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Pro-social behavior in rats is modulated by social experience

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Cite as: eLife 2014;3:e01385 doi: 10.7554/eLife.01385

Abstract

In mammals, helping is preferentially provided to members of one’s own group. Yet, it remains unclear how social experience shapes pro-social motivation. We found that rats helped trapped strangers by releasing them from a restrainer, just as they did cagemates. However, rats did not help strangers of a different strain, unless previously housed with the trapped rat. Moreover, pair-housing with one rat of a different strain prompted rats to help strangers of that strain, evidence that rats expand pro-social motivation from one individual to phenotypically similar others. To test if genetic relatedness alone can motivate helping, rats were fostered from birth with another strain and were not exposed to their own strain. As adults, fostered rats helped strangers of the fostering strain but not rats of their own strain. Thus, strain familiarity, even to one’s own strain, is required for the expression of pro-social behavior.

https://doi.org/10.7554/eLife.01385.001

eLife digest

Humans help family members and friends under circumstances where they may not help strangers. However, they also help complete strangers through both direct actions, such as helping someone who has stumbled, and indirect actions, such as giving to charity. Ben-Ami Bartal et al. have now explored the biological basis of such socially selective helping by testing whether rats help strangers, and if so, under what circumstances.

In the experiments a free rat was exposed to another rat trapped inside a plastic tube with an outward-facing door for 12 one-hour sessions. When tested with a cagemate trapped inside the tube, most free rats learned within a few days to release the trapped rat by opening the door. Ben-Ami Bartal et al. then exposed the free rats to strangers they had never met or seen before. Remarkably the rats consistently released the trapped stranger, acting toward strangers just as they had acted toward familiar cagemates. This result suggested that individual familiarity is not required for helping to occur.

To test the limits of rat benevolence, Ben-Ami Bartal et al. tested free rats (always white albino rats) with trapped rats from a different outbred strain (black-hooded rats). The rats helped cagemates of a different strain but not strangers of a different strain. These results could be explained by a requirement for strain familiarity or individual familiarity. To distinguish between these possibilities, albino rats were housed for 2 weeks with a rat of a different strain, and then re-housed with another albino rat before being tested with a trapped rat belonging to a different strain. Consistent with a requirement for strain but not individual familiarity, the free rats now helped stranger rats from the different, but now familiar, strain.

To explore if there is any role for genetics or relatedness in socially selective helping, Ben-Ami Bartal et al. tested whether rats will help strangers of their own strain based on genetic relatedness alone. To do this albino pups were transferred to litters of a different strain on the day they were born, and never saw or interacted with another albino rat until testing. Remarkably, the albino rats helped strangers from the different strain that they were raised with, but they did not help strangers of their own strain because this strain was unfamiliar to them. The fact that the motivation to help other rats has its origins in social interactions rather than genetics provides the flexibility that is needed to navigate their way through social environments that often change unexpectedly.

https://doi.org/10.7554/eLife.01385.002

Introduction

Pro-social behavior comprises actions that improve the well-being of others (Eisenberg et al., 1989) and is found widely in the animal world (Owens and Owens, 1984; Wilkinson, 1984; Lee, 1987; Heinrich and Marzluff, 1995; Warneken and Tomasello, 2006; Nowbahari et al., 2009; Yamamoto et al., 2012; Baden et al., 2013; Clay and de Waal, 2013; Hatchwell et al., 2013). Pro-sociality within a group promotes individual survival and reproduction (Hamilton, 1984; Preston and de Waal, 2002; Decety and Svetlova, 2012). On the other hand, acting pro-socially towards individuals from other groups may not be adaptive, as other groups often compete for valuable and limited resources. Thus animals can be motivated to act pro-socially or aggressively depending on the social context.

In humans, pro-social behavior is modulated by the degree of affiliation and is extended preferentially towards in-group members and less often toward unaffiliated others (Hornstein, 1978; Cialdini et al., 1997; Preston and de Waal, 2002; Levine et al., 2005; Sturmer et al., 2006; Lamm et al., 2010; Echols and Correll, 2012). Yet humans can and often do act pro-socially towards strangers (Batson et al., 2005). This human capacity to help unfamiliar individuals is often viewed as a cognitively complex behavior that depends on high cognitive capacities and cultural transmission (Levine et al., 2001).

Rodents have emerged as a valuable model system for social behavior and communication (Decety and Svetlova, 2012; Mogil, 2012; Panksepp and Panksepp, 2013; Preston, 2013). Rodents manifest emotional contagion (Langford et al., 2006; Chen et al., 2009; Jeon et al., 2010; Knapska et al., 2010; Panksepp and Lahvis, 2011; Akyazi and Eraslan, 2014; Atsak et al., 2011), cooperation (Rutte and Taborsky, 2007; Viana et al., 2010; Tsoory et al., 2012), and helping (Ben-Ami Bartal et al., 2011; Church, 1959; Rice and Gainer, 1962). We previously found that Sprague-Dawley (SD) rats learn to release SD cagemates trapped in a restrainer (Ben-Ami Bartal et al., 2011). Here, we set out to investigate how previous social experience acquired during development and adulthood influences helping behavior. First, we tested whether rats will help unfamiliar individuals, strangers. Rats showed helping behavior equally towards cagemates and strangers, if the strangers were of their own strain. However, rats did not help strangers of an unfamiliar strain, suggesting that helping in rats may be innately biased towards the helper’s own strain. Yet, further experiments demonstrated that a short period of pair-housing with a rat of the unfamiliar strain was sufficient to motivate helping for that individual. Moreover, rats that had previously lived with a rat of a different strain were as motivated to help strangers of that strain as they were to help strangers of their own strain. This finding demonstrates that rats choose to help others depending on the social context, and extend pro-social motivation beyond individual identity, to groups defined by strain. Finally, we sought to determine if genetic relatedness is at all capable of influencing pro-social motivation towards an unfamiliar rat. We found that rats that were fostered from birth with another strain did not help strangers of their own strain as adults. Fostered rats only acted pro-socially towards rats of the foster strain, demonstrating that genetic relatedness alone is not capable of producing pro-social motivation.

In each hour-long session, a free rat of the SD strain was placed in an arena containing another rat trapped inside a centrally located restrainer. (A rodent stock, colloquially referred to as an outbred strain, is a colony of conspecifics derived from a small group of founder animals. Individuals are not genetically identical. Here we refer to stocks as strains.) The door to the restrainer could only be opened from the outside and thus only by the free rat. Door-opening by the free rat led to the trapped rat’s release from the restrainer. If a free rat failed to open the door within 40 min, the experimenter opened it halfway, allowing the trapped rat to exit the restrainer. Only door-openings resulting from the free rat’s action, during the first 40 min of each session, were counted as such. Regardless of which rat opened the restrainer door and whether that occurred before or after halfway opening, both rats remained in the arena for the entire hour of every session. All rats were males and all free rats were from the SD strain. Sessions were repeated for 12 days.

Results

To determine whether rats help unfamiliar individuals, free SD rats were placed with trapped SD rats that were either strangers (n = 12, SD stranger condition) or cagemates (n = 8, SD cagemate condition) in the helping behavior test described above. Cagemates were pair-housed for 2 weeks prior to the experiment. Strangers were SD males from a different cohort, born in the same week but on a different day as the test rats. On each day of testing, a different stranger, to whom the free rat had no prior exposure, was trapped in the restrainer. Most rats in both SD cagemate (6/8, 75%) and SD stranger (10/12, 83%) conditions acted pro-socially, learning to release the trapped rat, and becoming openers (Figure 1; see Methods for opener definition). Rats in the two conditions were similarly active as measured by velocity (two-tailed Student’s t test, p>0.05), spent similar amount of time near the trapped rat (two-tailed Student’s t test, p>0.05), and began to open the door on around the same day (4.0 ± 1.1 days for trapped cagemates; 3.7 ± 0.8 for trapped strangers, mean ± SEM). Thus, rats were as motivated to help strangers as they were to help cagemates, showing that individual familiarity is not required for pro-social behavior in rats.

Rats were as motivated to help strangers of the same strain (SD stranger; right) as they were to help cagemates (SD cagemate; left).

In both experimental conditions (diagrammed at top), SD rats were housed with another SD rat. However in the cagemate condition, the cagemate served as the trapped rat (‘a’) whereas in the stranger condition, the trapped rat was a stranger (‘?’) and not the cagemate (‘b’). Across the days of testing, the median latency to door-opening (bottom) decreased for both conditions.

https://doi.org/10.7554/eLife.01385.003

The results above indicate that rats are motivated to help unfamiliar rats of their own strain. To determine if rats extend help to rats from a different strain, free SD rats were tested in the helping behavior test with trapped rats of the black-caped Long-Evans (LE) strain, which were either cagemates (n = 7, LE cagemate condition) or strangers (n = 16, LE stranger condition). SD rats did not release LE strangers with only a minority becoming openers (4/16, 25% openers; Figure 2; Video 1). In contrast, most SD rats in the LE cagemate condition became openers (5/7, 71%; χ2, p=0.04), learning to open the restrainer on average on day 3.8 ± 1.7 (mean ± SEM, Figure 2). Thus, rats help strangers of their own strain but not strangers of a different strain, suggestive of an in-strain bias for pro-social behavior. Moreover, short-term, paired-housing with a rat from a different strain is sufficient to motivate helping for that individual rat.

Rats of the SD strain helped trapped rats of the LE strain only if they were familiar with an LE individual.

Three experimental conditions are diagrammed at top. The free rat was always from the SD strain and was housed with a cagemate, denoted at top, from either the SD (white) or LE (black and white) strain. The free rat was then tested with an LE rat that was either the cagemate (‘a’) or a stranger (‘?’). Note that in the LE familiar condition (right), rats had previously housed with an LE rat (illustrated) but were housed with an SD rat (not illustrated) at the time of testing. Across the days of testing, the median latency to door-opening (bottom) decreased for SD rats tested with trapped LE cagemates (left), but not those tested with LE strangers (middle). Like rats in the LE cagemate condtion, rats in the LE familiar condition (right) also became openers.

https://doi.org/10.7554/eLife.01385.004
Video 1
Rats help rats of a familiar strain.

Rats that are familiar with at least one LE rat show interest in, open the restrainer door for, and rarely fight with trapped LE rats. Rats that are unfamiliar with LE rats show little interest in, do not help, and often fight with an LE rat.

https://doi.org/10.7554/eLife.01385.005

We then examined whether SD rats that were familiar with one LE individual would be motivated to help LE strangers. SD rats were pair-housed with an LE cagemate for 2 weeks, then re-housed with an SD rat, and a week later, tested in the helping behavior test with trapped LE strangers (n = 12, LE familiar condition; Figure 2). In contrast to SD rats with no exposure to the LE strain, most SD rats familiar with one LE individual became openers for LE strangers (8/12, 67%; χ2, p=0.03). They learned to open the door on average on day 4.0 ± 1.0 despite a lack of individual familiarity with the trapped LE rat (Figure 2). This experiment shows that social experience with one individual rat of a different strain is sufficient to motivate helping towards unfamiliar members of that strain. This provides further evidence that individual familiarity is not required for helping.

Finally, to determine if helping behavior is at all influenced by the strain of the trapped rat (SD, LE), or if strain bias in helping is entirely due to strain familiarity (familiar, unfamiliar), SD rats were fostered and raised with LE rats from birth, in an environment that effectively prevented exposure to others of their own strain (fostered conditions, Figure 3). If rats are innately motivated to help rats of their own strain, then SD rats raised exclusively with LE rats (fostered) should act pro-socially towards other SDs. In contrast, if strain familiarity is the only determinant of pro-social behavior, these rats should not help their own kind. At 2 months of age, fostered rats were tested with trapped rats that were SD strangers (n = 8, fostered+SD) or LE strangers (n = 8, fostered+LE). Fostered SD rats did not help trapped SD strangers (Figure 4; Video 2). Only 1 of 8 fostered SD rats (12.5%) became an opener, establishing that rats are not innately motivated to help their own strain. In contrast, fostered rats did help LE strangers (5/8, 62.5%; χ2, p=0.04) as expected from the pro-social influence of strain familiarity. Thus, strain familiarity, even to one’s own strain, is required for the expression of helping behavior.

Fostered SD rats were minimally exposed to other SD rats from birth.

This diagram indicates how many LE pups (black dots) and SD pups (red dots) were present in each litter during postnatal (P) days 0–14 (x-axis) for rats in the fostered+LE (A) and fostered+SD (B) conditions (see Figure 4). On P0–P1, two SD pups (n = 32) were transferred into each LE litter (n = 16). If both pups survived one of the two SD pups was removed at P6, leaving a single SD pup with an LE dam and LE littermates. As red dots indicate other SD pups, the number of red dots is representative of the amount of exposure each fostered SD rat had to other SD pups during their lives. Individual rats are arranged from the most exposure to SD rats (upper left in each group) to the least exposure (bottom right). Following P11, no fostered rat was exposed to other SD rats until testing. In two cases (both in the fostered+SD condition illustrated in B), the SD pup in the LE litter died at a later date (P11, P12). In these cases, an SD male who had been removed from an LE litter 5 or 7 days before was then re-added to the LE litter. Note that only one animal (upper left in B) was exposed to SD rats with his eyes open; this rat proved to be the only door-opener in the fostered+SD condition. Lines above the dots represent the strain of the dam (red: SD; black: LE).

https://doi.org/10.7554/eLife.01385.006
Strain familiarity, even to one’s own strain, is required for the expression of helping behavior.

Fostered SD rats were raised with LE rats from birth (top diagram) and were not exposed to or able to interact with other SD rats prior to testing. When fostered SD rats were adults, they were tested with trapped stranger rats (‘?’) of either the SD (left) or LE (right) strain. Fostered SD rats did not help trapped SD strangers. In contrast, fostered SD rats helped trapped LE strangers.

https://doi.org/10.7554/eLife.01385.007
Video 2
Fostered rats do not help rats of their own strain.

Normally rats show pro-social behavior toward rats of their own strain. However, SD rats raised exclusively with LE rats and in isolation from other SD rats did not show interest in or open for a trapped SD stranger.

https://doi.org/10.7554/eLife.01385.008

The finding that fostered rats did not help SD strangers indicates that a rat’s familiarity with himself is insufficient to motivate helping for individuals of his strain. Instead, social interaction with another rat, such as that occurring while two rats live together, is critical to shaping pro-social motivation. Moreover, since rats tested with strangers from unfamiliar strains did not become openers across the days of testing, the exposure and interactions afforded by testing sessions appear insufficient to produce pro-social motivation.

Fostered rats were noticeably more anxious than naturally reared rats. In open-field testing conducted prior to the experiment, fostered rats spent more time in the corners of the arena than rats in other conditions (33.1 ± 2.7 s/min for fostered rats; 25.0 ± 1.9 s/min for naturally reared rats; files with open field data for SD cagemate and SD strangers were corrupted and therefore not included; two-tailed Student’s t test, p<0.02; Figure 5A). Moreover, fostered rats that opened the restrainer began to do so significantly later (day 8.0 ± 1.1 for fostered rats; 4.5 ± 0.5 for all other rats; two-tailed Student’s t test, p<0.01; Figures 1–2, 4B). Fostered rats were tested for an extra day, confirming that opening behavior persisted. These data suggest that fostering rats in a different-strain environment is associated with increased anxiety. A same-strain, cross-fostering control condition is needed to distinguish whether strain, fostering, or both drive this effect.

(A) Fostered rats (left of dashed line) spent more time at the arena corners during open field testing than naturally reared animals (right of dashed line).

(B) The proportion of rats that opened increased across the testing sessions for rats tested with familiar strains (open markers) but not for those tested with unfamiliar strains (filled markers). (C) No differences between rats tested with trapped rats from familiar strains (white bars) and those tested with rats from unfamiliar strains (gray bars) were observed in the average number of alarm calls or the amount of time spent at corners during open field testing.

https://doi.org/10.7554/eLife.01385.009

Across all conditions, the free rat’s familiarity with the strain of the trapped rat is the common determinant of whether helping behavior occurred (Figure 6A). Most rats in familiar strain conditions became openers (33/47, 74%) whereas only 21% (5/24) of rats in unfamiliar strain conditions did (χ2, p<0.001). Rats tested with a familiar strain experienced opening the restrainer door as rewarding, as they typically opened it on consecutive days (Figure 6B–C). In contrast, rats tested with unfamiliar strains rarely opened on consecutive days, suggesting that opening was not rewarding for these animals. As open field performance and alarm calls were not different for familiar and unfamiliar strain conditions (Figure 5C), it is unlikely that trait anxiety or the trapped rat’s distress accounts for the results.

Rats experience helping rats of a familiar strain as rewarding.

(A) The latency to door-opening decreased along the days of testing for rats tested with familiar strains (open circles) but not for those tested with unfamiliar strains (filled squares). (B) The proportion of openings that were followed by a repeated opening on the next day of testing was higher for trapped rats from familiar strains (left of dashed line) than from unfamiliar strains (right of dashed line). (C) Opening data from representative rats of each condition are illustrated. Fostered SD rats were tested for 13 days.

https://doi.org/10.7554/eLife.01385.010

When tested with familiar strains, helping behavior was demonstrated equally for strangers and cagemates. Yet, the underlying affective response might differ in these conditions. In support of this idea, rats displayed a different movement pattern when tested with a trapped cagemate or stranger. Rats tested with a trapped cagemate were significantly more active prior to door-opening than rats tested with a stranger (MMA, p<0.05, Figure 7A). However, there was no difference in the distance from the restrainer in these conditions (Figure 7B). These results demonstrate that rats were equally motivated to help, but not equally aroused by, a trapped cagemate and stranger.

Prior to door-opening, rats are equally motivated to help, but show more activity for trapped cagemates than for strangers from a familiar strain.

(A) Across conditions and testing days, rats tested with cagemates (white circles) were more active in the period before door-opening than were rats tested with strangers from a familiar strain (black circles). (B) The distance from the restrainer was not different for rats tested with cagemates (white circles) and strangers from a familiar strain (black circles).

https://doi.org/10.7554/eLife.01385.011

Door-opening behavior overlapped greatly, but not completely, with strain familiarity. Across all conditions, rats who became openers demonstrated an affiliative behavioral pattern. Prior to door-opening, openers spent more time around the closed restrainer than did non-openers (two-tailed Student’s t test, p<0.01; Figure 8A; Video 1). Following the trapped rat’s exit from the restrainer, significantly less fights were observed for openers than for non-openers (two-tailed Student’s t test, p<0.01; Figure 8A). Baseline anxiety level was assessed by the time spent in the corners of the arena during open-field testing. Only for openers, baseline anxiety was positively correlated with the first day of opening (Figure 8B), suggesting that anxiety negatively impacts successful helping in the presence of pro-social motivation. Rats were less active before door-opening compared to after door-opening (repeated measures ANOVA, main effect, p<0.001; Figure 8C). Pairwise comparisons found that this effect was due to significantly reduced activity in non-openers prior to door opening, which was not the case for openers (pairwise comparisons, p<0.05; Figure 8C). This difference may result from either non-openers’ greater anxiety or reduced interest in the trapped rat.

Openers showed an affiliative behavioral pattern.

(A) Rats that became openers (left) spent more time around the closed restrainer (open bars) and fought less with the trapped rat (gray bars) than did non-openers (right). (B) The first day of repeated opening was correlated with time spent at the corners during open-field testing for openers (top) but not for non-openers (bottom). (C) Averaged across the 12 days of testing and regardless of strain familiarity, non-openers were less active when the restrainer was closed (white bars) than when it was open (gray bars).

https://doi.org/10.7554/eLife.01385.012

Discussion

This study demonstrates that helping another rat, by releasing it from a restrainer, is flexibly applied to select others based on previous social experience. It is neither the individual identity nor the particular strain of the rat in need that motivates helping. Rather, it is the prior social experience of the free rat with any member of the trapped rat’s strain that determines the target group for helping. Thus, rats help another rat from a given strain (same or different) only if they have previously lived with a member of that strain.

Rats pick up sensory cues from their cagemates that are retained and utilized to discriminate between strangers that share those cues and those that do not. This information then plays a key role in eliciting pro-social behavior, and could potentially be used for other behavioral decisions as well, when encountering an unfamiliar individual, effectively forming group categories. The experiments delineated above are not informative of the sensory modalities participating in the classification of strangers as similar or different to the cagemate. Previous research points to the importance of vision, olfaction and audition for affective communication between mice (Langford et al., 2006; Jeon et al., 2010). It is possible that helping behavior in rats similarly relies on visual, olfactory, auditory, and also tactile cues as the clear and perforated restrainer allows for full sensory communication between the free and trapped rats.

The present study effectively demonstrates that strain identity is meaningless without social experience during development. Fostered rats raised without social interactions with their own strain were not motivated to help strangers of their strain, compelling evidence against an innate bias for pro-social behavior towards one’s own kind. This finding is congruent with studies showing that neither kinship nor perceived similarity is needed to motivate pro-social behavior in primates (Batson et al., 2005; Horner et al., 2011; Baden et al., 2013). In nature, social animals typically live with related others, and thus a pro-social bias favoring familiar others would also favor genetically similar others. Yet, relying on social experience rather than genetic similarity for guiding pro-social behavior has an added value in that it allows animals to flexibly adapt to different circumstances (Dugatkin, 2002). Assigning an affective meaning to a group category following social experience would be an efficient mechanism for appropriately extending pro-social behavior towards unknown individuals belonging to that same group.

While door-opening has an obvious pro-social outcome, a variety of motivations could contribute to this behavior. We have previously excluded the possibilities that door-opening depends on reward from either motor mastery or social contact (Ben-Ami Bartal et al., 2011). In addition it is unlikely that rats were motivated to open the restrainer by aggressive or anti-social motivations (Myer and White, 1965) because conflict between the free and trapped rats was minimal for opener pairs. As non-openers tested with an unfamiliar strain were not less active before door-opening than openers tested with a familiar strain (Figure 8C), differences in general arousal cannot account for the observed results. Given the greater interest in the trapped rat shown by openers and the lack of aggressive interactions between opener pairs, the authors favor the interpretation that rats open the restrainer door in order to terminate the trapped rat’s distress.

While we did not see any difference in helping behavior expressed toward strangers and cagemates, the underlying affective responses may have differed. Indeed, movement velocity was greater for rats tested with cagemates compared to those tested with strangers, indicative of different motivational states or magnitudes. Nonetheless, rats were sufficiently motivated to help strangers. This result is consistent with previous demonstrations of helping behavior expressed toward strangers in other animals, including humans (Warneken and Tomasello, 2006; Batson et al., 2007; Loggia et al., 2008; Custance and Mayer, 2012; Tan and Hare, 2013). While emotional contagion has been demonstrated for both individually familiar and unfamiliar mouse pairs (Chen et al., 2009; Jeon et al., 2010), it may be facilitated by individual familiarity (Langford et al., 2006; Jeon et al., 2010). Work in dogs and primates further supports a familiarity effect for emotional contagion (Palagi et al., 2009; Campbell and de Waal, 2011; Silva et al., 2012).

Empathy, the capacity to share and recognize the emotional states of another (Decety, 2011), often motivates approach and pro-social behavior and caring in humans (Eisenberg and Miller, 1987). The empathic response involves activation of common neural networks for processing one’s own and another’s distress (Decety et al., 2012). Some forms of empathy are primarily dependent on subcortical neural structures that are phylogenetically conserved across mammalian species (Decety and Svetlova, 2012). Emotional contagion, a fundamental component of empathy resulting when the emotion of one individual evokes a matching emotional state in another individual (Preston and de Waal, 2002), has been demonstrated in rodents (Chen et al., 2009; Langford et al., 2006). Thus, rodents, and mammals in general, may share a mechanism for mobilizing pro-social motivation in response to the distress of another individual. We would argue that a rodent form of empathy, as defined above, is the main motivation for the helping behavior observed in our studies. Moreover, the absence of door-opening for rats of unfamiliar strains likely reflects a reduced empathic arousal of the free rats in these conditions.

Rats that are not familiar with a different strain do not act pro-socially towards others of that strain. The selectivity with which rats engage in helping behavior is further evidence that releasing a trapped rat is an intentional social behavior, congruent with an empathic drive to help some rats, but not others. Yet, social experience can modify this behavior. We conclude that through social interactions, rats form affective bonds that elicit empathy and motivate helping. This motivation to help is extended to strangers of familiar strains, showing that rats form groups based on social experience. As is the case for humans (Xu et al., 2009; Mathur et al., 2010), rats base the bias for pro-social behavior on group membership. And as for humans, a diverse social experience was effective in mitigating such bias (Chiao et al., 2008; Elfenbein and Ambady, 2003; Madsen et al., 2007; Telzer et al., 2013; Zuo and Han, 2013). Moreover, we have demonstrated that for rats, genetic similarity does not influence pro-social motivation. Rather, groups are socially defined. Whereas social bias is hard to overcome through cognitive effort (Dovidio et al., 2002; Johnson et al., 2002; Amodio et al., 2004; Correll et al., 2007), our results support the existence of at least one mechanism for altering group membership that does not require complex cognition.

Materials and methods

Subjects

Sprague-Dawley (SD) and Long-Evans (LE) male rats (Charles River, Portage, MI) were used for all studies. Rats were 8–11 weeks old at the start of the experiment. Rats were housed in pairs with ad libitum access to chow and water in a 12:12 light-dark cycle. Animals were allowed 2–3 weeks to acclimate to the housing environment. Stranger rats were always housed either in a separate room or on a separate rack than the free rats with which they were to be tested. Stranger rats were also handled separately, such that there was never any contact between strangers and free rats before the start of testing. The fostered SD rats were bred in-house from pregnant females purchased from outside (Charles River, Portage, MI; see more details below).

Set up

Above every Plexiglas arena (50 × 50 cm, 32–60 cm high), a CCD color camera (KT&C Co, Seoul, Korea) was mounted. The cameras were connected to a video card (Geovision, Irvine, CA) in a dedicated PC.

Sound recordings of ultrasonic (15–70 kHz) vocalizations were recorded (Avisoft Bioacoustics, Berlin, Germany) through a single microphone in each testing room. Because recorded ultrasonic vocalizations could not be ascribed to individual rats and instead had to be assigned to each condition, all rats tested at one time in one room were always from the same condition.

Restrainers

A Plexiglas rodent restrainer (25 by 8.75 by 7.5 cm, Harvard Apparatus, Holliston, MA) was placed in the center of the arena. The body of the restrainer had several small slits and the back end had a large slit, allowing for olfactory and tactile communication between rats. At the other end, a customized door had two panes that were attached with three screws, and a pole (5 cm) supporting two weights (25 g each). The weights were included in order to facilitate the door falling off to the side once the free rat pushed on the door. The door was designed to be opened only from the outside. The free rat could open the door from the top, from the side, or by pushing up on the door with its snout.

Handling

Animals were habituated to the experimenters (who were kept constant for each cohort of rats) and the arenas prior to being tested. On day 1, rats were transported to the testing room and left undisturbed in their home cages. On day 2, rats were briefly handled and tested for ‘time-out’ (see below). Starting with the second day of habituation, rats were weighed three times each week for the duration of the experiment; no animal lost weight during the experiment. On days 3–5, rats were tested for time-out, marked, and handled for 5 min by each experimenter. Rats were then placed with their cagemate in the testing arenas for 30 min. All free rats and all cagemates were always placed in the same arena for habituation and testing. Rats that were used as strangers were placed in the same arena for habituation but in different arenas during testing. Habituation of free rats and strangers never occurred at the same time. After each habituation session, rats were returned to their home cages and to the housing room. All sessions were run during the rats’ light cycle between 0800 and 1730. Order of testing was counterbalanced between sessions to control for effects of time of day on behavior.

Time-out measurements

Time-out was measured as the latency from opening the homecage lid halfway to the time that the rat approached the front edge of the cage, reared up, and placed its paws on the ledge. This measurement was recorded 3–5 times for each rat in every cage during habituation.

Open field testing

On the day following completion of habituation, rats were placed individually in an arena for 30 min and their activity recorded. Note that the arenas were the same as were used during habituation but that open field testing was the first time each rat had been in the arena alone.

Testing procedures

At the start of testing, the trapped rat was placed in the restrainer, the door closed, and the restrainer placed in the center of the arena. The free rat was then placed in the arena. If the free rat did not open the restrainer door within 40 min, the investigator opened the restrainer door halfway, to a 45° angle, greatly facilitating door-opening by either rat. Regardless of whether the door was opened before or after the 40 min mark, both rats always remained in the arena for the full hour-long session. After each session, the arena and restrainer were washed with 1% acetic acid followed by surface cleaner. Rats were tested once daily for 12 days.

If a trapped rat succeeded in opening the door from inside the restrainer (∼30% of rats), the trapped rat was placed immediately back in the restrainer, and a Plexiglas blocker was inserted, preventing his access to the door. If the free rat subsequently opened the door, the blocker was removed, allowing the trapped rat to exit the restrainer. The blocker was then used for that trapped rat on all following test days.

Protocols

The free rat was a male SD rat in all conditions. The strain of the male trapped rat was either SD (SD cagemate, SD stranger, fostered+SD) or LE (LE cagemate, LE stranger, LE familiar, fostered+LE) as described further below. All rats used in all conditions were pair-housed.

SD cagemate and SD stranger conditions

Rats for the SD cagemate conditions were housed together; the rat with the shorter time-out latency was used as the free rat. The time-out latency was not used to determine the free rat in any other condition. Stranger rats, destined to serve as trapped rats, were housed and habituated separately from the free rats. Following habituation, free rats were tested with trapped cagemates (n = 8) or strangers (n = 12). In the stranger condition, each free rat was exposed to a different stranger on every day of testing.

LE cagemate and LE stranger conditions

On the day of arrival from the vendor, each SD rat in the LE cagemate condition (n = 8) was housed with an LE rat of comparable size. Rats were observed for an hour after this pairing and no vigorous fighting or injuries occurred. In most pairs, the rats were asleep in a huddle before the hour was up. Rats in the LE cagemate condition remained pair-housed until the end of testing. One LE rat died and the remaining SD rat was excluded from the experiment. Hence, there were only seven rat pairs tested in the LE cagemate condition.

In the LE stranger condition, SD rats (n = 16) were pair-housed with each other and separately from the LE trapped strangers. Habituation to the testing environment occurred separately so that free SD rats were never exposed to LE rats prior to testing. Half of the LE strangers were pair-housed with other LE rats, and half were pair-housed with an SD rat. There was no difference in helping behavior between the two treatments and therefore, the data were pooled.

LE familiar condition

Each free rat (SD males, n = 12) was pair-housed with an LE rat for 2 weeks. On day 14, SD rats were re-housed with each other and allowed to readjust for 1 week prior to habituation. Rats were then tested with trapped LE strangers as described above. Two cages of SD rats (four rats) arrived 4 days late and were housed with LE rats for 10 days instead of 14. Two of those rats became openers and the other two were non-openers.

Fostered SD conditions

Multiparous pregnant LE (n = 16) and SD (n = 7) dams were purchased (Charles River, Portage, MI). Two SD pups were placed into each LE litter on either P0 or P1 (average 0.3 ± 0.1). At this time, which corresponded to P0 of the LE litter (0.3 ± 0.2), LE litters were culled to seven LE pups except for one litter which had only three LE pups. When the SD pups were on average 6.4 ± 0.2 days old, one SD pup was removed, leaving a single male SD pup in the LE litter. Thus, most SD male pups (14/16) were exposed to one SD littermate for no more than 7 days (days when the pups’ eyes were closed). However, in two cases, the SD pup in the LE litter died at a later date (P11, P12). In these cases, an SD male who had been removed from an LE litter 5 or 7 days before was then re-added to the LE litter. In sum, the average exposure to one or more SD pups was 7.2 ± 0.4 days. The pup with the maximal exposure to at least one SD pup (11 days) was transferred when his eyes were open; this rat became the sole opener in the fostered+SD condition.

Pups in the fostered SD conditions were weaned at 28 days of age and pair housed with a male LE littermate. At 2 months of age, the SD rats were tested with either SD strangers (n = 8) or LE strangers (n = 8). Both SD and LE strangers were purchased as adults and allowed to acclimate for 2 weeks prior to habituation and testing.

Video analysis

Ethovision tracking software (Noldus Information Technology, Inc. Leesburg, VA) was used to track the rats’ movements in the arena. To enable tracking both rats’ movements, free rats were colored red and trapped rats colored blue. The rat’s location was converted into x, y coordinates denoting the rat’s location at each frame at a rate of 7.5 FPS. These data were then used to calculate movement velocity and location in the arena (time around the restrainer, time at arena corners).

Behavioral coding

Freeware (Jaywatcher V1.0) was used to manually code the rats’ interactions for 15 min following door-opening. If the door was opened less than 15 min before the end of session, only the remaining time in the arena was coded. Data were not analyzed for five rats (four SD stranger, one LE cagemate) due to technical problems. Coded behaviors included anogenital sniffing, pinning, wrestling, and boxing. Coding was performed by four judges. There was an 84% agreement between judges. Boxing was the behavior that was selected to represent fighting, and analyzed. One rat (a non-opener in the SD cagemate condition) boxed on 95 occasions, a total that was more than five standard deviations over the mean for all rats tested. Additionally, this rat boxed on 10 of the 12 testing days, more than two standard deviations over the mean. This rat was therefore considered an outlier and was excluded from the boxing analysis.

Audio analysis

Vocalizations during the first 40 minutes of the session were analyzed for days 1, 3, and 9 of testing using Avisoft SASLab Pro (Berlin, Germany). Audio data was not available for rats in the SD cagemate and SD stranger conditions (data were lost due to disk failure). Thus, audio data came from rats in the LE cagemate, LE stranger, LE familiar, and both fostered conditions.

Mothering style analysis

The mothering behavior of all dams was recorded at 5 min intervals for three hours (an hour each, starting at 0900, 1200, and 1600) between P1 and P10 (methods adapted from Champagne et al., 2003). The nursing behaviors recorded were (1) arched back nursing; (2) blanket nursing; or (3) passive nursing. In addition, we recorded if the dam was either (4) not in contact with pups; or (5) licking or grooming a pup. The proportion of time bins within each of the five categories was calculated for each dam on each day. The proportions of each behavior recorded were not different between rats in fostered+LE and fostered+SD conditions (1-way repeated measures ANOVAs, p>0.11).

Door-opening latencies

Time to door-opening was calculated as the minute when the restrainer door was opened minus the start time. For rats that never opened, a cutoff time of 40 min was assigned.

Definition of openers

Rats that opened the restrainer on two sequential days, and did so at least three times were termed ‘openers’. Thus, a rat that opened the restrainer 4 days in a row was considered an opener. In all cases except one, once this criterion was met, the rat continued to open the restrainer until the end of the experiment. In the one exception, the rat opened the restrainer on days 4–8 but not on days 9–11. This rat (in the LE familiar group) was considered a non-opener. Three rats (SD cagemate, LE cagemate, and fostered+LE conditions) opened on the final 3 days of testing and did so at decreasing latencies. These rats were considered openers.

Definition of first day of learned opening

The day on which opener rats learned to open the restrainer was defined as the first opening that was repeated the next day.

Statistical analysis

Door-opening latencies, velocity, time in arena corners, and time around the restrainer were averaged per rat across all sessions and all days. ANOVA and two-tailed Student’s t tests were used to determine differences between groups. Fischer PLSD was used for all post-hoc analyses. A chi-square analysis was used to compare the proportions of openers and non-openers. In all cases, α <0.05 was used as criterion for significance. Door-opening latencies are displayed using the median since door-opening was not a normally distributed variable. Statistical comparisons were conducted using SPSS (PASW 18).

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
    Emotional reactions of rats to the pain of others
    1. RM Church
    (1959)
    Journal of Comparative and Physiological Psychology 52:132–134.
    https://doi.org/10.1037/h0043531
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
    The neuroevolution of empathy
    1. J Decety
    (2011)
    Annals of the New York Academy of Sciences 1231:35–45.
    https://doi.org/10.1111/j.1749-6632.2011.06027.x
  18. 18
  19. 19
  20. 20
  21. 21
  22. 22
    It’s more than skin deep: empathy and helping behavior across social groups
    1. S Echols
    2. J Correll
    (2012)
    In: J Decety, editors. Empathy: from bench to bedside. Cambridge: MIT Press. pp. 55–71.
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
    Why ravens share
    1. B Heinrich
    2. J Marzluff
    (1995)
    American Scientist 83:342–349.
  29. 29
    Spontaneous prosocial choice by chimpanzees
    1. V Horner
    2. JD Carter
    3. M Suchak
    4. FB de Waal
    (2011)
    Proceedings of the National Academy of Sciences of the United States of America 108:13847–13851.
    https://doi.org/10.1073/pnas.1111088108
  30. 30
    Promotive tension and prosocial behavior: a Lewinian analysis
    1. HA Hornstein
    (1978)
    In: L Wispé, editors. Altruism, sympathy, and helping: psychological and sociological principles. New York: Academic. pp. 177–207.
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
    Contagious yawning in gelada baboons as a possible expression of empathy
    1. E Palagi
    2. A Leone
    3. G Mancini
    4. PF Ferrari
    (2009)
    Proceedings of the National Academy of Sciences of the United States of America 106:19262–19267.
    https://doi.org/10.1073/pnas.0910891106
  47. 47
  48. 48
  49. 49
  50. 50
    Empathy: Its ultimate and proximate bases
    1. SD Preston
    2. FBM de Waal
    (2002)
    The Behavioral and Brain Sciences 25:1–20.
  51. 51
    ‘Altruism’ in the albino rat
    1. GE Rice
    2. P Gainer
    (1962)
    Journal of Comparative and Physiological Psychology 55:123–125.
    https://doi.org/10.1037/h0042276
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63

Decision letter

  1. Russ Fernald
    Reviewing Editor; Stanford University, United States

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Kindness to strangers depends on knowing their kind: Pro-social behavior in rats is modulated by social experience” for consideration at eLife. Your article has been favorably evaluated by a Senior editor, a Reviewing editor, and 3 reviewers.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

We have received three reviews of your submitted manuscript, all of which found the evidence you presented that rats preferentially help other rats from the same group plausible but not necessarily the only possible interpretation. There are several important concerns that need to be addressed based on the reviewer comments. A consistent, central theme of the reviews that needs to be addressed is the confounding of description with explanation as outlined in the first conceptual issue. The conceptual and practical issues are enumerated below.

Conceptual issues:

1) The authors state in the Introduction that the “release of cage mates … provides evidence for empathically motivated helping…” In subsequent prose, the results presented in the present paper are described in terms of “empathy” and “helping”, confounding description with explanation. The empirical facts are that rats, under some situations, will release another rat from an enclosure, however, that this is described as an act of ‘prosocial empathy and with the aim of helping the other animal’ which constitutes an explanatory hypothesis. The evidence in neither the previous paper nor this one convinces me that ‘empathy and helping’ are the most likely explanations. A lot more work is needed to tease out simpler explanations. For example, rats will work for the opportunity to engage another rat in a fight (Myer & White, 1965, Animal Behaviour, 13, 430-433) or in affiliative play (Humphreys & Einon, 1981, Anim Behav, 29, 259-270), and in unfamiliar settings adult males will engage in a form of play that incorporates elements of aggression (Smith et al. 1999, Aggress Behav, 25, 141-152). Thus, a mixture of prosocial and anti-social motivations may be involved in the paradigm used in this paper. I am only half convinced that the release of a conspecific is unambiguously due to prosocial motivation. Even less convincing is that this releasing behavior arises from empathy. There are a large number of added cognitive processes that need to be in place for this to be the case. If the trapped rat is truly in discomfort in the enclosure, it is likely to be emitting many 22kHz calls (as recorded in this paper as alarm calls). While rats are attracted to 50kHz calls (Wohr & Schwarting, 2007, PLOS ONE, 2, e1365), hearing 22-kHz induces freezing and avoidance (Brudzynski & Chiu, 1995, Physiol Behav, 57, 1039-1044), so what is it about the “trapped rat” that is attracting the performer? Empathy and helping may account for the performer’s coming to the rescue, but to do so, it would need to somehow suppress its own defensive reaction to the alarm calls, as is implied by the authors’ use of the words empathy and helping. But surely, this is just one of the many possible explanations for the releasing behavior. The authors need to revise the manuscript to reflect a more nuanced explication of the data in descriptive terms that are more neutral. Moreover, the concerns of alternative explanations described above need to be addressed.

2) An alternative explanation for the results that “the target rats are biased to release those rats with which they are familiar with not because of any empathic feelings towards the trapped rat, but that they are compelled to release these rats so that they can play with them, which then serves as a social reward.” The videos provided by the authors show playful interactions, including pouncing, pinning, and other measures of play in the rat. The authors should address this possibility and describe how they view this alternative explanation.

3) Citations that seem relevant are missing, including those reporting co-residence duration and maternal perinatal association known to be major cues for human helping behavior and moral judgment (see Lieberman et al., “The Architecture of Human Kin Detection”, 2007; Fessler and Navarrete “Third-party attitudes toward sibling incest”, 2004; Lieberman and Lobel, “Kinship on the Kibbutz: co-residence duration predicts altruism, personal sexual aversions and moral attitudes among communally reared peers”, 2012 for good examples ). Similar effects have been observed in non-human species as well, such as long-tailed tits that tend to help other long-tailed tits (by aiding in the rearing of non-offspring) they have associated with during the nesting phase (see West et al. “Evolutionary Explanations for Cooperation”, 2007 for a review). Specific social cues (especially those perceived during rearing) can be more important than genetic similarity for predicting helping behavior. Clarify how the reported results relate to this literature and what is novel in this context.

4) The authors should acknowledge that previous reports suggest rats would help their cagemates more than strangers, which is contrary to what was shown here.

5) Some literature was at least slightly misrepresented, for example, though the Van Bavel et al. 2008 study did show some interesting effects of social experience on hemodynamic responses to ingroup/outgroup faces, it did not claim to show anything about empathy or pro-social behavior. These data need to be correctly represented.

Practical issues:

1) The authors use “stock” throughout most of the paper but then use the term “strain” in Figures 4 and 5. Please make this consistent by using strain that is the more generally understood term.

2) Some terms are used loosely, including describing a rat approaching the front edge of the cage as “bold” but it could also be curious. Since “boldness” was used to choose rats to be “free” this needs to be more carefully defined. Also, boxing is used a measure of fighting yet is described among other behaviors that seem somewhat playful. These should be distinguished. Similarly, clear definitions of intentional, empathy, altruism and pro-social need to be provided.

3) Explain why the audio recordings from only testing days 1, 3, and 9 were analyzed.

4) The 1st panel of Figure 6 is very confusing, given that the “opener” rats would have opened the door at different times – is the claim that each rat’s locomotion decreased immediately after they opened the restrainer, or rather, that it decreased mainly during the last twenty minutes of the session?

5) Figure 3 is confusing. Is the fostered rat that will be tested not depicted in each illustrated litter? What is happening with the first 2 litters that switch from mostly LE to all SD and then back to LE in panel B? Please clarify.

[Editors' note: further clarifications were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled “Pro-social behavior in rats depends on social experience not genetic relatedness” for further consideration at eLife. Your revised article has been evaluated by a Senior editor, a member of the Board of Reviewing Editors, and the three original reviewers.

Your revised manuscript has been considerably improved but still has several issues needing attention. Overall, the reviewers continue to feel that the data do not support an interpretation of empathy and therefore some of the writing suggesting empathetic motivation remains and needs to be eliminated. Specific comments are summarized here:

1) General arousal seems to be a very reasonable alternative to the pro-social interpretation of your results. This explanation needs to be presented as an alternative and discussed.

2) The present study doesn’t really contrast an ‘experience’ versus a ‘genetic’ hypothesis. What is really interesting is that a rat will treat strangers that look like the rats with whom they were reared as if they were familiars, or least ones that they will preferentially release. The rats single out some cue(s) about their cage mates that they can then apply to strangers and discriminate among them as those that share the cues with cage mates and those that do not. The authors should discuss this and consider what cues might be being used.

3) A related concern is that there is no explanation of the observation that the door was opened for cagemates as often as for strangers. Every paper published thus far across species (including mice, rats, monkeys, and humans) shows increased “helping” or “cooperation” or “empathy” for cagemates vs non-cagemates, and there is a lot of theory justifying why this should be the case. This suggests that there is something about the paradigm that blocks social cues relevant for individual recognition. If you want to suggest something so out of line with previous results you either need to rule out alternative explanations with more experiments or more honestly acknowledge/discuss why these results differ, especially when the claim is so central to the significance of the paper.

4) The authors dismiss the concerns raised that the releasing may be due to social reward because their previous findings found that releasing can occur in the absence of subsequent contact. For personally acquainted rats, immediate reward may not be needed because of the past association made between access to an individual and rewarding experiences. Similarly, by showing that unfamiliar rats with some cue identifier marking them as similar to cage mates are preferentially released, it may be simply a two-step association process. Release of a rat with a similar cue may occur because of a past association with social reward. Then, in the present experimental paradigm, the releasing rat may actually obtain the social reward, reinforcing the releasing behavior. That is, in this case the social reward may be important to release a rat that is not familiar, but resembles cage mates. The present findings may be accounted by simple association mechanisms, not requiring higher order processes involving empathy. You should discuss the role of association learning in interpreting your results.

5) “…the most parsimonious explanation is that rats open the restrainer door in order to terminate the trapped rat’s distress”. This sentence should be replaced with something like: “A possible interpretation of the data is that rats open the restrainer door in order to terminate the trapped rat’s distress or in order to terminate their own vicarious arousal.”

6) “The selectivity with which rats engage in helping behavior is further evidence that releasing a trapped rat is an intentional social behavior, congruent with an empathic drive to help some rats, but not others… We conclude that rats form affective bonds through social interactions that motivate empathy and helping. The motivation to help is extended to strangers of familiar strains…” This is an example of how the current version of the paper still concludes that the observed behavior represents rats consciously and intentionally helping others (altruism). As all the reviewers agreed, this conclusion is not warranted.

7) Please cite the Rutte and Taborsky (2007) paper on how social experience modulates generalized reciprocity in rats, and modify the relevant language in the abstract and discussion to give proper credit to this prior study. The generalized reciprocity mechanism discussed by Rutte and Taborsky (2007) may be a useful explanation for the social experience-modulated door-opening behavior described here as well.

https://doi.org/10.7554/eLife.01385.013

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Conceptual issues:

1) The authors state in the Introduction that the “release of cage mates … provides evidence for empathically motivated helping…” In subsequent prose, the results presented in the present paper are described in terms of “empathy” and “helping”, confounding description with explanation. The empirical facts are that rats, under some situations, will release another rat from an enclosure, however, that this is described as an act of ‘prosocial empathy and with the aim of helping the other animal’ which constitutes an explanatory hypothesis. The evidence in neither the previous paper nor this one convinces me that ‘empathy and helping’ are the most likely explanations.

The reviewers were concerned that while we describe a pro-social act that is directed in a socially selective way, we then explain that behavior as resulting from empathic motivation. We completely agree that the data presented here, on their own, do not prove that empathy motivates the rats’ helping behavior. Further, attributing a specific motivation to door-opening is unnecessary for our central conclusion that rats show an experience-based rather than a genetic- based social selectivity for helping. Therefore, we have carefully revised the text to focus on the social modulation of pro-social behavior, rather than on the underlying affective motivation for this behavior.

In the Discussion we briefly review potential motivations for the observed behavior and argue that the balance of available evidence suggests that a rodent form of empathy is the most likely motivation for door-opening.

A lot more work is needed to tease out simpler explanations. For example, rats will work for the opportunity to engage another rat in a fight (Myer & White, 1965, Animal Behaviour, 13, 430-433) or in affiliative play (Humphreys & Einon, 1981, Anim Behav, 29, 259-270), and in unfamiliar settings adult males will engage in a form of play that incorporates elements of aggression (Smith et al. 1999, Aggress Behav, 25, 141-152). Thus, a mixture of prosocial and anti-social motivations may be involved in the paradigm used in this paper.

This comment raises a very important conceptual point that we now discuss explicitly in the manuscript.

While it is true that a variety of motivations could contribute to door-opening, it is unlikely that rats were motivated to open the restrainer by aggressive or anti- social motivations. Our results show that conflict between the free and trapped rat was minimal between opener pairs. Moreover, openers exhibited a behavioral pattern indicative of a pro-social motivation, such as constantly circling the restrainer (indexed by proximity to the restrainer), creating tactile contact with the trapped rat, digging and biting the restrainer, and making multiple attempts to release the trapped rat. The free rat’s behavior was often reciprocated by the movements of the trapped rat, as the latter changed his position in the restrainer to face the free rat. The non-openers, on the other hand, spent less time around the restrainer and were more aggressive when the trapped rat exited the restrainer following half-way door-opening. Thus, we find it unlikely that free rats released the trapped rat in order to engage in a fight.

The rats might have released the trapped rat in order to obtain social contact such as playing. However, we have previously established that social reward is not required for helping. Thus, at least some other source of motivation must be present. We would argue that this other motivation is to terminate the distress of the other rat, a rodent form of empathy. Yet, as mentioned above, the evidence provided by this study alone does not provide stand-alone evidence for this claim, and we have revised the manuscript to reflect this.

I am only half convinced that the release of a conspecific is unambiguously due to prosocial motivation. Even less convincing is that this releasing behavior arises from empathy. There are a large number of added cognitive processes that need to be in place for this to be the case.

We completely agree that rodents do not possess the complex empathic abilities manifested in humans, which include perspective taking and other functions of abstract cognition. However, the existence of a simple, primitive form of empathy in many mammals is by now widely accepted (Rice and Gainer, 1962; Langford et al, 2006; Rutte and Taborsky, 2007; Chen et al, 2009; Jeon et al, 2010; Panksepp and Lahvis, 2011; Decety and Svetlova, 2012; Mogil, 2012; Panksepp and Panksepp 2013; Preston 2013). It should be noted that fundamental empathic responses, such as emotional contagion and emotional resonance, are believed to rely on subcortical circuits rather than complex cognition (Decety and Svetlova, 2012).

If the trapped rat is truly in discomfort in the enclosure, it is likely to be emitting many 22kHz calls (as recorded in this paper as alarm calls). While rats are attracted to 50kHz calls (Wohr & Schwarting, 2007, PLOS ONE, 2, e1365), hearing 22-kHz induces freezing and avoidance (Brudzynski & Chiu, 1995, Physiol Behav, 57, 1039-1044), so what is it about the “trapped rat” that is attracting the performer? Empathy and helping may account for the performer’s coming to the rescue, but to do so, it would need to somehow suppress its own defensive reaction to the alarm calls, as is implied by the authors’ use of the words empathy and helping.

The reviewers appear concerned that the trapped rat is simultaneously not in discomfort and also frozen with distress. In fact, the truth appears to lie somewhere in the middle. The “restraint” in this study does not cause physical pain or extreme distress. The rats move and turn around within the restrainer. And they are never trapped for more than 40 mins. Nonetheless, the rats’ attempts to escape (30% of the trapped rats managed to open the door from the inside) make it clear that they find being in the restrainer aversive. Unpublished experiments show that all trapped rats given the opportunity to get themselves out (the door was reversed) of the restrainer do indeed take this opportunity.

It is our impression that the moderate stress level produced by the restraint prevents the extreme reaction of freezing immobility. This impression is supported by the low rates of ultrasonic vocalizations in the 22 kHz range (<6 min of alarm calls per 40 min session per 8-12 trapped rats) recorded.

The question of what feature of the trapped rat triggers the free rat’s actions is an interesting one. Research in mice suggests that emotional contagion is dependent on vision (Langford et al., 2006). We do not at present have any evidence regarding the modality by which the trapped rat communicates his distress.

But surely, this is just one of the many possible explanations for the releasing behavior. The authors need to revise the manuscript to reflect a more nuanced explication of the data in descriptive terms that are more neutral. Moreover, the concerns of alternative explanations described above need to be addressed.

As discussed above, we have carefully revised the manuscript to speak of pro- social behavior without committing to the motivation involved. Additionally, we have added a paragraph (see paragraph beginning, “While door-opening has an obvious pro-social outcome, a variety of motivations could contribute to this behavior…”) to the Discussion that addresses potential alternative explanations.

2) An alternative explanation for the results that “the target rats are biased to release those rats with which they are familiar with not because of any empathic feelings towards the trapped rat, but that they are compelled to release these rats so that they can play with them, which then serves as a social reward.” The videos provided by the authors show playful interactions, including pouncing, pinning, and other measures of play in the rat. The authors should address this possibility and describe how they view this alternative explanation.

Social reward is a fundamental underpinning of all social behavior. We agree that at root, behavior involving more than one individual is fundamentally motivated by social reward. However, as mentioned above, our previous results show that pro-social behavior occurs even in the absence of the possibility of social play (Ben-Ami Bartal et al., 2011). This is mentioned in the revised manuscript.

3) Citations that seem relevant are missing, including those reporting co-residence duration and maternal perinatal association known to be major cues for human helping behavior and moral judgment (see Lieberman et al., “The Architecture of Human Kin Detection”, 2007; Fessler and Navarrete “Third-party attitudes toward sibling incest”, 2004; Lieberman and Lobel, “Kinship on the Kibbutz: co-residence duration predicts altruism, personal sexual aversions and moral attitudes among communally reared peers”, 2012 for good examples ). Similar effects have been observed in non-human species as well, such as long-tailed tits that tend to help other long-tailed tits (by aiding in the rearing of non-offspring) they have associated with during the nesting phase (see West et al. “Evolutionary Explanations for Cooperation”, 2007 for a review). Specific social cues (especially those perceived during rearing) can be more important than genetic similarity for predicting helping behavior. Clarify how the reported results relate to this literature and what is novel in this context.

The kibbutz studies show that people expand their pro-social behavior toward individuals that they were reared with and that this occurs even for non-kin. Our findings are different in two very important ways from what was found in the kibbutz studies:

• The kibbutz studies show that living with another during childhood increases the likelihood of altruism expressed towards that one individual. In contrast, we show that social interaction (during either rearing and adulthood or adulthood only) with members of a strain leads rats to help strangers of that strain. The generalization from one individual to others of that individual’s strain is novel.

• The kibbutz studies show that early experience can influence pro-social motivation but do not rule out the possibility that genetic relatedness could influence helping behavior. We show that for rats, the genetic identity of the rat in need does not in any way predicate helping. The only determinant of helping is social experience, i.e., creating a social bond with a type of rat. The demonstration that genetic relatedness alone is not capable of producing pro-social motivation is novel.

There are numerous additional differences between our study and the kibbutz studies (timing of co-habitation, highly socialistic ideology, and culture in kibbutzim, and so on). Therefore, although we share the reviewers’ enthusiasm for these very interesting papers, we do not believe they are directly relevant and do not cite them.

Cooperation between tits is now cited in the Introduction (Hatchwell et al., 2013).

4) The authors should acknowledge that previous reports suggest rats would help their cagemates more than strangers, which is contrary to what was shown here.

As far as helping behavior in rodents, we are not aware of studies that directly compared pro-social behavior toward cagemates and strangers (e.g., Church, 1959; Rutte and Taborsky, 2007). However, there is a robust literature that emotional contagion in both rats and mice is greater for familiar conspecifics than for unfamiliar ones. The revised manuscript includes a sentence referencing this issue: “…it is important to note that although we did not observe differences in helping towards strangers and cagemates of a familiar strain, the underlying affective response to strangers and cagemates may differ, as is suggested by research in apes (Campbell and de Waal, 2011) and mice (Langford et al., 2006).”

5) Some literature was at least slightly misrepresented, for example, though the Van Bavel et al. 2008 study did show some interesting effects of social experience on hemodynamic responses to ingroup/outgroup faces, it did not claim to show anything about empathy or pro-social behavior. These data need to be correctly represented.

We thank the reviewers for pointing this out, and we have corrected the manuscript with regards to the Van Bavel paper. We have also double-checked our representations of other results.

Practical issues:

1) The authors use “stock” throughout most of the paper but then use the term “strain” in Figures 4 and 5. Please make this consistent by using strain that is the more generally understood term.

Done.

2) Some terms are used loosely, including describing a rat approaching the front edge of the cage as “bold” but it could also be curious. Since “boldness” was used to choose rats to be “free” this needs to be more carefully defined.

We agree completely that the meaning of the latency to approach the front of the cage is ambiguous. We now use the neutral term “time-out” to refer to this latency.

To be clear, the time-out measure was used in our previous study (Ben-Ami Bartal et al., 2011) but was only used to choose the free rat in the SD cagemate condition in the present study. In the other 6 conditions of the current study, the chosen rat was either the only SD rat in the cage (LE cagemate, 2 fostered conditions) or both rats in the cage were tested (SD stranger, LE stranger, LE familiar).

Note that the opening behavior of rats in SD stranger, LE cagemate, fostered+SD, and LE familiar conditions was not different from that of the only condition in which time-out was used to choose the free rat. Therefore, a short time-out is clearly not a requirement for motivating door-opening.

Also, boxing is used a measure of fighting yet is described among other behaviors that seem somewhat playful. These should be distinguished.

Most component movements in playing and fighting are the same (Meaney and Stewart, 1981). For example, pinning and wrestling can occur in bouts that appear to be “playful” and result in no injury as well as in interactions that escalate into boxing, biting and injury. We chose to analyze boxing which: 1) tended to appear only after intense, escalating interactions; 2) was often preceded by a lateral threat; 3) was always present in interactions that resulted in bites.

Similarly, clear definitions of intentional, empathy, altruism and pro-social need to be provided.

We now provide definitions of empathy and pro-social behavior:

• pro-social behavior comprises actions that improve the well-being of others

• empathy is defined as recognizing and sharing the emotional state of another

We do not use the terms altruism or intentional and therefore did not define them.

3) Explain why the audio recordings from only testing days 1, 3, and 9 were analyzed.

Rats were tested in a single room and not in soundproof chambers. A single microphone was used to collect the acoustic output of the group. Therefore, the audio recordings provide information about condition but not about individual rats. Because the audio recordings cannot be assigned to individual rats or even to one pair, examining the calls emitted before a pair’s first door-opening, for example, would not be informative. Since different pairs open on different days, there is no one day that reliably captures, for example, the day before first opening.

Given that only condition information can be gleaned from these recordings, we chose to sample calls that occurred early on (days 1 and 3) as well as later in testing (day 9). Sampling more days would be unlikely to provide novel information or insight and would likely overestimate the information content of the data.

4) The 1st panel of Figure 6 is very confusing, given that the “opener” rats would have opened the door at different times – is the claim that each rat’s locomotion decreased immediately after they opened the restrainer, or rather, that it decreased mainly during the last twenty minutes of the session?

We agree with the reviewers and removed panel 6A.

5) Figure 3 is confusing. Is the fostered rat that will be tested not depicted in each illustrated litter? What is happening with the first 2 litters that switch from mostly LE to all SD and then back to LE in panel B? Please clarify.

We apologize for the confusion. We have made several changes to this figure to improve clarity and have also rewritten the figure legend.

[Editors’ note: the author responses to the re-review follow.]

1) General arousal seems to be a very reasonable alternative to the pro-social interpretation of your results. This explanation needs to be presented as an alternative and discussed.

We appreciate the reviewers’ efforts to raise multiple alternative explanations, and now include this idea in the Discussion. The reviewers suggest that a non-specific response underlies door-opening behavior. If we understand correctly, the idea is that 1) rats from familiar strains elicit more “general” arousal than do rats from unfamiliar strains; and 2) “generally” aroused rats then move more and thereby open the door accidentally. While it is important to ponder every alternative explanation, our data do not support a general arousal mechanism. Further analysis shows that non-openers tested with unfamiliar strains were as active before door-opening as openers (new Figure 8C). These data indicate that general arousal cannot account for differential door-opening.

That being said, we do not doubt that the different conditions elicit different states of arousal for the free rat. Yet, we would not term this “general arousal”. Rather we perceive it as a state that combines arousal with affective valence. This form of specific arousal is elicited by the distress of another rat.

Finally, the general arousal hypothesis would suggest that rats are not acting in a goal-directed manner when they open the restrainer. We have previously demonstrated that door-opening is a goal-directed behavior. For the benefit of reviewers who may not have had a chance to read the previous publication (Ben-Ami Bartal, et al. 2011), we found that rats demonstrate a classic learning curve for door-opening latencies. In fact, door-opening is hard, and rats have to work at it even when the restrainer contains coveted chocolate chips. Rats overcome their initial aversion of the arena center and demonstrate a specific movement pattern that involves circling the restrainer, biting and digging under it, and making multiple attempts to open the restrainer. Rats only cease such restrainer-focused activity after learning to open the restrainer. As the rats learn to open the door, the method for door-opening becomes uniform, and the initial freezing response to sound of the door falling over disappears. Rats did not show these behaviors when the restrainer was empty, contained a toy, or when a non-trapped cagemate was present. As such, general arousal is an unlikely and non-specific explanation for door-opening behavior in this paradigm, much like it would be an uninformative interpretation of a rat locating the underwater platform in the Morris water maze.

2) The present study doesn’t really contrast an ‘experience’ versus a ‘genetic’ hypothesis.

We have taken into account the reviewers’ advice, and accordingly de-emphasized the discussion of genetic relatedness in the manuscript. It is true that members of an outbred strain (or stock) are not genetically identical. Yet, it is also true that genetic similarity is higher within strains than between strains. Thus it is safe to say that our findings demonstrate that genetic similarity cannot, on its own, motivate helping behavior, as rats fostered with a different strain do not help members of their own strain.

What is really interesting is that a rat will treat strangers that look like the rats with whom they were reared as if they were familiars, or least ones that they will preferentially release. The rats single out some cue(s) about their cage mates that they can then apply to strangers and discriminate among them as those that share the cues with cage mates and those that do not. The authors should discuss this and consider what cues might be being used.

We agree that our finding that rats treat strangers that resemble rats with whom they are familiar, as if they were themselves familiar is really interesting. We have changed our title to better reflect this core finding: “My kind of rat: helping behavior in rats is directed towards groups defined by social experience.”

[Editors’ note: an earlier version of the title, “Pro-social behavior in rats is modulated by social experience”, was chosen for publication.]

As the reviewers state, we find that rats help strangers only when they have social experience with another rat of the stranger’s strain. We agree that it would be very interesting to know the identity of the cue or cues upon which rats base a determination of familiarity. Addressing this issue has the potential to illuminate how mammals construct group identity and social attachment, which are considered critical for the development and expression of empathy in both humans and non-human animals. The possible cues that rats could use are now explicitly enumerated in the Discussion. We have no doubt that publication of our fundamental finding will stimulate exactly the type of follow-up studies suggested by the reviewers.

3) A related concern is that there is no explanation of the observation that the door was opened for cagemates as often as for strangers. Every paper published thus far across species (including mice, rats, monkeys, and humans) shows increased “helping” or “cooperation” or “empathy” for cagemates vs non-cagemates, and there is a lot of theory justifying why this should be the case.

While there are quite a few studies of the effects of familiarity on pro-social behavior, we do not believe they provide direct evidence regarding rodent helping behavior. Earlier studies on rodent helping did not detail the relationship between the helper and the recipient of help (Church 1959; Rice and Gainer 1962). Our previous work only tested cagemates. Thus, to the best of our knowledge there is no published evidence to support the idea that rodents preferentially help individually familiar animals.

With regard to cooperative behavior, Rutte and Taborsky (2007, 2008) demonstrated cooperative behavior between individually familiar as well as individually unfamiliar animals.

Regarding emotional contagion, there is evidence for affective communication between both individually familiar (Langford, et al. 2006; Jeon, et al. 2010) and individually unfamiliar (Chen, et al. 2009) rodents. Of note, Langford, et al. (2006) studied both familiar and stranger dyads. While they concluded that mice experience emotional contagion for cagemates but not for strangers, the data are open to other interpretations. When mice injected with acetic acid (to induce writhing, a visceral pain behavior) were tested with an uninjected stranger, they showed less pain behavior than did mice tested in isolation. While this result could stem from emotional contagion of the uninjected mouse’s low pain state, the authors interpreted it as stemming from stress analgesia. Further, no comparisons were made between rats tested in the presence of a stranger that was either injected or not injected; comparisons were made only with isolated mice. The error bars in Figure 1A of Langford, et al. (2006) would suggest that a mouse in the presence of a stranger injected with acetic acid shows significantly more writhing behavior than one tested with an un-injected stranger.

With regards to non-rodent species, it should be mentioned that studies in primates and dogs show mixed results regarding a familiarity effect on pro-social behavior, inclusive of evidence for pro-social behavior directed at strangers (e.g., Custance, et al. 2012; Tan, et al. 2013; Warneken and Tomasello 2006). Finally, humans routinely express empathy toward strangers such as unknown actors (e.g., Loggia, et al. 2008) or fictitious characters (e.g., Batson, et al. 2007 and Batson 2005 for a review of possible underlying mechanisms).

This suggests that there is something about the paradigm that blocks social cues relevant for individual recognition. If you want to suggest something so out of line with previous results you either need to rule out alternative explanations with more experiments or more honestly acknowledge/discuss why these results differ, especially when the claim is so central to the significance of the paper.

As detailed above, we do not agree that our results are “so out of line with previous results.” Further, we do not interpret our findings as evidence that there was no individual recognition between the rats studied. First, there was no obstacle to full sensory communication between the free and trapped rat, as the clear restrainer is perforated. Moreover, we have added a new analysis to the manuscript demonstrating differences in how the rats behaved with cagemates and strangers (see below and new Figure 7). We conclude that although rats could distinguish strangers from cagemates, they were motivated to help release both.

Prior to door-opening, rats moved faster when the trapped rat was familiar than when he was a stranger. After door-opening, the velocity was not different. A higher velocity before door-opening is evidence that rats recognize individually familiar vs. individually unfamiliar others. Our interpretation of this result is that familiar and unfamiliar rats elicit either different motivational states or different magnitudes of the same motivational state. Most importantly, despite these differences, the behavioral output of rats tested with familiar or unfamiliar rats from a familiar strain was the same: helping!

In sum, we do not believe that our data stand in direct contrast to consistent published findings that we are aware of. If indeed current theory requires that empathy occurs only between familiar individuals, then perhaps our findings should serve as an impetus to refine theory to better match reality.

4) The authors dismiss the concerns raised that the releasing may be due to social reward because their previous findings found that releasing can occur in the absence of subsequent contact.

We apologize if we came off as dismissive of the role of social reward. Of course, social reward is at the root of many social behaviors. If being with other individuals did not elicit positive affect, then it is unlikely that individuals would ever be motivated to interact and form groups. Yet, in our previous study, we found that rats opened the door of a restrainer containing a trapped rat even when they could not play with the released rat. Rats did not open the door of an empty restrainer under these same conditions. These findings led us to conclude that immediate social reward provided by the opportunity to play with a released rat is not required for helping behavior.

For personally acquainted rats, immediate reward may not be needed because of the past association made between access to an individual and rewarding experiences. Similarly, by showing that unfamiliar rats with some cue identifier marking them as similar to cage mates are preferentially released, it may be simply a two-step association process. Release of a rat with a similar cue may occur because of a past association with social reward. Then, in the present experimental paradigm, the releasing rat may actually obtain the social reward, reinforcing the releasing behavior. That is, in this case the social reward may be important to release a rat that is not familiar, but resembles cage mates. The present findings may be accounted by simple association mechanisms, not requiring higher order processes involving empathy. You should discuss the role of association learning in interpreting your results.

We largely agree. However, in contrast to the reviewers, we do not view empathy as a higher-order process (often synonymous for some scholars with theory of mind, perspective-taking or understanding of another’s state). Rather, empathy is a basic affective capacity that has deep evolutionary roots in mammalian species associated with affective communication, social attachment, and maternal caregiving (see for instance Decety, et al. 2012).

Finally, the principal point of this paper is that motivated helping behavior is different toward rats of familiar vs unfamiliar strains. While we would be remiss if we did not discuss the motivation for helping, we also believe that returning to defend in detail the core conclusion of our previous work would be inappropriate.

5) “…the most parsimonious explanation is that rats open the restrainer door in order to terminate the trapped rat’s distress”. This sentence should be replaced with something like: “A possible interpretation of the data is that rats open the restrainer door in order to terminate the trapped rat’s distress or in order to terminate their own vicarious arousal.

We have revised the sentence to read “Given the greater interest in the trapped rat shown by openers and the lack of aggressive interactions between opener pairs, the authors favor the interpretation that rats open the restrainer door in order to terminate the trapped rat’s distress.”

6) “The selectivity with which rats engage in helping behavior is further evidence that releasing a trapped rat is an intentional social behavior, congruent with an empathic drive to help some rats, but not others... We conclude that rats form affective bonds through social interactions that motivate empathy and helping. The motivation to help is extended to strangers of familiar strains...” This is an example of how the current version of the paper still concludes that the observed behavior represents rats consciously and intentionally helping others (altruism). As all the reviewers agreed, this conclusion is not warranted.

This remains our interpretation of the data and we respectfully ask to present our interpretation even if it is at variance with that of “all the reviewers.” We have limited the discussion of possible empathic motivation to the final two paragraphs of the Discussion, and have considerably elaborated on possible alternative explanations so that the thoughtful reader can choose their own interpretation.

It is interesting to note that while other demonstrations of affectively-motivated social behaviors, such as emotional contagion and pair-bonding, are widely accepted in the field, the reviewers find it very difficult to accept the possibility that rodents find the distress of others aversive, and that they experience a positive affect when they have an active role in eliminating the distress of another rat. Yet, basic findings support the conclusion that neural and hormonal mechanisms involved in empathy are evolutionarily conserved across species and that the same affective influences that operate in rodents also drive helping in other mammals, including humans.

7) Please cite the Rutte and Taborsky (2007) paper on how social experience modulates generalized reciprocity in rats, and modify the relevant language in the abstract and discussion to give proper credit to this prior study. The generalized reciprocity mechanism discussed by Rutte and Taborsky (2007) may be a useful explanation for the social experience-modulated door-opening behavior described here as well.

This paper was and is cited in the Introduction. Yet how that study would explain our findings is unclear to us. Rutte and Taborsky found that rats were more likely to give food when they were given food before, a result that the authors interpreted as reciprocated cooperative behavior. In our paradigm, the free rats were never trapped themselves, and there was no indication to them that they would ever require the other rat to release them.

At the risk of perseverating, we want to point out once again that the results of the present paper are novel and exciting because they demonstrate that motivated helping behavior is different toward rats of familiar vs. unfamiliar strains and begins to illuminate the biological basis of group identity. Returning to a full discussion of data published previously digresses from this exciting point.

https://doi.org/10.7554/eLife.01385.014

Article and author information

Author details

  1. Inbal Ben-Ami Bartal

    Department of Neurobiology, University of Chicago, Chicago, United States
    Contribution
    IB-AB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  2. David A Rodgers

    Department of Neurobiology, University of Chicago, Chicago, United States
    Contribution
    DAR, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    No competing interests declared.
  3. Maria Sol Bernardez Sarria

    Department of Neurobiology, University of Chicago, Chicago, United States
    Contribution
    MSBS, Acquisition of data, Analysis and interpretation of data
    Competing interests
    No competing interests declared.
  4. Jean Decety

    1. Department of Psychology, University of Chicago, Chicago, United States
    2. Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, United States
    3. Committee on Neurobiology, University of Chicago, Chicago, United States
    Contribution
    JD, Contributed critically to the conceptual and theoretical framework for this work, Drafting or revising the article
    Competing interests
    No competing interests declared.
  5. Peggy Mason

    1. Department of Neurobiology, University of Chicago, Chicago, United States
    2. Committee on Neurobiology, University of Chicago, Chicago, United States
    Contribution
    PM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    pmason@uchicago.edu
    Competing interests
    PM: Reviewing editor, eLife.

Funding

National Institutes of Health (DA022978, DA022429)

  • Peggy Mason

The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The assistance of Nora Molasky, Tony Logli, Michelle Yun, Kirill Karlin, Joshua Saucedo, Calvin Krogh, and Victoria Huang is gratefully acknowledged.

Ethics

Animal experimentation: All animal husbandry and all experiments were performed in accordance with the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committee of the University of Chicago (Animal Care and Use Protocol #71967). Every effort was made to minimize the number of rats used.

Reviewing Editor

  1. Russ Fernald, Reviewing Editor, Stanford University, United States

Publication history

  1. Received: August 19, 2013
  2. Accepted: November 29, 2013
  3. Version of Record published: January 14, 2014 (version 1)

Copyright

© 2014, Ben-Ami Bartal et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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