Novel competition test for food rewards reveals stable dominance status in adult male rats

1 Vol.:(0123456789) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports Novel competition test for food rewards reveals stable dominance status in adult male rats Diana F. Costa1, Marta A. Moita2 & Cristina Márquez1* Social hierarchy is a potent modulator of behavior, that is typically established through overt agonistic interactions between individuals in the group. Once established, social ranks are maintained through subtler interactions allowing the redirection of energy away from agonistic interactions towards other needs. The available tasks for assessing social rank in rats allow the study of the mechanisms by which social hierarches are formed in early phases but fail to assess the maintenance of established hierarchies between stable pairs of animals, which might rely on distinct neurobiological mechanisms. Here we present and validate a novel trial-based dominancy assay, the modified Food Competition test, where established social hierarchies can be identified in the home cage of non-food deprived pairs of male rats. In this task, we introduce a small conflict in the home cage, where access to a new feeder containing palatable pellets can only be gained by one animal at a time. We found that this subtle conflict triggered asymmetric social interactions and resulted in higher consumption of food by one of the animals in the pair, which reliably predicted hierarchy in other tests. Our findings reveal stable dominance status in pair-housed rats and provide a novel tool for the evaluation of established social hierarchies, the modified Food Competition test, that is robust and easy to implement. Social hierarchy is a multidimensional trait that has a profound impact on emotion and cognition, not only for humans1,2 but also other social species (see3 for review), having important consequences for social organization, survival, reproductive success, and health of animals in a group4. Indeed, adapting behavioral responses based on the social status of the interacting partner can be cost-effective and, in some cases, a crucial survival strategy. The most established view is that social hierarchy is built upon aggressive interactions5, and serve as a mechanism of resource management and minimization of energy expenditure by groups of animals: once a hierarchy is established, priority access to resources is organized allowing the reduction of aggressive levels between the interacting animals6. Following this view, the behavioral paradigms available for measuring social hierarchy in laboratory animals are based in the nature of agonistic interactions while defending access to resources, whether a sexual partner, food or water when they are scarce, or the defense of a territory (see3 for review). Of note, most recent advances on the identification of the neural circuits underlying the establishment of social hierarchies have been performed in mice, as a reflection of a general tendency in the field which favors the use of this species due to the exceptional genetic tools available7,8. However, important contributions have been also performed using rats9–11, and importantly, Norway rats live in complex social groups in the wild. This, together with the fact of being a model system amenable to monitoring, mapping and perturbation of neuronal circuits, has motivated a wave of recent laboratory studies uncovering the diversity and sophistication of rat’s social skills (see12for review). Regarding their social status, the visible burrow system (VBS) has been widely used to study the formation of hierarchies in large groups of animals, where mixed-sex rat groups living in a complex environment compete chronically for territory and resources13. The VBS generates very rich behavioral data sets but is difficult to implement in most laboratories, hence, other behavioral tasks are commonly used, where animals compete for food or water under deprivation states14–17. Typically, social isolation of variable durations is performed prior to testing as a means to increase territoriality, favoring strong agonistic interactions during the establishment of new hierarchies. Therefore, these tasks evaluate how a new hierarchy is established between pairs of unfamiliar, frequently isolated animals, in neutral arenas where subjects display very evident agonistic behaviors to establish dominance. However, there are no tools available enabling to assess already established hierarchies. Focusing on the early establishment of a hierarchy is neglecting a very important and rich part of this type of social interactions: OPEN 1Neural Circuits of Social Behavior Laboratory, Instituto de Neurociencias (CSIC-UMH), Avenida Ramon y Cajal s/n, Sant Joan d’Alacant, 03550 Alicante, Spain. 2Behavioral Neuroscience Laboratory, Champalimaud Research, Champalimaud Centre for the Unknown, Av. Brasilia, 1400-038 Lisbon, Portugal. *email: [email protected] 2 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ how are they maintained in stable conditions. The establishment of social hierarchy might not rely on the same mechanisms as the expression of dominance when a hierarchy is already established, and recent reports in mice indicate that this might be indeed the case18,19. However, to our knowledge, the study of the possible differences between de novo and already established social hierarchies in rats has been virtually unexplored. To this end new behavioral paradigms that evaluate social status of animals living in stable dyads are urgently needed. Preferably, the evaluation of the hierarchy would avoid manipulations that could induce long-term alterations in internal state (i.e. isolation, deprivation) that, in turn, could affect how social interactions of interest are displayed. This is a challenge, as once hierarchies are established, agonistic behaviors are minimized and the opportunities to observe dominance interactions are reduced and likely subtle. With this aim, we developed the modified Food Competition task, where a small conflict for access to a discrete number of palatable pellets was introduced in the homecage of non-deprived adult male Sprague–Dawley cage mate rats. We decided to focus our study on this specific population as most of the available literature on dominance and aggressive behavior in rats has been performed in adult males13–17 being Sprague Dawley rats among the most used laboratory strains in behavioral neuroscience. In order to validate this new tool, we compared the dyads’ behavior to that observed in other competition tests: (1) competition for 1% sucrose solution, (2) modified standard tests used in the field, that involve deprivation, where animals compete for food or water and (3) the Tube Test. We performed a detailed analysis of behavior in each test, and although no aggressive interactions were observed, our results indicate that stable hierarchies in rats are indeed detectable by the modified Food Competition test which is especially suited for their identification, based on its trial structure and the degree of conflict induced. Results Behavioral profiles differ across the social competition tests used. To identify social status within pairs of cage mates we performed the modified Food Competition test and other behavioral tasks in which the animals needed to compete for resources, either for palatable pellets, sucrose solution, water or tube occupation (Fig.1 and Supplemental Figure1). All the tasks, except for the Tube Test, were performed in the animals’ home cage. Animals displayed different behavioral profiles depending on the configuration of the test, whether it had a trial structure, the amount of reward available, and their internal state (satiated vs deprived) (Figs.2 and 3). In order to control for possible effects of winning history we created two independent groups where we counterbalanced the order of the tests. No differences were observed between the groups, suggesting that hierarchy was already established (Kruskal–Wallis test comparing the duration of consumption in the two counterbalanced groups: modified Food Competition test (mFC) Day1: X2(2) = 0.026, p = 0.871; mFC Day2: X2(2) = 0.007, p = 0.935; SC Day1: X2(2) = 1.516, p = 0.218; Sucrose Competition with continuous access to reward (SC) Day2: X2(2) = 1.904, p = 0.168; Sucrose Competititon with Intermittent access to reward (SCI) Day1: X2(2) = 0.457, p = 0.499; SCI Day2: X2(2) = 2.055, p = 0.152; mFCD: X2(2) < 0.0001, p = 0.989; Water Competition (WC): X2(2) = 0.293, p = 0.589; Tube Test (TT) Day1: X2(2) = 0.00, p = 1.0; TT Day2: X2(2) < 0.0001, p = 1.0). Data from both groups was thus merged for the rest of the analysis. In the modified Food Competition task (Fig.2A, Fig.3, Movie 1), the limited number of available food pellets (10 per trial) led to a very fast consumption of resources which lasted a few seconds (Fig.3B-C). Interestingly, although pellets were consumed in the first seconds of each trial, animals displayed high levels of exploration of the feeder and displayed notable amounts of pushing, suggesting high expectation of reward (Fig.3A). On the contrary, during the Sucrose Competition task, exploration of the reward location and pushing levels were low (Fig.2B and Fig.3A), but exploration of the bottle location increased when access to the sucrose bottle was presented in an intermittent manner (Fig.2C and Fig.3A). As expected, modulation of internal state (food or water deprivation) affected the behavior of the animals. In the modified Food Competition task with deprivation (Fig.2D) animals consumed the pellets faster (Fig.3C, Wilcoxon signed rank tests against non-deprived Food Competition day 1: z = − 3.659 p = 0.0003; day 2: z = − 3.136 p = 0.002) and spent significantly more time investigating the feeder (Fig.3A), although the amount of pushing did not differ from that displayed in non-deprived animals (Fig.3A). When competing for access to water under deprivation (Fig.2E), animals dramatically increased the time they spent drinking compared to consumption displayed in the other tests (Fig.3A), performing long bouts of drinking and alternating between animals (Fig.2E). Levels of exploration of the water bottle were low compared to the rest of the tests. Surprisingly, although the motivation to drink was high, revealed by the long water consumption time, pushing levels did not increase proportionally (Fig.3A). Finally, the dyads established a very stable winner/loser relationship in the Tube Test (Fig.2F), where most of the animals that would start winning in the first trials would continue doing so over the remaining trials. This winner/loser structure was maintained across the two testing days (Fig.2F middle panel, raster plot of winning history for all pairs). Interestingly, on the second day of testing, the loser partner of some pairs showed reluctance to enter in the tube (9% of the total trials), suggesting a strong subordination towards the partner. The time to solve the conflict in the Tube test, measured as the latency from the moment the partition at the center of the tube was removed until one of the rats was pushed out of the tube, rapidly decreased after the first trial, reaching then fast and stable latencies of around 3.5s on average (Fig.2F, right panel). Social hierarchy as priority access to resources. We categorized the animals of each pair as dominant (D) or submissive (S) according to the amount of resources they would consume within each test (pellets eaten in the Food Competition tests, time spent drinking in the Sucrose or Water Competition tests, and the number of wins in the case of the Tube Test). According to this criterion, as expected, animals categorized as dominant consumed significantly more resources than their partners in every task (Fig.4A–E) (Wilcoxon signed-ranks 3 Vol.:(0123456789) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ Sliding door Pellets 20 cm 6 cm Opening to place pellets 5 cm Bottle 5 cm 5 cm 5 cm ABC DEF GH 60 cm 8 cm Transparent Partition Modified Food Competition Water/Sucrose Competition Tube Test 44 cm 49 cm 44 cm 49 cm Figure1. Design and measurements of the behavioral tests used for identification of established dominance status. (A) Schematic illustration of the transparent lid and feeder in the modified Food Competition apparatus. (B) Detailed schematic illustration of the feeder used in Food Competition protocols (with and without deprivation) including the measurements to fit the bottom part of a Rat IVC cage (Sealsafe PLUS Green Line ventilated cages, Techniplast). The sliding door can be opened leaving a 5cm high access which only allows one animal to eat at a time, and allows a trial structure for the task. A small opening on the top of the feeder allows to refill new pellets during inter trial interval. If adaptation of measurements to another type of home cage is needed, we advise to leave 3–4cm from the end of the feeder and the bottom of the home cage. This prevents bedding to go into the feeder, which difficults the visibility of the pellets while animals are consuming. (C) Two cage mates can be observed at the feeder area, where one is consuming the pellets while the other is pushing to get access to the food. (D) Schematic illustration of the lid and bottle holder for the Water and Sucrose Competitions protocols (with continuous or intermittent access) protocols (E) Detailed schematic illustration of the bottle holder. A 5 × 5cm restraining tube around the lick spout was created to prevent simultaneous access of both animals to the resource. (F) Two rats behaving in the Water Competition task, where one of the rats is drinking while the other is pushing to have access to the bottle (G) Schematic of the Tube Test with measurements used in this task, the transparent partition in the middle of the tube is removed at the beginning of a trial once both animals reach this area. Laser-cut acrylic holders were used to give stability to the set up (H) Two rats interacting inside the tube during the initial moments of a trial. 4 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ Trial 1Trial 2Trial 3Trial 4Trial 5 0 200 400 600 B C D E F A Trial duration (sec) Day 1D ay 2 Pairs of rats Trials Trials Deprivation 10 min 10 trials A wins B wins Resistant trial Day 1 Day 2 0 200 400 600 Consumption Pushing Feeder exploration Grooming Animal AAnimal B Trial 1Trial 2Trial 3Trial 4Trial 5 0 200 400 600 5 trials Consumption Pushing Feeder exploration Grooming Duration (sec) Trial 1Trial 2Trial 3Trial 4Trial 5 0 200 400 600 Deprivation 5 trials Consumption Pushing Feeder exploration Grooming Duration (sec) Duration (sec) 010 01 0 0 20 10 Complete trials Resistent trials 9% Complete trials Consumption Exploration of the feeder Pushing Grooming 10 min 5 trials Duration (sec) 0 200 400 600 Consumption Pushing Feeder exploration Grooming Consumption Pushing Feeder exploration Grooming Duration (sec) Duration (sec) Duration (sec) Duration (sec) Duration (sec) 10 0 1 20 10 0 Modified Food Competition Sucrose Competition Sucrose Competition Modified Food Competition Water Competition Tube Test Duration (sec) 0100 200 300 400 0100 200 30 04 00 0100 200300 400 0100 200 300 400 0100 200 30 04 00 5 Vol.:(0123456789) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ for consumption in mFC: z = − 3.92, p < 0.0001; SC: z = − 3.82, p < 0.0001; SCI: z = − 3.92, p < 0.0001; mFCD: z = − 3.83, p < 0.0001; WC: z = − 3.92, p < 0.0001). In the same line, one animal always won more encounters than the other in the Tube Test (Fig.4F, Wilcoxon signed-ranks for TT Day1: z = − 4.06, p < 0.0001 and for TT Day2: z = − 4.12, p < 0.0001), with the exception of one pair of animals in Day 1 and another in Day 2, where both animals of the pair won the same number of trials, thus no categorization as dominant or submissive was possible in these cases. We decided to investigate how body weight would relate to dominance in established hierarchies of rats. Intriguingly, we did not observe differences in the body weight between dominant and submissive animals in any of the behavioral tests where animals would compete for food, sucrose or water (Fig.4A–F; Paired Sample T-test for weights in mFC: t = 1.208, p = 0.242; SC: t = − 0.309, p = 0.761; SCI: t = − 0.843, p = 0.410; mFCD: t = 0.522, p = 0.608; WC: t = 0.067, p = 0.947). In contrast when they had to compete for territory for the first time in the Tube Test, a significant relationship between dominance and body weight was observed (Fig.4F; Paired Sample T-test for weights in TTDay1: t = 2.529, p = 0.021). In conclusion, although no relation was observed between social hierarchy and body weight in the rest of the tasks, this was not the case in the Tube Test, where bigger rats had a higher probability of winning in the first encounters. We then asked whether hierarchy following this criterion, amount of resources consumed, would also translate to differences in other behaviours within each test. General exploration of the resource location during the whole session did not differ between dominant or submissive animals when consumption in the same test was taken as criterion. However, we did observe that submissive animals would spend more time self-grooming in the modified Food Competition test (z = − 2.016, p = 0.044) and that time spent pushing was modulated by dominance in some tests. Dominant animals tended to display more pushing in the Sucrose Competition with intermittent access (z = − 1.867, p = 0.062) and surprisingly, submissive animals were the ones that pushed more in the Sucrose Competition with continued access to the bottle and the Food Competition under deprivation (SC: z = − 2.722, p = 0.006 and FD z = − 3.472, p = 0.0005). As this observation was unexpected, we next explored this further. Dominant rats are more efficient displacing their partners to gain access to resources. Although the time animals spent pushing their partner should be a good measure of amount of conflict between the interacting animals, qualitative differences might be more informative of dominance status. One possibility is that even if a dominant rat pushes less often, its thrusts may be more successful in removing the partner from the resource. Thus, we calculated for each animal the percentage of successful pushing from the total number of pushing bouts, i.e. the fraction of pushing epochs that actually displaced the partner and allowed access to the resource. Strikingly, dominant animals were more successful in displacing their partners in the modified Food Competition under deprivation, while submissive animals would push often but failed to displace their partner (Fig.5, Wilcoxon signed rank test z = − 1.979 p = 0.048). However, this was not observed in the Water Competition test (WC: z = − 0.821 p = 0.411) nor in the tasks not involving deprivation (mFCD: z = − 0.933 p = 0.351; SC: z = − 1.014 p = 0.310; SCI: z = − 0.563 p = 0.573). The lack of differences in successful pushing between dominant and subordinate rats in the water competition test, could result from a limited window within the test where asymmetric interactions are apparent. To investigate this possibility, we identified the epochs with highest conflict in the Water Competition test, i.e., those where the most drinking and pushing behavior was observed for each pair of animals (Supplemental Figure2A). We then classified the animals as dominant and submissive according to the duration of drinking in that epoch and quantified pushing displayed by either dominant or subordinates. This new categorization led to a change in the hierarchy in 55% of the pairs (11 out of 20). Here, although submissive Figure2. Behavioral profiles change according to the specifics of each social competition test. (A) modified Food Competition test, (B) Sucrose Competition with continuous access to the bottle, (C) Sucrose Competition with intermittent access to the solution, where animals could only drink during 2min and the bottle was absent during 1min inter-trial interval; (D) modified Food Competition test with deprived animals, (E) Water Competition test, (F) Tube test. For all tests, left panel shows the schematic representation of the task. Cartoons with a shaded circle as background indicate that tests were performed under deprivation. (A–F) middle panel: raster plots showing frequency and duration of behaviors of interest in an example pair of animals (animal A in blue, animal B in grey), except for (F) where the animal wining each trial is depicted for the 20 pairs of animals in all trials. White coloured trials in the Tube Test correspond to “resistant trials” where the loser of the pair resisted to enter the tube and trial could not be completed. In those tasks with a trial structure, (A, C, D) grey shaded areas indicate the moments where access to the resources where available, while intertrial interval, during which animals could explore the feeders but not access the food or sucrose bottle, are indicated with white background. (A–E) right panel: Boxplot representation of the duration of each behavior of interest for each individual animal. When more than one day of testing was performed, data represents average of the two days. For those tests with a trial structure, values represent behavior throughout the entire test period averaged over the 5 trials (or 10 trials, when two days of testing were performed), being one trial defined as the last 40s of the intertrial interval and first 80s of access to reward. Median, quartile 1 and 3 are represented, whiskers indicate minimum and maximum values and extreme values are signaled as a dot. Consumption in red, Pushing in dark green, Exploration of the Feeder in light green, Grooming in yellow. (F) right: Trial duration in the Tube Tests in the two testing days. Black line represents the median duration that animals took to push their partner out of the tube, and grey shadows depicts 95% confidence interval. As an insert in each day, pie charts represents the percentage of trials that were completed. Note that 9% of trials in the second day of the Tube Tests were not completed as one animal (the loser) refused to enter the tube. ◂ 6 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ animals spent longer time pushing their cage mate (Fig.5E middle plot, z = − 3.920, p < 0.0001), again dominant animals displayed higher efficiency in displacing their subordinates, being successful practically 100% of the times (Fig.5E plot on the right, z = − 3.627 p = 0.0002). This fine grained behavioral analysis, where pushing is categorized into successful or unsuccessful, thus revealed that although in some tasks submissive animals displayed higher duration of pushing, they rarely managed to get the access to the resource, being dominant rats more successful to displace their cage mates. Social hierarchy as a stable trait between tests. To examine whether social hierarchy is a stable trait in familiar animals, we analyzed reliability across the performed tests. To this end, we computed the Dominance Index (DI) for each test where the difference in resource consumption across partner animals was normalized by the summed resource consumption of the pair. A DI close to 0 means that animals did not have a strong hierarchy. Positive values indicate that animal A consumed more, while negative values indicate that animal B was the one having priority access to resources or won more trials in the Tube Test. Provided that behavioral measures were stable across testing days (Supplemental Figure3) data was averaged for this analysis. Consumption Exploration of the feederPushing Grooming A BC 0 20 40 60 Day 1 Day 2 Day 1 Test Session (trials) Time to eat all pellets (trials) Non Food Deprived Food Deprived 0 20 40 60 Day 1Day 2Day 1 Test Session Time to eat all pellets (session) abc Total time (%) 0204060 Food Competition Suc.Competition Suc.Intermitent Food Deprivation Water Competitio n a a b c d b b a a c c d a b a 02 550 a b b a b 01020 02550 Figure3. Descriptives of the behavioral analysis for all social tasks involving competition for resources. (A) Comparison of the behaviors of interest displayed by each animal across all behavioral tests. Data is presented as percentage of time performing a specific behavior related to the total duration of the task, enabling comparison between tests differing on duration. For those tasks with a trial structure, consumption corresponds to the first 80s of reward availability for each trial, while pushing, exploration of the feeder and grooming durations correspond to the displays performed during the 40s before and 80s after reward availability. When more than one day of testing was performed, data represents average of the two days. Each competition test provided very different behavioral profiles, where the duration of consumption and exploration of the feeder or pushing behaviors to access the resources differed clearly over the tests. Note that pellets in the modified Food Competition test were consumed during the first seconds, but still high levels of pushing and exploration of the feeder where observed. However, the time spent consuming sucrose and the levels of pushing for accessing the bottle were low. Boxplots depict the median and quartile 1 and 3, whiskers indicate minimum and maximum values and extreme values are signaled as a dot. Consumption in red, Pushing in dark green, Exploration of the Feeder in light green, Grooming in yellow. Letters denote statistically significant differences between behavioral tests after one-way ANOVA with Tukey posthoc comparisons. Time spent grooming was not normally distributed, thus non-parametric analysis was performed. (B) Latency to eat all pellets for each trial is shown in the Food Competition tests, with or without deprivation where median and 95% CI for each trial are represented. Animals only took around 20s to eat all pellets available, decreasing this latency when food deprived. Shaded background indicates session performed under food-deprivation. (C) Boxplots representing the average time to eat all pellets over testing days. Wilcoxon rank test revealed that latency to eat the pellets decreased on the second day of testing of the modified Food Competition test, and did further under deprivation state. **p < 0.01, ***p < 0.001. 7 Vol.:(0123456789) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ DI for positive reinforcers led to a highly variable distribution across dyads, where in some dyads animals would strongly differ in their consumption and in others differences were subtle (Fig.6A). This was not the case in the Water Competition, where DI was mostly around 0 for all animals, indicating that both animals drank very similar amounts of water during the test. In contrast, the Tube Test gave very polarized DIs, where most of the pairs had one animal winning almost 100% of the trials. To refine our understanding of the hierarchy in the Tube Test, we computed a Conflict Resolution Index (CRI), which would take into account not only who won a trial, but also how long it took for the conflict to be resolved (i.e., the latency for one of the animals to be pushed out of the tube). The conflict resolution index revealed a more continuous and fine-grained measure of hierarchy strength in each pair (Fig.6B). If social hierarchy was a stable trait, then dominance indexes should be correlated across tests. We found DIs from tests with competition for positive reinforcers were positively correlated (Fig.6C). Moreover, the modified Food Competition test correlated with the DI index in the Tube Test, when conflict resolution time was taken into account. Water competition DI was not correlated with any of the other tests. Water deprivation could have challenged the homeostasis of the interacting animals bringing them to a very different internal state that disrupted the stable hierarchies revealed by the other tests. Alternatively, computing the DI for the water competition test using the whole test duration, may have diluted differences in water consumption across animals within the pair. Hence we re-calculated DI for different time windows, just as we observed for the pushing behavior (see above). No correlation was found between water competition test and the other test when other time windows were used to compute DI (Supplemental Figure2B). Still, we have shown that during this test, the dominant animals accessed the water bottle in a qualitatively different manner, by successfully displacing their subordinates (Fig.5E). This highlights the importance of considering multiple behaviors simultaneously and suggests that social status in the Water Competition task could be better assessed by finer behaviors rather than water consumption. Modified Food Competition tests as a tool to measure stable hierarchies. Last, we calculated for each task a Conflict Index as a measure of the degree of conflict that our manipulations introduced in the home-cage, by dividing the time animals spent pushing in one test by either the time spent consuming in case of the Sucrose and Water Competitions, or latency to eat all rewards in the case of the modified Food Competitions. Food competition with and without deprivation were the tasks with higher conflict, as animals displayed high amounts of pushing and the time available to eat the resources in each trial was very short (Supplemental Figure4A). Since the modified Food Competition test yielded significant levels of conflict but did not involve food deprivation, we next evaluated whether attributing dominance within dyads using this test, would allow correct identification of the dominant rat in the other tests. Specifically, animals were classified as Dominant or Submissive according to their DI in the second day of testing in this task, as the conflict index was higher in this day (Supplemental Figure4B). Those pairs where the difference of the number of pellets eaten was small (less than 5% difference compared to equal consumption between the animals) were considered to have an unstable or unclear hierarchy (n = 4) and were not included in this last analysis. As expected, dominant animals consumed more pellets in the Food Competition test (Fig.7A, Wilcoxon signed-ranks of average Consumption in mFC of both days z = − 3.362, p = 0.001). Interestingly, they also successfully pushed their partner away from the feeder more (z = − 2.275, p = 0.023), explored the feeder more during inter the trial interval, where the pellets were present but not accessible (z = − 2.844, p = 0.004), and groomed less than their submissive pairs during the trial period (z = − 2.275, p = 0.023). Moreover, dominant animals according to the modified Food Competition did consumed more sucrose, both when access was continuous (Fig.7B, SC z = − 2.499, p = 0.012) or intermittent (Fig.7C, SCI z = − 2.275, p = 0.023). Attributing dominance found in the modified Food Competition, to the same test run under food deprivation, revealed similar dominance interactions (Fig.7D, Consumption in mFCD z = − 2.619, p = 0.009; Successful Pushing in mFCD z = − 2.534, p = 0.011; Anticipatory exploration of the feeder in mFCD z = − 2.902, p = 0.034, Anticipatory grooming in mFCD z = − 1.992, p = 0.046). Moreover, dominant animals also successfully pushed away their partner more in the Water Competition test (Fig.7E, z = − 2.379, p = 0.017). In the case of the Tube Test, the amount of wins did not differ between dominant and submissive animals, as determined by the modified food competition test (Supplemental Figure4C). In Fig.3 we show that differences in body weight affects the probability of winning in the Tube Test, especially on the first day. This was however not the case for the other tests. Thus, weight may dominate the outcome of the tube test, overshadowing the dynamics of social interactions within stable pairs. Therefore, we decided to examine the relationship between the hierarchy in the modified Food Competition and the Tube Test while controlling for the effect of the body weight. To this end, we first regressed the number of pellets eaten against the animals’ body weight and calculated the residuals. Next, we calculated, in the same manner, the residuals when regressing out body weight from the conflict resolution index (see above) of the first day of the Tube Test. Interestingly, the linear regression of these residuals was statistically significant (p = 0.010) indicating that indeed, when correcting for the effect of body weight, consumption in the modified Food Competition predicts who will win in the first interactions of the Tube Test (Fig.7F). Thus, dominant animal in the modified Food Competition test also won more trials in the Tube Test when the influence of the body weight was controlled for. Discussion Here, we developed and validated the modified Food Competition task, a new tool designed to provide, for the first time, the means to evaluate established hierarchies in pairs of rats, and with the added value of doing so in the home cage, without inducing aggressive behaviors nor requiring food-deprivation. In our task, cage mates with a stable hierarchy competed for access to positive reinforcers. The introduction of a small conflict in the home cage, where consumption of appetitive food is only possible for one animal at a 8 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ time, led to subtle competition which translated into increased consumption by the dominant rat. This measure reliably predicted differences in other behaviors observed during the competition test. These differences held across the different social tasks evaluated, such as competition for sucrose solution or food and water competition under deprivation. Importantly, testing was performed in the home cage of the animals, thus minimizing the influence of the experimenter in the social interactions displayed, and interference by other factors such as anxiety or exploratory behaviors usually displayed in novel environments. Conflict in the modified Food Competition increased over days, providing the second day a clearer picture of the established hierarchy, most probably because during the first of competition in the home cage, differences in attention to the appearance of the conflict situation might be modulating the interactions. In future experiments, it might be desirable to include a third day of testing to confirm whether conflict continues increasing or is stable after the second day. Interestingly, although competition was observed and hierarchy could be identified, no agonistic behaviors (biting, boxing, keep down, lateral threat) were observed between the cage mates in our task. This is in accordance with previous reports indicating that once social hierarchies are established, the number of agonistic encounters decreases6. Moreover, the fact that no food deprivation was required, nor aggressive behaviors were observed, can be considered as an added value of our task. Aggressive behaviors induce robust stress reactions in rodents20, and food deprivation, although widely used in neuroscience to increase motivational salience during behavioral testing, modifies internal state21 and social behavior22. Minimizing the possible long term effects of these manipulations on the internal state of tested animals is particularly important for studies regarding the impact of social hierarchy on other behavioral, physiological and brain functions. Moreover, the fine behavioral characterization across the different social tasks used, allowed us to identify very interesting social patterns, that to our knowledge have not been reported before. In this direction, we describe that social hierarchy tasks with a trial structure (modified Food Competition tasks and Sucrose Competition with Intermittent access), where access to resources was presented in a repeated and intermittent manner, promoted competition. Moreover, we describe that when measuring conflict, the time spent pushing by the animals is not indicative of dominance, but rather how efficient an animal is to displace its partner while pushing. Although the dominance index in tasks that involved competition for positive reinforcers reliably revealed the strength of social hierarchy within a pair, this was not the case in the Water Competition test under deprivation, where the animals drank around the same amount of water (dominance index around 0), nor in the case of the Tube Test, where very polarized results were observed. In the Water Competition task, animals tended to perform long bouts of drinking, alternating consumption until both animals were satiated, which resulted in very similar final consumption levels in both animals. We asked whether an analysis with finer temporal resolution could unveil structured dynamics of water consumption in this task, such that asymmetries across rats in the dyad would emerge during bouts of conflict, and these asymmetries would correlate with other social competition tests. However, to the extent that we could quantify, we did not observe such a pattern. Strikingly, we found that although consumption between animals was very similar, other behaviors displayed while approaching the water bottle were clearly different. Although submissive animals in this task spent more time pushing their cage mate in moments of high conflict, they were rarely successful in accessing the water if the dominant was already drinking. Indeed, the ability of dominant rats to successfully displace their partners from the resource location was not limited to the Water Competition, being reliable in those tests where more conflict (pushing) was observed. In the case of the tube test, we showed that computing conflict resolution index which takes into account multidimensional behavioral measures, such as the winner of a trial and conflict duration (latency for one of the animals to be pushed out of the tube) revealed a more granular view of the strength of social hierarchy in this Figure4. Categorization of animals as dominant and submissive according to behavior within each task. (A–E) Behavioral profiles when consumption of the resources within each test is used to define dominant and submissive animals. For all panels, a schematic cartoon with experimental design is provided. Cartoons with shaded background indicate that tests were performed under deprivation. For each test, differences between dominant and submissive animals are represented regarding body weight, time spent consuming the resource (or number of pellets eaten in the case of the food competition tasks), duration of pushing, exploration of the feeder and grooming is provided, where median and 95% interval confidence are displayed and individual values are showed with light lines. Color coded raster plots of behaviors of interests display raw data in a testing session, where pairs of animals are sorted according to the stronger differences in hierarchy for each test. For those tests with a trial structure, the five trials of each pair are plotted in separate lines, all aligned to time 0 (when access to reward was possible) and reflecting the behavioral data of the 40s before and 80s after that moment. When more than one day of testing was performed, data represents average of the two days and rasters display data of day 2. In those cases where the animals of one pair had identical values in the categorizing criteria (same amount of pellets eaten, duration of consumption or trials won in the Tube Test), no hierarchy was assumed for this specific task, and these pairs were removed from this analysis (one pair for the Sucrose Test, one for the Tube Test and one for the Food Competition under deprivation). For all graphs and rasters, Consumption is represented in red, Pushing in dark green, Exploration of the Feeder in light green and Grooming in yellow. Time of consumption in each test was significantly different between animals defined as dominant or submissive, and in some cases, these differences also were translated to a differential amount of grooming or pushing behavior. Interestingly, no differences in body weight were found in these tests, indicating that priority access to resources in established hierarchies is not influenced by the size of the animals. (F) In the Tube Test, the amount of winnings was clearly different between dominant and submissive rats, and was related to differences in body weight, which reached significance in the first days of testing, being bigger animals those more likely to win. *p < 0.05, **p < 0.01, ***p < 0.001. ▸ 9 Vol.:(0123456789) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ 16 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ Moreover, Python Video Annotator allowed easy post hoc categorisation of the two types of exploration of the feeder (anticipatory or during the presence of the resource) and pushing behavior (successful or unsuccessful) which can only be identified once the bouts of pushing behaviour are finished and is thus not possible to analyse with online video analysis. Data analysis. Data was parsed and processed with Python (Python Software Foundation, v.2.7). In addition to comparing raw data obtained, we calculated several indexes to compare hierarchies across tests. Dominance Index. The Dominance Index (DI) was calculated for each test, where the difference in resource consumption across partner animals was normalized by the summed resource consumption of the pair, following this formula: Consumption corresponded to the number of pellets in the modified Food Competition tests, the duration of drinking in the Sucrose and Water Competition tests, and the number of wins displayed by an animal in the case of the Tube Test. The sign of this index would indicate whether animal A or B would consume more, i.e. positive values would indicate that animal A consumed more, and negative values that animal B consumed more. DIs close to 0 would indicate no differences in consumption between the animals of a pair. Differences of 5% to equal consumption, i.e. DI ranging from − 10 and + 10, were considered noise and indicative of no reliable hierarchy. Conflict Resolution Index in the tube test. The Conflict Resolution Index (CRI) was calculated taking into account not only who won a trial, but also how long it took for the conflict to be resolved (i.e., the latency for one of the animals to be pushed out of the tube): Conflict Index. The Conflict Index (CI) was calculated by dividing the time animals spent pushing in a specific test by either: (1) the time spent consuming, in case of the Sucrose and Water Competitions, or (2) the latency to eat all rewards in the case of the modified Food Competition with and without food deprivation. Statistical analysis. The statistical analysis was performed using IBM SPSS Statistics version 24.0 for Windows. The normality of the data was tested using Kolmogorov–Smirnov normality test, and when normality was not observed non-parametric tests were applied and median and 95% confidence interval were chosen to represent data in figures. Wilcoxon signed-rank tests with Bonferroni correction were used to study differences between counterbalanced groups in each protocol, of each behavior across the tasks, and to study differences between dominant and submissive animals on behavior. Paired t-test were performed to assess differences in the weights between dominant and submissive animals. One-Way ANOVA followed by post-hoc test Tukey was used to compare behaviors of interest across tasks. Here, when normality was not observed, a Kruskal–Wallis test with post-hoc Dunn-Bonferroni correction was performed. Bivariate Pearson Correlation was performed to measure the strength and direction of association between the Dominance Index of all the tasks and linear regressions for assessing predictive value of Food Competition and Tube Tests controlling for body weight. Statistical significance was set at p < 0.05. Data availability All data generated to support the findings of this study are available from the corresponding author upon reasonable request. Received: 29 April 2021; Accepted: 24 June 2021 References 1. Sapolsky, R. M. Social status and health in humans and other animals. Annu. Rev. Anthropol. 33, 393–418 (2004). 2. da Cruz, J. et al. Dominant men are faster in decision-making situations and exhibit a distinct neural signal for promptness. Cereb. Cortex 28, 3740–3751 (2018). 3. Zhou, T., Sandi, C. & Hu, H. Advances in understanding neural mechanisms of social dominance. Curr. Opin. Neurobiol. 49, 99–107 (2018). 4. Sapolsky, R. M. The influence of social hierarchy on primate health. 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Acknowledgements This work was supported by grants of the NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation under the grant number 26478 to C.M., the Spanish Agency of Research (grant RTI2018097843-B-100 to C.M.), the “Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2013-0317 and SEV-2017-0723) and the Champalimaud Foundation. D.F.C. was further supported by the Ministerio de Ciencia e Innovación (BES-2016-07674) and C.M. by a Ramon y Cajal contract (RYC-2014-16450). We thank the Marquez lab for fruitful discussions and specially, Kevin Caref for insightful comments on the manuscript. We also express our gratitude to Gonçalo Lopes for his help with Bonsai workflow for annotation of behavior, Antonio Dias for feedback on the python scripts and to Cristina Savin for discussion of the data analysis. 18 Vol:.(1234567890) Scientific Reports | (2021) 11:14599 | https://doi.org/10.1038/s41598-021-93818-0 www.nature.com/scientificreports/ Author contributions DF Costa: Conceptualization, Investigation, Methodology, Formal analysis, Visualization, Writing original draft and Review and editing the manuscript; MA Moita: Methodology, Review and editing manuscript, Funding acquisition; C Márquez: Conceptualization, Supervision, Investigation, Methodology, Formal analysis, Visualization, Writing—original draft, Writing—review & editing, Funding acquisition. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https:// doi. org/ 10. 1038/ s4159802193818-0. 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