Empathy, the ability to share another individual’s emotional state and/or experience, has been suggested to be a source of prosocial motivation by attributing negative value to actions that harm others. The neural underpinnings and evolution of such harm aversion remain poorly understood. Here, we characterize an animal model of harm aversion in which a rat can choose between two levers providing equal amounts of food but one additionally delivering a footshock to a neighboring rat. We find that independently of sex and familiarity, rats reduce their usage of the preferred lever when it causes harm to a conspecific, displaying an individually varying degree of harm aversion. Prior experience with pain increases this effect. In additional experiments, we show that rats reduce the usage of the harm-inducing lever when it delivers twice, but not thrice, the number of pellets than the no-harm lever, setting boundaries on the magnitude of harm aversion. Finally, we show that pharmacological deactivation of the anterior cingulate cortex, a region we have shown to be essential for emotional contagion, reduces harm aversion while leaving behavioral flexibility unaffected. This model of harm aversion might help shed light onto the neural basis of psychiatric disorders characterized by reduced harm aversion, including psychopathy and conduct disorders with reduced empathy, and provides an assay for the development of pharmacological treatments of such disorders.

We show that (1) male and female Sprague-Dawley rats switch significantly away from the shock-delivering lever, (2) this effect is stronger in shock-pre-exposed actors, and (3) deactivating the ACC reduces this effect. By altering the timing of shock delivery, we show that contingency between lever pressing and shock delivery is essential. By varying the reward value of the levers, we show rats switch from an easier to a harder lever and from one that provides two pellets to one that provides one pellet to prevent harm to another. However, rats were unwilling to switch from a lever that provides three pellets to one that provides one pellet. We additionally report and explore substantial individual differences in switching across rats.

Inspired by classic studies, here, we refine a paradigm to study instrumental harm aversion in rats. A rat called the “actor” can press one of two levers for sucrose pellets. After a baseline phase revealing the rat’s preference for one of the levers, we pair this preferred lever with a shock to a second rat (“victim”), located in an adjacent compartment ( Figure 1 ). We then measure how much actors switch away from the preferred lever as a behavioral index of harm aversion.

(G) Trial structure for the shock session. Trial structure for baseline and food is identical to shock except that shocks are never delivered to the victim, and three pellets are delivered to the actor when the non-preferred lever is pressed in the food session. Light blue boxes denote events and lavender boxes the rat’s responses. Latency nosepoke and latency lever are used as criteria to determine valid trials, although latency and duration food hopper are dependent variables reported in Figures 3 and S2

(F) Design of training, exposure, baseline, shock, and food sessions. During step 1 of the training, the animal is free to press any lever and each lever press delivers three pellets. In step 2, the animal is exposed to 5 blocks, each starting with four forced trials (only one lever at the time is presented, which has to be pressed within 20 s) and finishing with 6 free trials (both levers presented and one has to be pressed within 20 s). Only one pellet is given at each lever press. In step 3, a difference in strength necessary to operate one of the levers is introduced and nose spoke is required in order to initiate a trial. Again, animals are exposed to 50 trials each session, with the same number of forced and free trials as in step 2. During exposure, the actor receives 4 shocks alone in the victim compartment. During baseline, pressing one lever (left column) or the other (right column) leads to one pellet for the actor. The lever preferred during baseline then additionally triggers a shock to the victim during the 3 days of the shock phase. During food sessions, shocks are no longer delivered, but the non-preferred lever from shock session 3 now leads to 3 pellets. Note that the food session is only present in some conditions ( Tables 1 and 2 ).

(E) Experimental timeline from the training to the end of the experiment. The numbers within each block indicate the number of lever presses (in training step 1) or trials (in training steps 2 to 3, baseline, shock, and food sessions) maximally allowed (training step 1) or required (training steps 2 and 3, baseline, shock, and food sessions). Training steps 1–3 were repeated until 70% of the maximum number of possible lever presses per session in the free trials was reached (i.e., 35 in Step1 and 21 in Step2–3, gray numbers). A time out for the lever press was introduced in step 2. An additional time out based on the duration of the nose poke was introduced in step 3. This duration went from a minimum of 10 ms to a minimum of 400 ms in three separate sessions.

(D) Close up of the left wall with one of the two levers and food hoppers available to the actor.

An increasing number of studies show that rodents display affective reactions to the distress of conspecifics []. These reactions are observed as increased freezing and modulation of pain sensitivity of the witness while attending to the other conspecific in pain [] or when the witness is re-exposed to cues associated with the other’s pain []. Recent studies in rats identified emotional mirror neurons in the anterior cingulate cortex (ACC; area 24 in particular) [], which respond to the observer experiencing pain and to witnessing a conspecific’s distress. Reducing activity in the ACC reduces emotional contagion []. However, in these paradigms, the observing rat is not the cause of the witnessed pain, and whether vicarious activity in area 24 is associated with harm aversion thus remains unclear.

Learning to avoid actions that harm others is an important aspect of human development [], and callousness to others’ harm is a hallmark of antisocial psychiatric disorders, including psychopathy and conduct disorder with reduced empathy []. What could motivate humans and other animals to refrain from harming others? An influential theory posits that vicarious emotions (i.e., emotions felt by a witness, in the stead of the witnessed individual), including emotional contagion and empathy, trigger harm aversion []. Put simply, harming other people is unpleasant, because we vicariously share the pain we inflict. Accordingly, it has been argued that psychiatric disorders characterized by antisocial behavior [] might stem from malfunctioning or biased vicarious emotions [].

Muscimol injection did not appear to alter goal-directed behavior more generally in terms of latency to enter and duration in the food hopper, percentage of time spent close to the divider, and the amount of freezing ( Figure 4 F). We also started including food sessions in later animals (saline n = 7 and muscimol n = 6; Table 3 ), continuing the injection of the respective drug during those sessions. In that small subsample, FIs were not significantly different between muscimol and saline conditions ( Figure 4 E), but the Bayesian analysis suggests a larger sample is necessary to exclude small effects on flexibility. If we repeat the ANOVA on the subsample of n = 7+6 animals for which we performed food session, we still find that switching is reduced after ACC deactivation (2 condition× 4 sessioninteraction; F= 5.6; p = 0.003; BF= 12.7).

A 2-condition× 4-sessionrmANOVA revealed a trend for an effect of session (F= 2.32; p = 0.08; η= 0.10; BF= 0.88) and a significant session × condition interaction (F= 3.33; p = 0.025; η= 0.14; BF= 2). Although baseline preferences for the shock lever were comparable between muscimol and saline, preferences for the shock lever were significantly higher for the muscimol- than saline-infused animals in shock sessions ( Figure 4 C), and switching scores were significantly reduced in the muscimol compared to the saline group (Mann-Whitney U = 30.5, p= 0.016; Mann-Whitney BF= 3.1; Figure 4 D).

Several studies performed in humans [] and rats [] suggest the ACC (including area 24a and b) [] is recruited during the observation of distress and maps other’s pain onto one’s own pain circuitry. To test whether the ACC is necessary for the switching in our paradigm, we infused muscimol bilaterally in the ACC in a group of rats (muscimol; Table 3 ) prior to baseline and shock sessions and compared the choice allocation to a saline-infused group (saline; Figure 4 A; Table 2 ). Infusions were centered at +1.8 mm from the bregma and had an anterior-posterior spread of [+1.95 mm; +1.45 mm] from bregma (muscimol group: n = 9 out of 11; M = 1.76 mm; SD = 0.26; saline group: n = 9 out of 12, M = 1.84; SD = 0.26; Figure 4 B), confirming that area 24a (approximately 0.6 mm dorsal to corpus callosum) and area 24b were targeted. The infusion spared midcingulate areas 24’, located closer to the bregma [], as well as deeper area 33 and the cingulum, located postero-ventral to most infusions [].

(F) Latency to enter and duration in hopper, percent of time spent close to the divider, and percent of freezing after shock and no shock trials for the muscimol (red) and saline (green) groups. Data are mean ± SEM, but Mann-Whitney U test has been reported as values for latency to reward poke and percent of freezing were not normally distributed. Student’s t tests, where applicable, confirmed the non-significance of all differences but time spent close to the divider on shock 3 (t(15) = 2.5; p = 0.025). Significant numbers in bold. p1 and p2 indicate one-tailed and two-tailed testing, respectively.

(B) Estimated anterior-posterior and dorsal-ventral coordinates of the infusions on a sagittal representation of the medial surface of the rat brain based on [] for muscimol (red) and saline (green) animals. Each dot is the average of the coordinates of the right and left cannula tip location in histological slices. Gray shading, estimate of likely spread based on a combination of published data [] and estimates from our own lab based on similar injections of fluorescent muscimol.

In our experiments, rats were not required to develop strong and stable preferences for a lever before associating the preferred lever with a shock. However, some animals showed a consistent preference for the same lever over the last training session and the baseline session and showed significant switching (see Figure S4 A). To investigate the impact of more pronounced preferences, we used data from two additional groups in which animals (n = 21) were trained to reach stable preference levels >80%, which took on average 480 additional trials compared to ContingentHarm, NoHarm, and RandomHarm conditions. In these “over-trained” animals, we did not find significant switching at the group level (see Figure S4 B). These results therefore suggest that the more habitual a behavior, the less it is sensitive to modification by social consequences.

To test whether actors would give up food to avoid another’s distress, we tested new groups of rats (1vs2Pellets and 1vs3Pellets; Table 3 ), where levers required the same effort (∼30 cN) but differed in rewards from the beginning of the baseline session. In the 1vs2 condition, the shock lever provided n = 2 pellets, although the no-shock lever provided n = 1 pellet. Actors decreased their preference for the 1vs2 option upon association with a shock (paired one-tailed t test; Figure 3 J, solid line), and 3 out of 7 actors in this group were detected as significant switchers. To explore whether this switching differed from the ContingentHarm, we computed a repeated-measures ANOVA using ContingentHarm and 1vs2Pellets conditions with 4 sessions each. We found a highly significant main effect of session (F= 7.03; p < 0.001; η= 0.20; BF= 175,403) but only a trend for an interaction (F= 1.82; p = 0.15; BF= 2.96). Hence, rats are willing to forgo one sucrose pellet to avoid the victim’s distress, but the effect tends to be slightly reduced compared to a difference in effort. The 1vs3 pellet condition, where the levers led to n = 1 versus n = 3 pellets ( Figure 3 J, dotted line) did not show a significant decrease of preference from baseline (paired one-tailed t test), none of the actors were significant switchers, and a rmANOVA (2 groups× 4 sessions) shows the effect was significantly smaller than in the ContingentHarm condition (interaction; F= 5.46; p = 0.002; BF= 148), suggesting that harm aversion may not be strong enough to counteract high costs.

During the piloting phase of the paradigm, we tested actors that were unfamiliar with their victims taken from unrelated cages (unfamiliar victims; Table 3 ). Actors showed a significant decrease from baseline preference levels also for shocks to these unfamiliar victims (paired one-tail t test; Figure 3 I), and 2 out of the 12 actors were detected as significant switchers. This effect was comparable to the one observed in the ContingentHarm animals of our main experiment (session× condition; F= 0.20; p = 0.66; η= 0.006; BF= 0.80). Accordingly, familiarity is not necessary for reducing lever preference, in line with data showing that rats freeze even when an unfamiliar conspecific gets a shock [] and free an unfamiliar trapped rat []. However, we cannot exclude that familiarity may have a subtle effect on the magnitude of switching, as shown in mice []. It is important to note that, during the piloting phase of the experiment, actors in the unfamiliar victims condition were also exposed to shocks prior to the experiment but in another context rather than in the victim compartment. Hence, this condition shows that the switch in preference observed in the ContingentHarm condition is not solely due to contextual fear formed during the exposure session in the victim’s compartment.

In summary, we thus identified two main factors associated with individual differences in switching. First, prior experience with footshocks increases switching. Second, animals that switch more show a stronger reaction to the shocks of the victim: they delay entering the food hopper, spend less time in the food hopper, and take longer to perform trials. In contrast, quantifications of the behavior of the victim appear not to predict switching. It thus appears, within our paradigm, as though the main determinant of individual differences stems from the actor, not the victim: among the variables we quantified, it is how the actor reacts to the victim and prior shock experience with shock, not how the victim reacts to the shocks, that are most associated with switching.

Prior experience with footshocks increases the sensitivity of rodents to witnessing footshocks in others []. Does prior experience also influence switching in our paradigm? During the exposure sessions, animals increased freezing ( Figure 3 F). We found a hint toward higher average freezing during the shock epochs of the pre-exposure in switchers compared to non-switchers (thick versus dotted lines in Figure 3 F; t= 1.55; p= 0.135; BF= 0.88). A similar trend was observed in actors tested with unfamiliar victims (t= 1.8; p= 0.102; BF= 1.2). However, these data remain inconclusive. To further test the importance of prior exposure, we tested a new group that followed the same procedure as the ContingentHarm condition, except that the actors received no shocks during the exposure session (NonExposed; Table 3 ). We observed no significant effect of session on lever preference in NonExposed animals ( Figure 3 G; one-way ANOVA; F= 1.45; p = 0.242; BF= 0.38), and a repeated measures ANOVA (rmANOVA) using both ContingentHarm and non-exposed conditions revealed a significant session (F= 8.71; p < 0.001; η= 0.20; BF= 10,819) and session × condition interaction (F= 3.51; p = 0.018; η= 0.09; BF= 5.9). Although baseline preference levels were comparable across both conditions, ContingentHarm animals showed significantly lower preference for the shock lever during the first shock session compared to the NonExposed condition ( Figure 3 G; pairwise comparisons). This difference becomes nonsignificant in the subsequent shock sessions. Switching was within chance levels in the NonExposed condition (n = 2 significant switcher; Figure 3 H, filled yellow dots; binomial; p = 0.12). Together, these analyses show that prior fear experience primes rats to a higher sensitivity to other’s pain.

In all but the food row, “/” indicates that the animal was not present, “yes” that the animal was present and received shocks, and “no” that the animal was present but received no shocks. For the food row, / indicates groups in which the food condition was not run. No (x) indicates that the condition was run in x of the N animals but that no shock was delivered. Sample size (N) reflects the number of actors included in the behavioral analyses.

To relate all these measures to SI (which is not normally distributed over the entire group), we used Kendall’s Tau rank order correlation as the measure of association. Table 2 and Figure S2 show these variables ranked by the evidence (Bayes factor) for an association. Focusing on associations with a BF> 3 (dark red lines in Figure S2 ) shows that animals that switched more spent less time in the food hopper and took longer to enter the food hopper after trials in which the victim received a shock, leading to longer overall trial duration. Figure 3 E illustrates that this effect is visible specifically in the Shock1 session, where switchers, but not non-switchers, delay their entry and accelerate their exit from the food hopper specifically on trials in which they delivered a shock to the other animal. This was confirmed by an ANOVA that revealed a session× trial× typeinteraction (significantly for log latency F= 5.9, p = 0.002, BF= 73 and a trend for log duration F= 2.37, p = 0.08, BF= 2.7). A similar effect was not apparent in the NoHarm or RandomHarm conditions ( Figure S3 ). As a result, switcher rats also took longer to perform trials ( Figure 3 E). This suggests witnessing the victim receive shocks interfered with the food-directed action of switchers, but not non-switchers. We also observed that dyads with more switching had victims that spent less time close to the divider. In contrast, variables that might have captured differences in distress signals (freezing, 22-kHz USV emissions, and loudness of pain squeaks) failed to reveal robust associations with switching ( Table 2 ). The same was true for weight and our measures of behavioral flexibility, as measured by changes in lever choice across training and baseline or in response to food rewards.

To explore what may determine these differences in switching in the ContingentHarm condition, we extracted a number of variables from the behavior of the actors and victims and examined which could predict the SI ( Table 2 Figure S2 ). To limit multiple comparisons, we focused on a limited number of variables that are meant to assess distress, attention, the ability to press the levers, and behavioral flexibility. Behavioral flexibility was assessed in two ways: (1) how much the preference for the lever that will later be paired with shocks changed from step 3c of the training session to baseline ( Figure 1 E) and (2) how much actors switched lever preferences after the shock session for food reward. The latter was measured after the end of the third shock session by turning off shock delivery, identifying which lever was less preferred, and baiting that lever with 3 pellets, in contrast to one pellet delivered by pressing the preferred lever (food session; Figures 1 E and 1F; Table 1 ). We computed individual food indices (FIs) ( Figures 3 C and 3D), which quantified the change of lever choice from the last shock session across the three successive food sessions. ContingentHarm animals did not show significant differences in FI from the NoHarm and RandomHarm animals ( Figure 3 C), and switchers and non-switcher animals showed comparable FI in the ContingentHarm condition ( Figure 3 C), suggesting that non-switchers switch as much as switchers for rewards, but not for shocks to others.

Variables ranked based on decreasing evidence of correlation (BF10) using the rank order correlation Kendall Tau. The horizontal lines separate variables based on whether there is (1) evidence for the presence of an association (top cases, BF> 3), (2) inconclusive evidence (middle, 0.33 < BF10 < 3), or (3) evidence for the absence of an association (BF10 < 0.33, bottom). See also Figure S2 . BF10, Bayes factor in favor of the presence of a correlation; CI, confidence interval; P2, two tailed frequentist probability for Tau = 0; Tau, Kendall’s tau.

where Sis the proportion of shock lever presses during baseline and Sthe average proportion of shock lever presses over all shock sessions (see STAR Methods ). Positive SIs reflect switching away from the shock lever and SI = 1 maximum possible switch given an individual’s baseline preference ( Figure 3 D). In the ContingentHarm condition, some animals showed substantial preference changes in shock sessions, and others remained indifferent. A permutation test revealed n = 9 actors (i.e., 38%) in the ContingentHarm condition (n = 4 males and n = 5 females) showed a significant switch (at p < 0.05; green solid circles in Figure 3 A; hereafter referred to as “switchers”). A binomial test showed that 9 out of 24 switchers are not explained by chance (binomial; n = 24; alpha = 0.05; p = 10). These switchers found across males and females showed a decrease between 25% and 80% from baseline. Switching rates were within chance level in the NoHarm (n = 1 significant switcher; blue colored in circle; binomial; p = 0.36) and absent in the RandomHarm condition (n = 0 significant switchers). A χsquare test revealed the ContingentHarm condition had more switchers than the NoHarm condition (χ= 4.20; p = 0.04) and the RandomHarm condition (χ= 4.17; p = 0.04). Figure 3 B shows the lever choices session per session for each ContingentHarm actor and the distribution of changes across sessions. For switchers, most changes in lever choice occurred in the first shock session, with little change occurring in the subsequent sessions.

Data in (E–G), (I), and (J) are mean ± SEM. Numbers in bold are significant; BF, Bayes factor; p1 and p2 indicate one-tailed and two-tailed testing, respectively. FDR, p values corrected using false discovery rate for 4 sessions; df, degree of freedom, which is lower in sessions in which some animals never chose the no-shock or the shock lever; See data.csv at https://osf.io/65j3g/ for the data that went into (C). See also Figures S2 and S3 and Tables S1 S2 , and S3

(F) Percentage freezing during the shock exposure sessions for the animals that will become switchers (thick line) and non-switchers (dotted line). The percent freezing quantifies the percentage freezing in the following inter-shock interval.

(E) Log-transformed latency to enter the food hopper, duration in the food hopper, as well as average trial duration for switchers and non-switchers and shock and no-shock trials in the ContingentHarm condition. See Figure S3 for similar data for the NoShock and RandomShock conditions.

(C) Food index for the different conditions. Boxplot of each distribution shows the median (red bar) and outliers (crosses). For the ContingentHarm condition, the boxplots have been computed separately for the switchers (green filling), the non-switchers (green contour), and the whole group (black filling). For the NoHarm and RandomHarm, only the group results are presented (black filling) because there are insufficient switchers. See also Figure S2 for related results and data.csv for the choice data.

(A) Switching index (SI) for the different conditions. The dots represent the SI values of each rat separately for the three main conditions. Filled dots indicate the rats that switched to the non-preferred lever. Boxplot of each distribution shows the median (red bar), outliers (crosses), and the 25% and 75% percentile values.

To prevent harm to their victim, actors could stop pressing any lever instead of switching to the no-shock lever. Across our three groups, all animals performed all their baseline trials. In the ContingentHarm shock sessions, six animals failed to press any lever within the 400 s allowed (missing 1, 1, 2, 10, 20, and 32 out of the 60 free choice trials over the 3 shock sessions, respectively). In contrast, all animals in the NoHarm condition performed all their 60 free choices over all sessions, and only one in the RandomHarm condition missed one trial. This illustrates witnessing contingent shocks to another rat can motivate agents to stop pressing levers altogether. However, given that, over all ContingentHarm animals, 95% of free trials were performed, we concentrate on the shift away from the shock lever as our dependent measure.

Male and females did not differ in their change in preference across sessions ( Figure 2 B; session × gender: F= 0.21; p = 0.89; η= 0.01; BF= 0.20), with both showing a significant main effect of session when analyzed individually (female: F= 5.9, p < 0.003, BF= 14.5; Male: F= 7.8, p < 0.001, BF= 64). For all subsequent analyses looking at the change of preference across sessions, we thus pool males and females into one single ContingentHarm condition (n = 24 actors). The Bayes factor for including a main effect of gender, however, was anecdotal (BF= 0.44).

At the group level, we compared preference changes from baseline to shock sessions across conditions. A 4-group× 4-sessionrepeated-measures ANOVA revealed a significant effect of session (F= 7.34; p < 0.001; η= 0.15; BF= 433) and session × group interaction (F= 1.93; p = 0.05; η= 0.12; BF= 3). We first concentrate on male actors, for which we have three groups (ContingentHarm, NoHarm♂, and RandomHarms♂), which showed similar preferences at baseline (i.e., comparable preference for the future shock lever; ANOVA; F= 1.29; p = 0.289; BF= 0.46). From baseline to all shock sessions, ContingentHarm male actors showed the expected decrease in shock lever pressing, with their preference for the shock lever lower than the NoHarm and RandomHarm control groups in all shock sessions (even if regressing out differences in baseline preference; Figure 2 A). Actors in the male ContingentHarm group thus shifted significantly away from a lever that causes shocks to a conspecific, and this was not simply due to the distress of the victim (which was matched, i.e., no significant differences in the amount of freezing and ultrasonic vocalizations (USVs), across ContingentHarm and RandomHarm groups; Figure S1 ) but to the contingency between the actions of the actor and the reactions of the victim.

(A) Percent choice for shock lever across sessions (baseline, Shock1, Shock2, and Shock3) and conditions (ContingentHarm, NoHarm, and RandomHarm). The numbers above the graph specify the t-statistic (t), the one-tailed false discovery rate (FDR) corrected p (p1,FDR) and the one-tailed Bayes factor (BF-0) for the comparisons between the two conditions indicated leftmost, separately for each session. In the lowest part of the graph, statistics for pairwise comparison between each shock session and the baseline are shown. Values in bold are significant. Bsl, baseline; Shk1–3, shock sessions 1–3. Gray rectangles on background help visually discriminate the sessions. Data are presented as mean ± SEM.

These 24 trials started with 4 forced trials (2 for each lever; pseudo-randomized) to force actors to sample both options, followed by 20 free choice trials to measure baseline lever preference ( Figure 1 E). In the ContingentHarm condition, on the 3 days following baseline (Shock1, Shock2, and Shock3 sessions; Figure 1 E), the actor performed 24 trials of the same task each day (4 forced + 20 free choice), similar to baseline trials, except that pressing the lever preferred during baseline triggered a footshock (0.8 mA; 1 s) to the victim in the adjacent compartment. In this condition, we had two groups: male (ContingentHarm) and female pairs (ContingentHarm). We compared this condition against a NoHarm control condition, in which pressing either lever never delivered a shock to the victim to control for spontaneous changes in preference. Finally, we created a RandomHarm control condition, in which the victim is exposed to the same shocks that triggered strong switching in the ContingentHarm condition but were administered independently of the choices of the actor. For this RandomHarm condition, we identified the 8 actors from the ContingentHarm condition (from all 24 animals) that showed the strongest switching away from the shock lever. For each, we recorded the sequence of shock and no-shock trials to the victim. In the RandomHarm condition, each victim then received the sequence of shocks from one of the switchers from the ContingentHarm condition, independently of what lever was pressed by the actor. Crucially, to break the action-outcome contingency, shocks were delayed randomly by 3–8 s after actors exited the food receptacle, i.e., before the start of the following trial.

We first compare the behavior of rats in three main conditions: ContingentHarm; NoHarm; and RandomHarm ( Table 1 ). In all three conditions, an actor was trained to press one of two levers for one sucrose pellet in the actor compartment ( Figures 1 A–1F). One lever required ∼60 cN (∼60 g) of force to be pressed, although the other required ∼30 cN (∼30 g), with the harder-to-press side randomized across animals. After initial training alone, all actors were exposed to 4 footshocks (exposure; Figures 1 E and 1F) in the adjacent victim’s compartment to maximize emotional contagion []. Actors were then placed back into their actor compartment and performed 24 trials of lever pressing with their cage mate in the victim compartment (baseline).

For each experimental condition (columns), the table specifies who got electrical shocks during the exposure, baseline, and shock sessions (yes) and who did not (no). A “/” indicates that the victim was not present during the exposure session. Sample size (n) reflects the number of actors included in the behavioral analyses. ♂, male; ♀, female.

Discussion

By characterizing the willingness of rats to reduce the use of a lever that triggers pain to another conspecific, we provide evidence that rats display a contingency-dependent harm aversion, which we show to be influenced by ACC deactivation. Despite substantial individual variability, harm aversion was replicated at the group level in 4 separate groups of animals that were tested with levers differing in effort (male ContingentHarm, female ContingentHarm, unfamiliar victims, and saline). Significant switching away from a lever harming another rat was also replicated in a condition in which switching involved using a lever delivering one instead of a lever delivering two pellets (2 versus 1 pellet), although this effect was statistically weaker. The willingness to switch was no longer significant when the difference in value across levers was too high (3 versus 1 pellet) or when animals were overtrained ( Figure S4 ).

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Jeste D. Empathic fear responses in mice are triggered by recognition of a shared experience. Our experiment has a number of important limitations to consider. First, we found that deactivating the ACC reduces switching. This demonstrates the potential of our paradigm to reveal the involvement of brain regions in harm aversion. Specifically, recent studies have suggested that both in rats and humans, the pain felt by a conspecific is mapped onto our own pain representation through emotional mirror neurons, located within the ACC []. Our deactivation data now suggest that this region may be important to prevent harm to others. However, we injected muscimol throughout all sessions, from baseline to shock3, and we thus cannot pinpoint in what phase of the task the ACC is important. Recording cellular activity during the task and using optogenetic deactivation at particular moments in the task will be essential to pinpoint when and how the ACC is important in harm aversion. Recent studies suggest that the medial prefrontal cortex [] and the amygdala [] are recruited during the discrimination of the emotions of conspecifics in mice. Whether these structures together with the ACC are involved in harm aversion in rats should be further explored. Second, one would be inclined to interpret our data as suggesting that switchers are willing to exert twice the force to save shocks to others. However, as observed in previous studies [], approximately 25% of the actors actually preferred the hard to the easy lever in baseline sessions, and for some, switching thus did not involve additional effort but actually a reduction of effort. Rather than showing a willingness to work harder for others, our data thus show that rats are willing to switch to a less-preferred lever. Third, we show that pre-exposure to shocks potentiates switching. It has been shown that freezing while observing another animal receive shocks is potentiated by prior shock experience [], but other arousing experiences, such as a forced swim test, do not have the same potentiating effect []. Moreover, although frequentist statistics suggest an effect of prior shock experience, the Bayes factor remains within a range where caution should be used in the interpretation of the data. Quantifying switching in our paradigm following a forced-swim test instead of footshock exposure would be necessary to explore whether switching also specifically depends on prior experience with a stimulus similar to that of the victim or, less specifically, on any heightened state of arousal or fear that sensitizes actors to any stimulus that could signal danger. Fourth, our unfamiliar group was tested with prior shock exposure in an environment that differed from the test environment although the rats in the main experiments received prior exposure in the test environment itself. That switching was significant at the group level in both cases shows that it is robust against changes in familiarity and context but makes it difficult to isolate the effect of either variable precisely.