodulation and stress, altering baseline levels and deviations from baseline. We also discuss the interpretation of the results in the context of non-linear systems in which sensitivity of the behavioral response to underlying variations may be modified by specific neuromodulatory and environmental perturbations, without assuming direct differences in neuromodulatory states over development or across individuals (p. 16).

    Reviewer #3 (Public Review):

    In this study, Nasser et al. aim to understand how early-life experience affects 1) developmental behavior trajectory and 2) individuality. They use early life starvation and longitudinal recording of C. elegans locomotion across development as a model to address these questions. They focus on one specific behavioral response (roaming vs. dwelling) and demonstrate that early life (right after embryo hatching) starvation reduces roaming in the first larval (L1) and adult stages. However, roaming/dwelling behavior during mid-larval stages (L2 through L4) is buffered from early life starvation. Using dopamine and serotonin biosynthesis null mutant animals, they demonstrated that dopamine is important for the buffering/protection of behavioral responses to starvation in mid-larval stages, while in contrast, serotonin contributes to early-life starvation's effects on reduced roaming in the L1 and adult stages. While the technique and analysis approaches used are mostly solid and support many of the conclusions made in the manuscript for part 1), there are some technical limitations (e.g., whether the method has sufficient resolution to analyze the behaviors of younger animals) and confounding factors (e.g., size of the animal) that the authors do not yet sufficient address, and can affect interpretation of the results. Additionally, much of the study is descriptive and lacks deep mechanistic insight. Furthermore, the focus on a single behavioral parameter (dwelling vs. roaming) limits the broad applicability of the study's conclusions. Lastly, the manuscript does not provide clear presentation or analysis to address part 2), the question of how early life experience affect individuality.

    We thank the reviewer for these important comments. As described below, in the revised manuscript we include new analyses (following extraction of size data), showing behavioral modifications across different conditions/genotypes also in size-matched individuals (within the same size range) (Figure 1 - figure supplement 1F; Figure 3 - figure supplement 1D,E; Figure 5 - figure supplement 1B,D). We also made edits to the text to describe these results (Methods p. 21 and Results section). In addition, while we can detect behavioral changes using our imaging method even in young L1 worms across conditions and genotypes (described in Stern et al. 2017 and this manuscript), as the reviewer correctly pointed out, we may miss some milder behavioral effects due to lower spatial imaging resolution in younger worms. We are now referring to this spatial resolution limitation in the revised manuscript (discussion part). Lastly, in the revised manuscript we added clearer and more direct analyses of changes in inter-individual variation in multiple PC dimensions following early stress, by directly comparing variation between starved and unstarved individuals within the mutant and wild-type populations (Figure 6; Figure 6 - figure supplement 1). These analyses show significant changes in inter-individual variation within specific PC individuality dimensions following early stress. Also, we made textual changes along the manuscript to increase the clarity of presentation of these results.

  3. eLife

    eLife assessment

    Early life stress can have profound effects on animal behavior, including potential influences on individuality. In this valuable work, the authors use a rich new dataset to solidly demonstrate that the behavioral consequences of early life stress in C. elegans can be buffered by neuromodulators previously implicated in patterns of individuality. While much remains to be learned about the mechanisms by which stress might influence individuality, these studies provide an important entry point that will be of interest to neurobiologists studying interactions between behavior, neuromodulation, stress, and individuality.

  4. eLife

    Reviewer #1 (Public Review):

    In this interesting manuscript, Nasser et al explore long-term patterns of behavior and individuality in C. elegans following early-life nutritional stress. Using a rigorous, highly quantitative, high-throughput approach, they track patterns of motor behavior in many individual nematodes from L1 to young adulthood. Interestingly, they find that early-life food deprivation leads to decreased activity in young larvae and adults, but that activity between these times, during L2-L4, is largely unaffected. Further, they show that this "buffering" of stress requires dopamine signaling, as L2-L4 activity is significantly reduced by early-life starvation in cat-2 mutants. The paper also provides evidence that serotonin signaling has a role in modulating sensitivity to stress in L1 larvae and adults, but the size of these effects is modest. To evaluate patterns of individuality, the authors use principal components analysis to find that three temporal patterns of activity account for much of the variation in the data. While the paper refers to these as "individuality types," it may be more reasonable to think of these as "dimensions of individuality." Further, they provide evidence that stress may alter the strength and/or features of these dimensions. Though the circuit mechanisms underlying individuality and stress-induced changes in behavior remain unknown, this paper lays an important foundation for evaluating these questions. As the authors note, the behaviors studied here represent only a small fraction of the behavioral repertoire of this system. As such, the findings here are an interesting and very promising entry point for a deeper understanding of behavioral individuality, particularly because of the cellular/synaptic-level analysis that is possible in this system. This paper should be of interest to those studying C. elegans behavior and also more generally to those interested in behavioral plasticity and individuality.

  5. eLife

    Reviewer #2 (Public Review):

    This paper set out to understand the impact of early life stress on the behavior and individuality of animals, and how that impact might be amplified or masked by neuromodulation. To do so, the authors built on a previously established assay (Stern et al 2017) to measure the roaming fraction and speed of individuals. This technique allowed the authors to assess the effects of early life starvation on behavior across the entire developmental trajectory of the individual. By combining this with strains with mutant neuromodulatory systems, this enabled the authors to produce a rich dataset ripe for analysis to analyze the complicated interactions between behavior, starvation intensity, developmental time, individuality, and neuromodulatory systems.

    The richness of this dataset - 2 behavioral measures continuous across 5 developmental stages, 3 different neuromodulatory conditions (with the dopamine system subject to decomposition by receptor types) and 4 different levels of starvation, with ~50-500 individuals in each condition-underlies the strength of this paper. This dataset enabled the authors to convincingly demonstrate that starvation triggers a behavioral effect in L1 and adult animals that is largely masked in intermediate stages, and that this effect becomes larger with increased severity of starvation. Furthermore, they convincingly show that the masking of the effect of starvation in L2-L4 animals depends on dopaminergic systems. The richness of the dataset also allowed a careful analysis of individuality, though only neuromodulatory mutants convincingly manipulated individuality, recapitulating earlier research. Nonetheless, a few caveats exist on some of their findings and conclusions:

    1. Lack of quantitative analysis for effects within developmental stages. In making the argument for buffered effects of starvation on behavior during periods of larval development, the authors make claims regarding the temporal structure of behavior within specific stages. However, no formal analysis is performed and and the traces are provided without confidence intervals, making it difficult to judge the significance of potential deviations between starvation conditions.

    2. Incorrect inferences from differences in significance demonstrating significant differences. The authors claim that there is an increase in PC1 inter-individual variation in tph-1 individuals, however the difference in significance is not evidence of a significant difference between conditions (see Nieuwenhuis et al. 2011). This undermines claims about an interaction of starvation, neuromodulators, and individuality.

    3. Sensitivity of analysis to baseline effects and assumptions of additive/proportional effects. The neuromodulatory and stress conditions in this paper have a mixture of effects on baseline activity and differences from baseline. The authors normalize to the roaming fraction without starvation, making the reasonable assumption that the effect due to starvation is proportional to baseline, rather than an additive effect. This confound is most visible in the adult subpanel of figure 5d, where an ~2-3 fold difference in relative roaming due to starvation is clearly noted, however, this is from a baseline roaming fraction in tph-1 animals that are ~2 fold higher, suggesting that the effect could plausibly be comparable in absolute terms.

    Unavoidably, any such assumptions on the expected interaction between multiple effects will be a gross simplification in complicated nonlinear systems, and the data are largely shown with sufficient clarity to allow the reader to make their own conclusions. However, some of the interpretations in the paper lean heavily on an assumption that the data support a direct interpretation (e.g. "neuronal mechanisms actively buffer behavioral alterations at specific development times") rather than an indirect interpretation (e.g. that serotonin reduces baseline roaming fraction which makes a fixed sized effect more noticeable). Parsing the differences requires either more detailed mechanistic study or careful characterization of the effect of different baselines on the sensitivity of behavior to perturbation-barring that it's worth noting that many of these interactions may be due to differences in biological and experimental sensitivity to change under different conditions, rather than a direct interaction of stress and neuromodulatory processes or evidence of differing neuromodulatory activity at different stages of development.

  6. eLife

    Reviewer #3 (Public Review):

    In this study, Nasser et al. aim to understand how early-life experience affects 1) developmental behavior trajectory and 2) individuality. They use early life starvation and longitudinal recording of C. elegans locomotion across development as a model to address these questions. They focus on one specific behavioral response (roaming vs. dwelling) and demonstrate that early life (right after embryo hatching) starvation reduces roaming in the first larval (L1) and adult stages. However, roaming/dwelling behavior during mid-larval stages (L2 through L4) is buffered from early life starvation. Using dopamine and serotonin biosynthesis null mutant animals, they demonstrated that dopamine is important for the buffering/protection of behavioral responses to starvation in mid-larval stages, while in contrast, serotonin contributes to early-life starvation's effects on reduced roaming in the L1 and adult stages. While the technique and analysis approaches used are mostly solid and support many of the conclusions made in the manuscript for part 1), there are some technical limitations (e.g., whether the method has sufficient resolution to analyze the behaviors of younger animals) and confounding factors (e.g., size of the animal) that the authors do not yet sufficient address, and can affect interpretation of the results. Additionally, much of the study is descriptive and lacks deep mechanistic insight. Furthermore, the focus on a single behavioral parameter (dwelling vs. roaming) limits the broad applicability of the study's conclusions. Lastly, the manuscript does not provide clear presentation or analysis to address part 2), the question of how early life experience affect individuality.

  7. Version published to 10.1101/2022.10.24.513603v1 on bioRxiv