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

Abstract

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation Early life stress can have profound effects on animal behavior, including potential influences on individuality. Here, the authors use a rich new dataset to convincingly 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 report important advances that will be of interest to neurobiologists studying interactions between behavior, neuromodulation, stress, and individuality. https://doi.org/10.7554/eLife.84312.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Long-term behavioral patterns across development are highly dynamic across and within developmental stages and are temporally synchronized with the individual’s developmental clock. For instance, flies show differences in foraging behavior that depend on their larval stage (Sokolowski et al., 1984), fish internally modify their startle response across life (Kimmel et al., 1974) and stomatogastric motor patterns are regulated across development (Rehm et al., 2008). In addition, fear-extinction learning is inhibited during adolescence compared to other life stages in humans and mice (Pattwell et al., 2012). Long-term behavioral outputs are influenced by the changes in the internal state of individuals, as well as by their past and current environmental exposures. In particular, animals may be transiently exposed to environmental perturbations at different stages of life, but experiences during early developmental windows, which are usually referred to as critical or sensitive periods, were shown to generate long-lasting effects (Lorenz, 1935; Korosi et al., 2012; Jin et al., 2016; Nevitt et al., 1994; Remy and Hobert, 2005). This stable imprinting of early memories has the potential to increase survival and reproduction during later life stages of the organism (Immelmann, 1975). However, a complete temporal view of the long-term effects of early experiences on behavior throughout development, across and within all stages, is still lacking. While long-lasting effects on behavior may be shared by many individuals, reflected by stereotypic behavioral responses following an early-life experience, individuals within the same population may also show unique patterns of long-term behavior that distinguish them from each other. This inter-individual variation in behavioral responses may be exposed even when animals are initially experiencing the same early conditions. Here we study how early-life experiences shape stage-specific behavioral patterns across development and how they affect the diversity in long-term behavioral responses among individuals. Consistent behavioral individuality within isogenic populations that were raised in the same environment has been previously described in various species, including in the pea aphid (Schuett et al., 2011), Drosophila melanogaster (Buchanan et al., 2015; Kain et al., 2012; Linneweber et al., 2020), clonal fish (Bierbach et al., 2017), and mice (Freund et al., 2013). The nematode Caenorhabditis elegans is an ideal system to study how early-life experiences shape long-term behavior and inter-individual variation across developmental timescales due to their short development time of 2.5 days and the homogeneous populations generated by the self-fertilizing reproduction mode of the hermaphrodite. It was previously shown that under normal growth conditions, C. elegans shows both stereotypic patterns of long-term behavior and consistent individual biases within the isogenic population (Stern et al., 2017). By continuously tracking the locomotory behavior of single individuals following transient periods of starvation early in life throughout their complete developmental trajectory, we show that early-life starvation exposes long-term behavioral plasticity that is discontinuous over development time. Temporal differences in long-term behavioral responses to early stress are reflected by strong behavioral modifications during early and late developmental stages and the buffering of behavioral effects during intermediate stages of development. We further found that dopamine maintains the buffering of behavioral responses during mid-development and serotonin promotes behavioral sensitivity to early starvation during early and late stages of development. Moreover, by performing unsupervised analysis of patterns of individual biases across development, we identified a spectrum of temporal individuality dimensions that are dominant within stressed and unstressed populations. Both the early-life history and neuromodulatory state of the population affect variation within specific individuality dimensions. These results show how a transient early-life environment shapes a long-term behavioral structure of stereotypic and variable responses across developmental stages. Results Early-life stress generates discontinuous and distinct behavioral effects at different stages of development To study how stressful environments early in life influence long-term behavioral patterns across and within all developmental stages we continuously tracked the behavior of individual animals following a transient period of starvation early in development. Imaging was performed using a custom-made multi-camera imaging system across the full developmental trajectory of C. elegans individuals (55 hr), at high spatiotemporal resolution (3 fps, ~10 µm) and in a tightly controlled environment (Stern et al., 2017). First larval stage (L1) animals that hatch into an environment that completely lacks a food source do not grow and their development is arrested (Greenwald and Horvitz, 1982; Johnson et al., 1984; Baugh, 2013). Following L1 arrest, when animals encounter food, they resume their normal developmental trajectory to reach adulthood. This early-stress paradigm allows us to maintain a homogeneous stress environment across individuals at their earliest stage of development, immediately after hatching. We continuously monitored single N2 wild-type individuals grown in isolation from their first larval stage to 16 hr of adulthood (n = 456) on defined concentrations of UV-killed OP50 bacteria, following periods of stress ranging from 1 to 4 days of early starvation (Figure 1A, B). In parallel, we tracked the behavior of individuals grown continuously on food, without experiencing starvation (Figure 1A, B). Animals exposed to early starvation required more time to complete their development (Figure 1—figure supplement 1A). To align developmental trajectories of different individuals in time, we age-normalized individuals by dividing each developmental stage, detected by the lethargus period during molting (Cassada and Russell, 1975), into 75 time windows (Figure 1—figure supplement 1C; Stern et al., 2017). Whilst growing in a food environment, C. elegans shifts between two behavioral states called roaming and dwelling that last seconds to minutes (Ben Arous et al., 2009; Flavell et al., 2013; Fujiwara et al., 2002; Stern et al., 2017). During a roaming episode animals explore a large area by high-speed forward movements, while in the dwelling episode they show dramatically less exploration due to low-speed movements coupled with frequent reorientations (Figure 1—figure supplement 1B). We quantified long-term patterns of locomotory behavior shown by individuals throughout development by measuring two behavioral parameters: fraction of time spent roaming, and speed during roaming episodes. Figure 1 with 1 supplement see all Download asset Open asset Long-term behavioral tracking of C. elegans following early starvation reveals discontinuous behavioral effects across developmental stages. (A) Multi-camera imaging system allows longitudinal behavioral tracking of multiple individual worms across all stages of development following early L1 starvation and without starvation, under tightly controlled environmental conditions. Shown are representative locomotion trajectories (middle) and age-normalized roaming activity (right) of post-starved (red) and well-fed (gray) individual worms across all four larval stages and adulthood. Normalization equally divides each stage into 75 time bins. (B) Roaming and dwelling behavior of wild-type N2 animals without early starvation (n = 123) and following 1-day (n = 99), 3-day (n = 119), and 4-day starvation (n = 115). Each row indicates the age-normalized behavior of one individual across all developmental stages. The different stages are separated by vertical white lines indicating the middle of the lethargus state. Color bar represents the fraction of time spent roaming in each of the 375 time bins. (C) Average roaming fraction of 1-day starved wild-type animals compared to the unstarved population. (D) Average roaming fraction of 3- and 4-day starved wild-type animals compared to the unstarved population. (E) Average roaming fraction relative to the unstarved population in each developmental stage. Shading highlights intermediate stages of development in which average behavioral effects within a stage are buffered. Error bars indicate standard error of the mean. Upper bars indicate statistical significance (Wilcoxon rank-sum test, False Discovery Rate (FDR) corrected) of difference in average roaming fraction between starved and unstarved populations (−log(p-value), indicated are p-values <0.01). Unstressed individuals hatching in a food environment show dynamic behavioral structures of roaming activity across development, as was previously shown (Stern et al., 2017; Figure 1B–D). We found that a transient exposure to early-life starvation generates alterations in long-term behavioral patterns throughout development that were distinct and discontinuous across and within developmental stages. A short early-starvation period of 1 day strongly decreased average roaming activity levels during the L1 and adult stages compared to unstressed individuals (Figure 1C, E; Figure 1—figure supplement 1D). In contrast, 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 (Figure 1C; Figure 1—figure supplement 1E), average roaming activity was not decreased during these stages (Figure 1E). Similarly, we found that average roaming activity level was also maintained during the L4 stage, following 1-day starvation (Figure 1C, E; Figure 1—figure supplement 1D). Interestingly, animals exposed to longer starvation periods of 3 and 4 days further showed strong roaming decrease during L1 and adulthood, but exhibited only minor effects on average roaming activity within the intermediate L2, L3, and L4 larval stages (Figure 1D, E; Figure 1—figure supplement 1D). Furthermore, while the average size of animals that experienced early starvation was slightly decreased (~10%), a comparison of starved and unstarved individuals within the same size range (size-matched) showed similar stage-specific effects on roaming activity during L1 and adulthood following early starvation (Figure 1—figure supplement 1F). These results indicate that, while the memory of early starvation is maintained throughout development to expose strong decrease in average roaming behavior during L1 and adulthood, behavioral effects are buffered across intermediate development times. Similar to the stage-specific effects of early starvation on the fraction of time spent roaming, instantaneous speed during roaming episodes in individuals exposed to early starvation was affected more strongly at the L1 and adult stages, compared to the intermediate stages (Figure 1—figure supplement 1G). In summary, transient early-life starvation discontinuously reshapes long-term behavioral patterns across development time by exposing strong behavioral alterations at early and late developmental stages and buffered effects during intermediate stages. Unsupervised analysis uncovers multiple individuality dimensions within stressed and unstressed populations The longitudinal measurements in single animals across development allow us to further quantify long-term inter-individual diversity within stressed and unstressed populations. Following early starvation, different individuals show substantial variation in long-term behavioral responses. For instance, during L1 and adulthood, a fraction of wild-type individuals that were exposed to early stress show 8- to 10-fold decrease in roaming activity relative to the average roaming level of the unstressed population, while other stressed individuals show roaming activity which is indistinguishable from unstressed animals (Figure 1—figure supplement 1D). Behavioral individuality is usually defined as a consistent tendency of an individual to show the same behavioral bias relative to the population across long time-periods (Bierbach et al., 2017; Buchanan et al., 2015; Kain et al., 2012; Stern et al., 2017; Schuett et al., 2011). However, individuals may also show alternative patterns of temporal behavioral biases relative to the population that are not random and represent more complex structures of individual biases over time. Here, we extend the ‘classic’ analysis of individuality and ask if alternative individuality dimensions coexist within C. elegans populations across development. To analyze long-term individual biases in behavior we first systematically rank individuals based on their roaming activity compared to all other individuals within the same experiment across developmental windows (50 time bins) (Figure 2A, B). The rank approach allows us to homogeneously compare between individuals at each developmental window. To take an unsupervised approach for detecting temporal patterns of individual biases that are dominant within stressed and unstressed populations we performed principal component analysis (PCA) of the temporal behavioral ranks of all wild-type individuals (n = 456). Following PCA, each individual within the population is represented by its score (value) in each of the PC dimensions. We identified statistically significant PC dimensions by comparing the variances in PC scores within each dimension to those obtained from PCA of a randomly shuffled rank dataset (Figure 2C; Figure 2—figure supplement 1A) or to score variances of a shuffled rank dataset within the same PCA space (Figure 2—figure supplement 1B). Figure 2 with 1 supplement see all Download asset Open asset Unsupervised analysis of temporal individuality dimensions across development. (A) Individual animals are ranked based on their roaming activity in each time window compared to other individuals within the same experiment. (B) Heat-map represents relative rank of all N2 wild-type individuals (n = 456) across 50 time windows (10 per developmental stage). (C) Variance explained by each of the first 20 PCs following principal component analysis (PCA; blue bars), compared to the variance explained by the first 20 PCs extracted from PCA of a shuffled dataset (500 repetitions, orange lines). (D–F) PC1–3 represent the first three temporal individuality dimensions. For each PC individuality dimension shown are its components in each time window (top) and individuals sorted based on their PC score (bottom). Heat map is smoothed (4-bins window) for visual clarity. Average relative rank is plotted for extreme individuals (top and bottom 15%) within each individuality dimension. Midline represents the population median. (G–I) Distributions of individual scores (blue) within starved and unstarved wild-type populations for PC1–3 individuality dimensions, compared to distributions of individual scores of a shuffled dataset in the same PCA space (orange). p-values above distributions were calculated using bootstrapping (see Methods) for significance of difference in PC1–3 variation to variation of a shuffled dataset in the same PCA space (bottom asterisks) or in a PCA space extracted from the shuffled dataset (upper asterisks). (J) Average roaming activity of top (red) and bottom (blue) 15% of extreme individuals within each of the PC1–3 individuality dimensions in 4-day starved and unstarved wild-type populations. *p < 0.05, ***p < 0.001 (FDR corrected). We found that among the significant PC dimensions (Figure 2D–F; Figure 2—figure supplement 1F), the three major PCs (PC1–3) captured three distinct dimensions of temporal individuality patterns within stressed and unstressed populations (Figure 2D–F). PC1, which explained the majority of temporal variation in individual biases over time had eigenvector components of the same sign, indicating an individuality dimension of animals that consistently roam more or less than the population homogeneously across all developmental stages (Figure 2D). The individuality dimension identified by PC1 unbiasedly recaptured a known mode of consistent individuality that was previously identified using a pre-defined index of long-term behavioral consistency across development (Stern et al., 2017; Figure 2—figure supplement 1C, D). This was further verified by the high correlation between the pre-defined consistency index and scores of PC1 across individuals (R = 0.9) (Figure 2—figure supplement 1E). Interestingly, other PC dimensions identified uncharacterized individuality patterns. PC2, which had opposite signs of eigenvector components before and after mid-development captured an individuality dimension that includes individuals that switch their behavioral bias once, during the L3 stage, from roaming more to roaming less than the population and vice versa (Figure 2E). In addition, PC3, which had signs of eigenvector components that switch twice during development (at the end of L1 and L4), identified individuals that show the same behavioral bias during L1 and adulthood, which is opposite to their behavioral bias during intermediate stages (Figure 2F). Other significant PC dimensions showed more complex dynamics of temporal individual biases across development, displaying multiple bias switching within developmental stages (Figure 2—figure supplement 1F). PC individual scores in these alternative PC dimensions did not correlate with the pre-defined consistency index (R = 0.003–0.09) (Figure 2—figure supplement 1E), further indicating that they indeed represent uncharacterized modes of temporal individuality. Inter-individual variation in PC scores within a specific PC dimension reflects how extreme individuals are within a population toward the identified individuality dimension. We found that wild-type populations with different early-life experiences show extreme inter-individual variation in multiple PC dimensions, compared to a randomly shuffled rank dataset within the same PCA space or within a PCA space generated from the shuffled rank dataset (Figure 2G–J; Figure 2—figure supplement 1G, H), indicating the coexistence of these alternative individuality dimensions. Altogether, these results demonstrate the use of unsupervised analysis for identifying multiple individuality dimensions across development and suggest a broad individuality space within isogenic populations. Dopamine buffers behavioral responses to early stress during intermediate developmental stages Neuromodulatory pathways are known to establish internal behavioral states and modify them based on the environmental context (Harris-Warrick and Marder, 1991; Bargmann, 2012; Marder, 2012; Kennedy et al., 2014; Taghert and Nitabach, 2012; Nusbaum and Blitz, 2012). In particular, the bioamine dopamine was implicated in controlling a wide array of behavioral outputs at various timescales, ranging from minutes and hours, to long-term behavioral patterns that are regulated across life stages (Marella et al., 2012; Omura et al., 2012; Sawin et al., 2000; Cermak et al., 2020; Stern et al., 2017). In C. elegans, dopamine is produced in a specific set of neuronal sites and its effects are known to be mediated by dopamine receptors that are localized to responding neurons (Sulston et al., 1975; Lints and Emmons, 1999; Chase et al., 2004; Tsalik et al., 2003). To ask if dopamine acts across different developmental stages to shape the discontinuous pattern of long-term behavioral responses to early stress and to dissect its temporal requirement, we tracked the behavior of dopamine-deficient cat-2 animals following exposure to L1 starvation (Figure 3A; Figure 3—figure supplement 1A). When continuously grown in a food environment, cat-2 individuals show a long-term roaming activity pattern that is similar to the wild-type population (Stern et al., 2017; Figure 1; Figure 3). However, we found that in contrast to stressed wild-type individuals that show buffering of behavioral responses during the L2, L3, and L4 intermediate stages, cat-2 individuals that were exposed to early starvation show reduction in average roaming activity across all developmental stages, including during the intermediate stages (Figure 3B, C). The behavioral effects of early starvation during mid-development in cat-2 individuals were not only restricted to animals that were exposed to long starvation periods, as 1 day of early starvation was sufficient to induce a strong reduction in roaming activity during the L2–L4 intermediate stages (Figure 3D; Figure 3—figure supplement 1B–D). Interestingly, during the L2 intermediate stage the effects on roaming activity patterns were more pronounced during earlier time windows of the stage, suggesting a potential within-stage regulation of behavioral response by dopamine (Figure 3B, C; Figure 3—figure supplement 1C). However, behavioral effects during L1 and adulthood following early stress were similar in cat-2 and wild-type (Figure 3B–D), implying that dopamine function is mainly required during intermediate developmental stages to buffer alterations in roaming activity in response to a transient early stress. Figure 3 with 1 supplement see all Download asset Open asset Dopamine buffers long-term behavioral effects during intermediate stages of development. (A) Roaming and dwelling behavior of cat-2 animals without early starvation (n = 124) and following 1-day (n = 98), 3-day (n = 124), and 4-day starvation (n = 85). Each row indicates the age-normalized behavior of one individual across all developmental stages. The different stages are separated by white lines indicating the middle of the lethargus state. Color bar represents the fraction of time spent roaming in each of the 375 time bins. (B) Average roaming fraction of 1-day starved cat-2 animals compared to the unstarved population. (C) Average roaming fraction of 3- and 4-day starved cat-2 animals compared to the unstarved population. (D) Average roaming fraction relative to the unstarved population in cat-2 and wild-type individuals, in each developmental stage. Error bar indicates standard error of the mean. Upper bars indicate statistical significance (Wilcoxon rank-sum test, FDR corrected) of the difference in behavioral effect following early stress between the cat-2 and wild-type populations (−log(p-value), indicated are p-values <0.01). It was previously shown that during L2 to adulthood, cat-2 animals have higher instantaneous speed during roaming episodes (Stern et al., 2017; Sawin et al., 2000; Figure 3—figure supplement 1E). We found that unlike stressed wild-type individuals in which roaming speed was decreased mainly during L1 and adulthood (Figure 1—figure supplement 1G), cat-2 mutants show lower speed also across the L2 and L3 stages following stress (Figure 3—figure supplement 1F). To further ask if dopamine supplementation can restore roaming activity following stress, during intermediate developmental stages, we exposed cat-2 individuals to exogenous dopamine (Figure 4A–C; Figure 4—figure supplement 1A–E). We found that supplementing dopamine was sufficient to increase only the roaming activity following stress in cat-2 individuals exposed to 1 and 3 days of starvation, during the L2, L3, and L4 stages (Figure 4A–C; Figure 4—figure supplement 1B, C, F, G). In contrast, following 1 day of starvation exogenous dopamine did not restore roaming activity during L1 and adulthood (Figure 4A, C; Figure 4—figure supplement 1A, B) and following 3 days of starvation it only slightly increased roaming in the L1 stage and did not affect roaming activity during adulthood (Figure 4B, C; Figure 4—figure supplement 1A, C). Overall, these results show that following early and transient starvation, dopamine acts to restrict long-term behavioral alterations in roaming activity, specifically during intermediate developmental windows. Figure 4 with 3 supplements see all Download asset Open asset Effects of exogenous dopamine and temporally restricted functions of dopamine receptors across intermediate developmental stages. (A) Average roaming fraction of unstarved (n = 124), 1-day starved (n = 98), and 1-day starved with exogenous DA (n = 46) cat-2 populations. (B) Average roaming fraction of unstarved (n = 124), 3-day starved (n = 124), and 3-day starved with exogenous DA (n = 50) cat-2 populations. (C) Average roaming fraction relative to the unstarved population in 1- and 3-day starved cat-2 populations, with or without exogenous DA, in each developmental stage. Upper bars indicate statistical significance (−log(p-value), Wilcoxon rank-sum test (FDR corrected), indicated are p-values <0.01). (D) Average roaming fraction of 3-day starved (n = 145) and unstarved (n = 111) dop-2 populations. (E) Average roaming fraction of 3-day starved (n = 134) and unstarved (n = 73) dop-1 populations. (F) Average roaming fraction of 3-day starved (n = 95) and unstarved (n = 82) dop-3 populations. (G) Average roaming fraction relative to the unstarved population in dop-2 and wild-type individuals, in each developmental stage. (H) Average roaming fraction relative to the unstarved population in dop-1 and wild-type individuals, in each developmental stage. (I) Average roaming fraction relative to the unstarved population in dop-3 and wild-type individuals, in each developmental stage. (J) Average roaming fraction of 1-day starved (n = 62), 3-day starved (n = 63), and unstarved (n = 69) dop-2;dop-3 populations. (K) Average roaming fraction relative to the unstarved population in dop-2;dop-3 and wild-type individuals, in each developmental stage. Upper bars in (G, H, I, K) indicate statistical significance (Wilcoxon rank-sum test, FDR corrected) of the difference in behavioral effect following early stress between the dopamine receptors mutants and N2 populations (−log(p-value), indicated are p-values <0.01). Error bars indicate standard error of the mean. Specific dopamine receptors function during mid-development to mediate buffering of long-term behavioral responses The buffering of behavioral effects during the L2, L3, and L4 intermediate developmental stages by dopamine led us to explore the temporal contribution of specific dopamine receptors during these development times. The C. elegans dopamine receptor DOP-1 is a D1-like receptor which signal through Gαs/olf to activate adenylyl cyclase and DOP-2 and DOP-3 receptors are D2-like receptors which signal via Gαi to suppress adenylyl cyclase (Chase et al., 2004; Sanyal et al., 2004; Sugiura et al., 2005; Suo et al., 2003). To study the independent function of dopamine receptors we analyzed the long-term behavioral effects of early starvation in animals mutant for each of the single dopamine receptors. These analyses showed that each receptor has a different temporal effect on behavioral responses within the intermediate L2–L4 stages (Figure 4D–I; Figure 4—figure supplement 2). In particular, following 3 days of early starvation, dop-2 animals showed strong roaming decrease during the L2 and L4 stages, but not during the L3 stage, compared to wild-type (Figure 4D, G; Figure 4—figure supplement 2). Interestingly, similar to cat-2 mutants, roaming was mainly decreased in dop-2 individuals during early time windows within the L2 stage. In addition, dop-1 individuals showed a roaming decrease during the L2 stage an