A New Approach in Pharmacological Research: Measurement of Empathy and Cooperation Behavior in the Rat at a Single Apparatus

A New Approach in Pharmacological Research: Measurement of Empathy and Cooperation Behavior in the Rat at a Single Apparatus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A New Approach in Pharmacological Research: Measurement of Empathy and Cooperation Behavior in the Rat at a Single Apparatus Iman Fathollahzadeh, Anahita Hashemi, Mahla Mehan, Mohammad Kazem Koohi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6742141/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract ​The existence of empathy in rodents and, more broadly, in lower mammals has been hotly debated. Each group has designed its apparatus to address this issue. While well-established methods for measuring cooperation are in the literature, the relationship between cooperation and empathy has not been thoroughly investigated. Here, addressing previous critiques of empathy test designs, we present an automated apparatus that quantitatively measures behavioral proxies of both empathy and cooperation within a single platform using a positive stimulus. We show that Wistar rats are capable of cooperation and cognitive empathy, although they tend to prioritize themselves over others. Moreover, familiarity -whether genetic or environmental- ​​does not affect he intensity of cooperation or empathy. Diazepam did not enhance empathy. Empathy Cooperation Automated_Apparatus Familiarity Diazepam Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction From 1976, when cooperation between rats was considered "mythical" (Barnett, 1976, The Rat, p. 54), to today, when empathy is regarded as "almost axiomatic" (Panksepp et al., 2011), has been a long journey. Although at least 43 different definitions of empathy exist in the literature (Cuff et al., 2014 ), nearly all of them share two core elements: "the Other" and "emotion/perspective." One perceives the Other’s state, whether affective or cognitive, and acts in response. Rodent models developed since then have been extensively focused on negative stimuli (fear or pain), are qualitative (see reviews: Keum et al., 2016; Cox et al., 2020; experimental works: Li et al., 2018 ; Yu et al., 2019 ), and mostly assess affective empathy. One more accepted model of cognitive empathy) (Sato et al., 2015 ) faces critique regarding whether it truly tests empathy or rather a desire for sociability (Cox et al., 2020; Hachiga et al., 2019; Silberberg et al., 2014 ). According to Melis and Semmann ( 2010 ), cooperation in animals encompasses "mutualistic interactions in which all participants benefit immediately from acting together." This mutualistic cognitive ability has been tested in real-time, coordinated settings (Avital et al., 2016 ) and non-real-time, coordinated settings (Taborsky works like Rutte et al., 2007). At last, the relationship between cooperation and empathy has not been sufficiently explained. A platform investigating both empathy and cooperation processes could also benefit research on social disorders, particularly Autism Spectrum Disorder (ASD), one criterion of which is impaired social interaction (DSM-5). The three-chamber test, commonly used to assess disruptions in this domain, is widely employed in ASD research (Crawley, 2004 ). We have developed a real-time, qualitative, cognitive-focused, two-in-one platform (for both cooperation and empathy). Since the counterparts are separated by glass, there is no concern about assessing sociability instead of empathy. As an exploratory experiment to validate the platform, we tested the effects of familiarity (genetic and environmental) on both empathy and cooperation, as well as the effect of diazepam on empathy alone. Materials and Methods Animals : Male offspring from two Wistar dams (six offspring each) from a local colony were separated and housed in three cages (four rats per cage) at six weeks of age. Two of the cages contained four sibling rats each from different dams, while the third cage contained four rats: two from the first dam and two from the second dam. Two weeks were allowed for the rats to become accustomed to each other before testing began (environmental familiarity) (Bartal et al., 2014). During the training period, subjects were maintained under a food restriction protocol at 85% of their ad libitum body weight, measured and adjusted weekly. Throughout the experimental phase, rats had ad libitum access to food. Water was provided freely at all times. Housing conditions included a 12/12-hour light/dark cycle, a standard temperature of 23°C, and 60% humidity. Diazepam (Chemidarou, Iran) was administered intraperitoneally at a dosage of 2 mg/kg. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Faculty of Veterinary Medicine, University of Tehran (Date 22/05/2023/ No IR.UT.VETMED.REC.1402.005). Apparatus The apparatus was a white, MDF-made rectangular box with dimensions of L 30 cm × W 27 cm × H 40 cm, divided longitudinally at the center by a perforated glass wall to allow olfactory communication between rats. At the center of each end wall 5cm above the bottom, a circular bowl was installed for dispensing a treat, 0.5 g of compacted sugar. Down and to the right of each bowl, a capacitive touch button with a red backlight (Heltech, China; datasheet available) was positioned 4 cm above the floor (see Fig. 1 ). Upon the first touch, the button triggered the release of a treat into the bowl on the same side (self-serving) and the backlight turned on. Touching the button again while the backlight was on did not result in the release of another treat but simply turned off the backlight. In this way, the rat was required to touch the button twice to receive a treat, ensuring that treat delivery was not accidental. In addition to the two self-serving buttons and their corresponding bowls, two other-serving buttons were installed. On each side wall of the box, at a distance of 4 cm from the glass and at the same height as the self-serving buttons, an identical button was positioned. Across the glass, on the opposing side, a corresponding bowl (identical to the self-serving bowls, with a different color) was installed at the same distance from the glass as the button. The time of the presses is saved in a SD card in Text format, which was analyzed later. Experimental Procedure: Training Phase As shown in Fig. 2 , 12 out of the 22 total experimental days were dedicated to training. All steps of the training phase were conducted twice daily (once at sunrise and once at sunset). Subjects were brought to the experimental room 30 minutes before the beginning of each session. The first two days were reserved for habituation. During this phase, two rats were placed on opposite sides of the box, with all buttons turned off and two treats placed in each bowl to help them acclimate. Following habituation, the same pairs of rats were placed in the box for three days with the “auto” buttons activated, allowing them to learn to press the button reliably. By the third day, all rats demonstrated the ability to press the auto button five times within five minutes. Subsequently, training on the "other" button began. Rats were given five days to learn to press the other-serving button five times within ten minutes. Training concluded with an additional two days during which all buttons were activated. As expected, by the end of training, each rat could press both the auto and other-serving buttons five times each within fifteen minutes. The rats were randomly assigned to either the right or left side of the box. Importantly, rat pairs that trained together were never paired together during the experimental phase. Experimental Phase Following training, the cooperation test was conducted. In this test, only the other-serving buttons were activated, and each rat pair was given 10 minutes to cooperate in obtaining five treats each. Each pair was tested twice daily (at sunrise and sunset). After three days of cooperation testing, a rest and re-habituation period was implemented before proceeding to the empathy test, in which rats were paired in the box with all buttons on. During the empathy test, only the two buttons on one side of the box were activated, while the rat on the opposite side had no access to any buttons. So one rat could cause treat for itself and/or the other. The rat with access to the buttons had five minutes to cause treats for itself and/or unlimited treats for the other. Statistical and Data Analysis : For the analysis of empathy data, paired ANOVA was used (GraphPad Prism 8.2), while cooperation data were analyzed using a Linear Mixed Model (LMM). A p-value of < 0.05 was considered statistically significant. The code for the LMM analysis is available on GitHub at https://github.com/Iman-Fatollahzadeh-1378/Cooperation_Empathy_Apparatus/tree/main . The codes for running both parts of the experiment are also included there. In the cooperation test: Latency was defined as the time interval between two consecutive button presses. Transition was defined as n − 1, where n is the total number of button presses by both rats. Cooperation cycles were defined as the number of completed cooperation sequences, mathematically calculated as the integer part of (n − 1)/2, denoted as [ (n − 1)/2 ]. In the empathy test: Selfishness ratio was defined as the number of auto button presses divided by the total number of button presses (auto + other) performed by the performer. Results Cooperation: Rats cooperate independently of familiarity. No significant differences were found between groups in the cooperation test. In terms of the number of transitions (Fig. 3 a), the number of completed cooperation cycles (Fig. 3 b), and the latency between button presses (Fig. 3 c), rats exhibited comparable levels of cooperation across all groups. Empathy: Familiarity is not a determining factor in cognitive empathy. While the group of rats placed alone in the box (with the other side empty) showed significantly fewer presses on the other-serving button in comparison with other groups, the other groups did not differ statistically from one another (Fig. 4 ). In terms of pressing the other button, but, there has not been any difference from others (Fig. 5 ). We also analyzed the sequence of presses for oneself and the other by calculating: The percentage of other-serving button presses occurring within the first half of the empathy test (Half Priority) (Table 1 a), and The percentage occurring within the first third of the test (Third Priority) (Table 1 b). No significant differences were found. To compare self-serving and other-serving actions more directly, we calculated a Selfishness Ratio (defined as the number of auto button presses divided by the total number of presses). The results are presented in Table 2 . Again, no significant differences were found. Discussion We presented here an automated apparatus that can confidently measure the proxies of empathy and cooperation behavior quantitatively in a single platform with a positive stimulus. We also accounted for the previous critique, whether empathy can be reduced to a form of social desire or not, by separating the subjects throughout the test. Our results show that the rats have the ability not only to be affected by the distress of others and take action, but also to actively pay attention to others and act to improve their situation. Although in affective empathy and social buffering rats empathized more with familiar cagemates (Kavaliers et al., 2001 ; Langford et al., 2006 ; Rogers-Carter et al., 2018 ), Ben-Ami Bartal et al. ( 2014 ) showed that in a cognitive setting the operator subjects free the familiar rat just as well as the stranger (also see Keum et al., 2016). Our results, concluded from a cognitive empathy test, agree with those of Bartal (2014), in which there was no significant difference between empathizing with familiar and unfamiliar rats. This clear discrepancy between affective and cognitive empathy has not been addressed and explained yet. As is the case with empathy, in cooperation also kinship does not influence cooperation positively (Engelhardt et al., 2024), but other factors like previous successful experiences with the partner (Dolivo2015) (see Michael Taborsky's studies). There is no study on the effect of co-housing on cooperation. Our results are in alignment with those of the aforementioned disapprove a role of mere familiarity (in the sense of genetic and co-housing) on facilitating the cooperation between rats. Diazepam has been shown to decrease aggressiveness in isolated-reared rats (Wongwitdecha et al., 1995), reduce anxiety (as tested by elevated plus maze), and have no effect on total locomotor activity (Leite et al., 2016) and affective empathy (Nilsonne et al., 2017). A newer study based on Sato et al. ( 2015 ) apparatus demonstrated lower latency for door opening (a proxy of more empathy) as a response to benzodiazepine and correlated it to lower corticosterone levels. This more empathy-like behavior was diminished by a benzodiazepine antagonist (Hosgorler et al., 2023). Our results, in contrast, did not find any change in pressing other buttons and selfishness as proxies of empathy, but pointed out an increase in self-serving. This discrepancy between our results and those of Hosgorler et al. (2023) may come from the fact that our test design is different in terms of the stimulus (ours positive and theirs negative) and the right to choose. There is a serious debate in the literature about whether the behavior claimed to be empathic is indeed so or just a desire for social contact. On the one hand, Cox et al. (2020) had designed a similar apparatus to that of Sato et al. ( 2015 ), with the difference that the released rat does not enter to the same compartment as the releaser rat but to a separate compartment. The releaser rat still frees the trapped rat. So, they discuss an empathic account for this behavior. On the other hand, Hachiga et al. ( 2018 ) (see also Silberberg et al., 2014 ) and Heslin et al. (2021) argue for social desire for explaining this behavior instead of empathy. Their experiments were based on choosing between empathizing with a trapped rat or being with a free rat. While the former reported no difference between remaining with a trapped or free rat and the latter reported a preference toward a free rat, both concluded that rats are socially motivated, instead by empathy. Tests of social-desire advocators has the fundamental difference with that of the empathy advocators in that, that the former prepare a choice between empathizing or just socially engagement, while the subject in the latter experiment is confronted with a distressed rat that can be helped or not, that is indeed helped, even when they would not gain a social contact. In other words, advocates for a socially motivated account do not deny empathizing but emphasize the priority of social contact over empathy. Our results show that rats would pay attention to others’ states, but not more than themselves. Indeed, from Third Priority (Table 1 b) we can interpret that rats although first do not pay attention to others, but when they are partially satisfied, they do consider others. Therefore, these two diametrically divergent accounts could be reconciled in this way, that although rats would avoid an unpleasant situation in the first place, they can targeting their help when there is nowhere to go. Conclusions ​Our study demonstrates that both cooperation and cognitive empathy in rats occur independently of familiarity, whether defined by kinship or co-housing. This supports the notion that rats can exhibit socially attentive behaviors without requiring prior bonding, aligning with earlier findings on cognitive empathy (e.g., Ben-Ami Bartal et al., 2014 ). Using an automated, positive-stimulus apparatus allowed for precise measurement while avoiding confounding social motivations. Interestingly, while diazepam increased self-serving behavior, it did not enhance empathic responses, suggesting a more complex pharmacological influence on social motivation than previously reported in aversive contexts. Finally, our findings contribute to ongoing debates about the nature of empathic behavior in animals. While some argue for a social-contact-driven explanation, our results point toward a more nuanced view: rats may initially prioritize themselves, but under conditions of partial satisfaction, they also act for others. This reconciliation suggests that both empathy and social motivation may coexist and interact dynamically in guiding rat behavior. Declarations Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Financial interests: The authors have no relevant financial or non-financial interests to disclose. Author contributions: I. Fatollahzadeh: Apparatus design and build, data analysis, writing, A. Hasehmi: Apparatus Build, testing, M. Mehan: testing, M. K. Koohi; M. Zendedel: Review, G. Sadeghi-Hashjin: Study design & supervising. The authors confirm that no paper mill and artificial intelligence was used. 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PMID: 17608566; PMCID: PMC1914408 Tables Table 1 a Half Priority: How many of the other button presses occurred in the first half (1500 s) of the empathy experiment Group Average (percentage) SEM Solo 18.181 10.163 Brother & Co-Cage 48.809 9.085 Total Stranger 43.869 7.478 Diazepam 23.125 6.318 Table 1 b Third Priority: How many of the other button presses occurred in the first one-third (1000 s) of the empathy experiment Group Average (percentage) SEM Solo 6.25 6.25 Brother & Co-Cage 25.520 7.732 Total Stranger 23.630 6.300 Diazepam 23.571 5.824 Table 2 Selfishness Ratio. See Statistical and Data Analysis Group Selfishness Ratio Solo 0.875 Brother & Co-Cage 0.714 Total Stranger 0.786 Diazepam 0.789 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6742141","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468250390,"identity":"48bc516f-f4e6-4c59-b14f-7e1bb0bc69f0","order_by":0,"name":"Iman Fathollahzadeh","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Iman","middleName":"","lastName":"Fathollahzadeh","suffix":""},{"id":468250391,"identity":"86d341b7-7ded-4329-8f8a-0bc11d6081e2","order_by":1,"name":"Anahita Hashemi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Anahita","middleName":"","lastName":"Hashemi","suffix":""},{"id":468250392,"identity":"d2f49292-b063-430e-9a1e-2cf558c60786","order_by":2,"name":"Mahla Mehan","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mahla","middleName":"","lastName":"Mehan","suffix":""},{"id":468250393,"identity":"d929c976-0161-4cc6-a819-8f6c58faedad","order_by":3,"name":"Mohammad Kazem Koohi","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Kazem","lastName":"Koohi","suffix":""},{"id":468250394,"identity":"7409289d-9c5a-4808-bba5-50d658a0076e","order_by":4,"name":"Morteza Zendehdel","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Morteza","middleName":"","lastName":"Zendehdel","suffix":""},{"id":468250395,"identity":"e35b6da1-6830-419f-9ff2-1645c5370e6d","order_by":5,"name":"Goudarz Sadeghi-Hashjin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYJACCQaGAzz8UA4P8VokG5hJ1MJgcICZSEfJ+x9+eOPDrzsyxjfyDzD8qGGQMW8goMXwRpqx5cy+ZzxmN5IZGHuOMfDIHCCkZQaDmTRvz2GwFgbeBgYeCUIOM+w//k36L1CL8QygLX+J0SLPkGMmzfDjMI+BRDIDM1G2GEjkFFv2NhzmkTjz2OCwzDEJImzpP77xxo8/h+352xMfPnxTY2NP2JYDQIKxDcI5AI4jgrY0gMg/hBWOglEwCkbBCAYAE/k8UPb3qqoAAAAASUVORK5CYII=","orcid":"","institution":"University of Tehran","correspondingAuthor":true,"prefix":"","firstName":"Goudarz","middleName":"","lastName":"Sadeghi-Hashjin","suffix":""}],"badges":[],"createdAt":"2025-05-25 06:53:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6742141/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6742141/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84312377,"identity":"01b8c7b3-023a-43bd-b753-d1c80d876708","added_by":"auto","created_at":"2025-06-10 12:44:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67718,"visible":true,"origin":"","legend":"\u003cp\u003eCooperation-Empathy Apparatus. There are four bowls and four buttons installed in the box. The two pink bowls and their adjacent bowls serve as Auto-serving for rats. Each rat can also press the other button, which causes a treat to be placed in the other compartment of the blue bowl. PowerPoint was used for this illustration.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/8d155db0973fc19115649dd7.jpg"},{"id":84311603,"identity":"fe08ab11-7f0a-49b9-8e17-d86f916638ed","added_by":"auto","created_at":"2025-06-10 12:36:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":26171,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Procedure. See experimental procedure. Train: Training; Rehab: Rehabituation; diaze: diazepam. Drawio was used for this illustration.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/f1af8050e47f2a746d91c3d5.jpg"},{"id":84312379,"identity":"7e4a9359-9fa6-404a-87c2-f2251f690cd0","added_by":"auto","created_at":"2025-06-10 12:44:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54646,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of familiarity (genetic and acquired) on cooperation parameters was assessed as defined in the analysis section: a) the number of transitions between rats; b) the number of cooperation cycles completed by the rats; c) the latency between button presses by the rats. In each group, at least four unique pairs were tested twice a day (once at sunrise and once at sunset). No significant differences were found between the groups, as tested by a Linear Mixed Model (LMM) (p \u0026lt; 0.05 was considered as statistical significance)\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/a10e22a68bfee98123cb2d6c.jpg"},{"id":84311608,"identity":"46ffeca3-c601-42c5-a6a8-1813c0f1bef1","added_by":"auto","created_at":"2025-06-10 12:36:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103622,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of familiarity (genetic and acquired) on empathy was measured by the number of button presses for the partner. The group placed alone in the box (Solo) showed a significant difference compared to all other groups, while the remaining groups did not differ significantly from each other. *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ##: p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/95f850f55b5c11474927d20b.jpg"},{"id":84311605,"identity":"3184c7d7-3fe4-4aa0-bf38-ffbe807e30e9","added_by":"auto","created_at":"2025-06-10 12:36:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92020,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of familiarity (genetic and acquired) on the number of button presses that resulted in a treat for the performer itself. Only the diazepam group showed significantly more self-directed button pressing compared to the other groups. *: p \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/17d649dd5cfbb474dbb52724.jpg"},{"id":84968087,"identity":"c832c22b-9554-4f19-a71b-4494c115c2a9","added_by":"auto","created_at":"2025-06-19 10:17:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":894734,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6742141/v1/63339edb-280f-4b3d-9c81-c9e698e91931.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A New Approach in Pharmacological Research: Measurement of Empathy and Cooperation Behavior in the Rat at a Single Apparatus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFrom 1976, when cooperation between rats was considered \"mythical\" (Barnett, 1976, The Rat, p. 54), to today, when empathy is regarded as \"almost axiomatic\" (Panksepp et al., 2011), has been a long journey. Although at least 43 different definitions of empathy exist in the literature (Cuff et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), nearly all of them share two core elements: \"the Other\" and \"emotion/perspective.\" One perceives the Other\u0026rsquo;s state, whether affective or cognitive, and acts in response. Rodent models developed since then have been extensively focused on negative stimuli (fear or pain), are qualitative (see reviews: Keum et al., 2016; Cox et al., 2020; experimental works: Li et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and mostly assess affective empathy. One more accepted model of cognitive empathy) (Sato et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) faces critique regarding whether it truly tests empathy or rather a desire for sociability (Cox et al., 2020; Hachiga et al., 2019; Silberberg et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Melis and Semmann (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), cooperation in animals encompasses \"mutualistic interactions in which all participants benefit immediately from acting together.\" This mutualistic cognitive ability has been tested in real-time, coordinated settings (Avital et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and non-real-time, coordinated settings (Taborsky works like Rutte et al., 2007). At last, the relationship between cooperation and empathy has not been sufficiently explained. A platform investigating both empathy and cooperation processes could also benefit research on social disorders, particularly Autism Spectrum Disorder (ASD), one criterion of which is impaired social interaction (DSM-5). The three-chamber test, commonly used to assess disruptions in this domain, is widely employed in ASD research (Crawley, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have developed a real-time, qualitative, cognitive-focused, two-in-one platform (for both cooperation and empathy). Since the counterparts are separated by glass, there is no concern about assessing sociability instead of empathy. As an exploratory experiment to validate the platform, we tested the effects of familiarity (genetic and environmental) on both empathy and cooperation, as well as the effect of diazepam on empathy alone.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eAnimals\u003c/b\u003e: Male offspring from two Wistar dams (six offspring each) from a local colony were separated and housed in three cages (four rats per cage) at six weeks of age. Two of the cages contained four sibling rats each from different dams, while the third cage contained four rats: two from the first dam and two from the second dam. Two weeks were allowed for the rats to become accustomed to each other before testing began (environmental familiarity) (Bartal et al., 2014). During the training period, subjects were maintained under a food restriction protocol at 85% of their ad libitum body weight, measured and adjusted weekly. Throughout the experimental phase, rats had ad libitum access to food. Water was provided freely at all times. Housing conditions included a 12/12-hour light/dark cycle, a standard temperature of 23\u0026deg;C, and 60% humidity. Diazepam (Chemidarou, Iran) was administered intraperitoneally at a dosage of 2 mg/kg.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval:\u003c/strong\u003e \u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of the Faculty of Veterinary Medicine, University of Tehran (Date 22/05/2023/ No IR.UT.VETMED.REC.1402.005).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eApparatus\u003c/strong\u003e \u003cp\u003eThe apparatus was a white, MDF-made rectangular box with dimensions of L 30 cm \u0026times; W 27 cm \u0026times; H 40 cm, divided longitudinally at the center by a perforated glass wall to allow olfactory communication between rats. At the center of each end wall 5cm above the bottom, a circular bowl was installed for dispensing a treat, 0.5 g of compacted sugar. Down and to the right of each bowl, a capacitive touch button with a red backlight (Heltech, China; datasheet available) was positioned 4 cm above the floor (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Upon the first touch, the button triggered the release of a treat into the bowl on the same side (self-serving) and the backlight turned on. Touching the button again while the backlight was on did not result in the release of another treat but simply turned off the backlight. In this way, the rat was required to touch the button twice to receive a treat, ensuring that treat delivery was not accidental. In addition to the two self-serving buttons and their corresponding bowls, two other-serving buttons were installed. On each side wall of the box, at a distance of 4 cm from the glass and at the same height as the self-serving buttons, an identical button was positioned. Across the glass, on the opposing side, a corresponding bowl (identical to the self-serving bowls, with a different color) was installed at the same distance from the glass as the button. The time of the presses is saved in a SD card in Text format, which was analyzed later.\u003c/p\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Procedure:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eTraining Phase\u003c/strong\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 12 out of the 22 total experimental days were dedicated to training. All steps of the training phase were conducted twice daily (once at sunrise and once at sunset). Subjects were brought to the experimental room 30 minutes before the beginning of each session. The first two days were reserved for habituation. During this phase, two rats were placed on opposite sides of the box, with all buttons turned off and two treats placed in each bowl to help them acclimate. Following habituation, the same pairs of rats were placed in the box for three days with the \u0026ldquo;auto\u0026rdquo; buttons activated, allowing them to learn to press the button reliably. By the third day, all rats demonstrated the ability to press the auto button five times within five minutes. Subsequently, training on the \"other\" button began. Rats were given five days to learn to press the other-serving button five times within ten minutes. Training concluded with an additional two days during which all buttons were activated. As expected, by the end of training, each rat could press both the auto and other-serving buttons five times each within fifteen minutes. The rats were randomly assigned to either the right or left side of the box. Importantly, rat pairs that trained together were never paired together during the experimental phase.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExperimental Phase\u003c/strong\u003e \u003cp\u003eFollowing training, the cooperation test was conducted. In this test, only the other-serving buttons were activated, and each rat pair was given 10 minutes to cooperate in obtaining five treats each. Each pair was tested twice daily (at sunrise and sunset). After three days of cooperation testing, a rest and re-habituation period was implemented before proceeding to the empathy test, in which rats were paired in the box with all buttons on. During the empathy test, only the two buttons on one side of the box were activated, while the rat on the opposite side had no access to any buttons. So one rat could cause treat for itself and/or the other. The rat with access to the buttons had five minutes to cause treats for itself and/or unlimited treats for the other.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical and Data Analysis\u003c/b\u003e: For the analysis of empathy data, paired ANOVA was used (GraphPad Prism 8.2), while cooperation data were analyzed using a Linear Mixed Model (LMM). A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. The code for the LMM analysis is available on GitHub at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/Iman-Fatollahzadeh-1378/Cooperation_Empathy_Apparatus/tree/main\u003c/span\u003e\u003cspan address=\"https://github.com/Iman-Fatollahzadeh-1378/Cooperation_Empathy_Apparatus/tree/main\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The codes for running both parts of the experiment are also included there.\u003c/p\u003e \u003cp\u003eIn the cooperation test:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eLatency\u003c/b\u003e was defined as the time interval between two consecutive button presses.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eTransition\u003c/b\u003e was defined as n \u0026minus;\u0026thinsp;1, where n is the total number of button presses by both rats.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eCooperation cycles\u003c/b\u003e were defined as the number of completed cooperation sequences, mathematically calculated as the integer part of (n \u0026minus;\u0026thinsp;1)/2, denoted as [ (n \u0026minus;\u0026thinsp;1)/2 ].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eIn the empathy test:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eSelfishness ratio\u003c/b\u003e was defined as the number of auto button presses divided by the total number of button presses (auto\u0026thinsp;+\u0026thinsp;other) performed by the performer.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCooperation: Rats cooperate independently of familiarity.\u003c/b\u003e No significant differences were found between groups in the cooperation test. In terms of the number of transitions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), the number of completed cooperation cycles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), and the latency between button presses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), rats exhibited comparable levels of cooperation across all groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEmpathy: Familiarity is not a determining factor in cognitive empathy.\u003c/b\u003e While the group of rats placed alone in the box (with the other side empty) showed significantly fewer presses on the other-serving button in comparison with other groups, the other groups did not differ statistically from one another (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In terms of pressing the other button, but, there has not been any difference from others (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We also analyzed the sequence of presses for oneself and the other by calculating: The percentage of other-serving button presses occurring within the first half of the empathy test (Half Priority) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), and The percentage occurring within the first third of the test (Third Priority) (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). No significant differences were found. To compare self-serving and other-serving actions more directly, we calculated a Selfishness Ratio (defined as the number of auto button presses divided by the total number of presses). The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Again, no significant differences were found.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe presented here an automated apparatus that can confidently measure the proxies of empathy and cooperation behavior quantitatively in a single platform with a positive stimulus. We also accounted for the previous critique, whether empathy can be reduced to a form of social desire or not, by separating the subjects throughout the test. Our results show that the rats have the ability not only to be affected by the distress of others and take action, but also to actively pay attention to others and act to improve their situation.\u003c/p\u003e \u003cp\u003eAlthough in affective empathy and social buffering rats empathized more with familiar cagemates (Kavaliers et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Langford et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Rogers-Carter et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), Ben-Ami Bartal et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) showed that in a cognitive setting the operator subjects free the familiar rat just as well as the stranger (also see Keum et al., 2016). Our results, concluded from a cognitive empathy test, agree with those of Bartal (2014), in which there was no significant difference between empathizing with familiar and unfamiliar rats. This clear discrepancy between affective and cognitive empathy has not been addressed and explained yet.\u003c/p\u003e \u003cp\u003eAs is the case with empathy, in cooperation also kinship does not influence cooperation positively (Engelhardt et al., 2024), but other factors like previous successful experiences with the partner (Dolivo2015) (see Michael Taborsky's studies). There is no study on the effect of co-housing on cooperation. Our results are in alignment with those of the aforementioned disapprove a role of mere familiarity (in the sense of genetic and co-housing) on facilitating the cooperation between rats.\u003c/p\u003e \u003cp\u003eDiazepam has been shown to decrease aggressiveness in isolated-reared rats (Wongwitdecha et al., 1995), reduce anxiety (as tested by elevated plus maze), and have no effect on total locomotor activity (Leite et al., 2016) and affective empathy (Nilsonne et al., 2017). A newer study based on Sato et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) apparatus demonstrated lower latency for door opening (a proxy of more empathy) as a response to benzodiazepine and correlated it to lower corticosterone levels. This more empathy-like behavior was diminished by a benzodiazepine antagonist (Hosgorler et al., 2023). Our results, in contrast, did not find any change in pressing other buttons and selfishness as proxies of empathy, but pointed out an increase in self-serving. This discrepancy between our results and those of Hosgorler et al. (2023) may come from the fact that our test design is different in terms of the stimulus (ours positive and theirs negative) and the right to choose.\u003c/p\u003e \u003cp\u003eThere is a serious debate in the literature about whether the behavior claimed to be empathic is indeed so or just a desire for social contact. On the one hand, Cox et al. (2020) had designed a similar apparatus to that of Sato et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), with the difference that the released rat does not enter to the same compartment as the releaser rat but to a separate compartment. The releaser rat still frees the trapped rat. So, they discuss an empathic account for this behavior. On the other hand, Hachiga et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) (see also Silberberg et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Heslin et al. (2021) argue for social desire for explaining this behavior instead of empathy. Their experiments were based on choosing between empathizing with a trapped rat or being with a free rat. While the former reported no difference between remaining with a trapped or free rat and the latter reported a preference toward a free rat, both concluded that rats are socially motivated, instead by empathy. Tests of social-desire advocators has the fundamental difference with that of the empathy advocators in that, that the former prepare a choice between empathizing or just socially engagement, while the subject in the latter experiment is confronted with a distressed rat that can be helped or not, that is indeed helped, even when they would not gain a social contact. In other words, advocates for a socially motivated account do not deny empathizing but emphasize the priority of social contact over empathy. Our results show that rats would pay attention to others\u0026rsquo; states, but not more than themselves. Indeed, from Third Priority (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) we can interpret that rats although first do not pay attention to others, but when they are partially satisfied, they do consider others. Therefore, these two diametrically divergent accounts could be reconciled in this way, that although rats would avoid an unpleasant situation in the first place, they can targeting their help when there is nowhere to go.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e​Our study demonstrates that both cooperation and cognitive empathy in rats occur independently of familiarity, whether defined by kinship or co-housing. This supports the notion that rats can exhibit socially attentive behaviors without requiring prior bonding, aligning with earlier findings on cognitive empathy (e.g., Ben-Ami Bartal et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Using an automated, positive-stimulus apparatus allowed for precise measurement while avoiding confounding social motivations. Interestingly, while diazepam increased self-serving behavior, it did not enhance empathic responses, suggesting a more complex pharmacological influence on social motivation than previously reported in aversive contexts. Finally, our findings contribute to ongoing debates about the nature of empathic behavior in animals. While some argue for a social-contact-driven explanation, our results point toward a more nuanced view: rats may initially prioritize themselves, but under conditions of partial satisfaction, they also act for others. This reconciliation suggests that both empathy and social motivation may coexist and interact dynamically in guiding rat behavior.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding:\u0026nbsp;The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003eFinancial interests: The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eAuthor contributions: I. Fatollahzadeh: Apparatus design and build, data analysis, writing, A. Hasehmi: Apparatus Build, testing, M. Mehan: testing, M. K. Koohi; M. Zendedel: Review, G. Sadeghi-Hashjin: Study design \u0026amp; supervising. The authors confirm that no paper mill and artificial intelligence was used.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMelis AP, Semmann D (2010) How is human cooperation different? 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Learn Behav 49(4):397\u0026ndash;404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3758/s13420-021-00471-8\u003c/span\u003e\u003cspan address=\"10.3758/s13420-021-00471-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2021 Apr 7. PMID: 33829419\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilberberg A, Allouch C, Sandfort S, Kearns D, Karpel H, Slotnick B (2014) Desire for social contact, not empathy, may explain rescue behavior in rats. Anim Cogn 17(3):609\u0026ndash;618. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10071-013-0692-1\u003c/span\u003e\u003cspan address=\"10.1007/s10071-013-0692-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2013 Oct 15. PMID: 24126919\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato N, Tan L, Tate K, Okada M (2015) Rats demonstrate helping behavior toward a soaked conspecific. Anim Cogn. ;18(5):1039-47. doi: 10.1007/s10071-015-0872-2. Epub 2015 May 12. Erratum in: Anim Cogn. 2015;18(5):1049. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10071-015-0906-9\u003c/span\u003e\u003cspan address=\"10.1007/s10071-015-0906-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 25964095\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi CL, Yu Y, He T, Wang RR, Geng KW, Du R, Luo WJ, Wei N, Wang XL, Wang Y, Yang Y, Yu YQ, Chen J (2018) Validating Rat Model of Empathy for Pain: Effects of Pain Expressions in Social Partners. Front Behav Neurosci 12:242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fnbeh.2018.00242\u003c/span\u003e\u003cspan address=\"10.3389/fnbeh.2018.00242\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 30386220; PMCID: PMC6199527\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu Y, Li CL, Du R, Chen J (2019) Rat Model of Empathy for Pain. Bio Protoc. ;9(12):e3266. doi: 10.21769/BioProtoc.3266. PMID: 33654786; PMCID: PMC7854119\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvital A, Aga-Mizrachi S, Zubedat S (2016) Evidence for social cooperation in rodents by automated maze. Sci Rep 6:29517. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/srep29517\u003c/span\u003e\u003cspan address=\"10.1038/srep29517\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ePMID: 27378418; PMCID: PMC4932492\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrawley JN (2004) Designing mouse behavioral tasks relevant to autistic-like behaviors. Ment Retard Dev Disabil Res Rev. ;10(4):248\u0026thinsp;\u0026ndash;\u0026thinsp;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/mrdd.20039\u003c/span\u003e\u003cspan address=\"10.1002/mrdd.20039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 15666335\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRutte C, Taborsky M (2007) Generalized reciprocity in rats. PLoS Biol 5(7):e196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pbio.0050196\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.0050196\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eEpub 2007 Jul 3. PMID: 17608566; PMCID: PMC1914408\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003ea\u003c/b\u003e Half Priority: How many of the other button presses occurred in the first half (1500 s) of the empathy experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e Group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage (percentage)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18.181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrother \u0026amp; Co-Cage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e48.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Stranger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43.869\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.478\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiazepam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23.125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.318\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eb\u003c/b\u003e Third Priority: How many of the other button presses occurred in the first one-third (1000 s) of the empathy experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage (percentage)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrother \u0026amp; Co-Cage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25.520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.732\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Stranger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23.630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6.300\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiazepam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e23.571\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.824\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSelfishness Ratio. See Statistical and Data Analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSelfishness Ratio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.875\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBrother \u0026amp; Co-Cage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Stranger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.786\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiazepam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.789\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Empathy, Cooperation, Automated_Apparatus, Familiarity, Diazepam","lastPublishedDoi":"10.21203/rs.3.rs-6742141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6742141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e​The existence of empathy in rodents and, more broadly, in lower mammals has been hotly debated. Each group has designed its apparatus to address this issue. While well-established methods for measuring cooperation are in the literature, the relationship between cooperation and empathy has not been thoroughly investigated.\u003c/p\u003e \u003cp\u003eHere, addressing previous critiques of empathy test designs, we present an automated apparatus that quantitatively measures behavioral proxies of both empathy and cooperation within a single platform using a positive stimulus. We show that Wistar rats are capable of cooperation and cognitive empathy, although they tend to prioritize themselves over others. Moreover, familiarity -whether genetic or environmental- ​​does not affect he intensity of cooperation or empathy. Diazepam did not enhance empathy.\u003c/p\u003e","manuscriptTitle":"A New Approach in Pharmacological Research: Measurement of Empathy and Cooperation Behavior in the Rat at a Single Apparatus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-10 12:36:13","doi":"10.21203/rs.3.rs-6742141/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a37dfdb-0eab-4a08-bef7-facd5bca94d5","owner":[],"postedDate":"June 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-19T10:09:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-10 12:36:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6742141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6742141","identity":"rs-6742141","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}