A new paradigm of learned cooperation reveals extensive social coordination and specific cortical activation in mice
Molecular Brain volume 16, Article number: 40 (2023)
Cooperation is a social behavior crucial for the survival of many species, including humans. Several experimental paradigms have been established to study cooperative behavior and related neural activity in different animal species. Although mice exhibit limited cooperative capacity in some behavioral paradigms, it is still interesting to explore their cooperative behavior and the underlying neural mechanisms. Here, we developed a new paradigm for training and testing cooperative behavior in mice based on coordinated lever-pressing and analyzed social interactions between the animals during cooperation. We observed extensive social contact and waiting behavior in cooperating animals, with the number of such events positively correlated with the success of cooperation. Using c-Fos immunostaining and a high-speed volumetric imaging with synchronized on-the-fly scan and readout (VISoR) system, we further mapped whole-brain neuronal activity trace following cooperation. Significantly higher levels of c-Fos expression were observed in cortical areas including the frontal pole, motor cortex, anterior cingulate area, and prelimbic area. These observations highlight social interaction and coordination in cooperative behavior and provide clues for further study of the underlying neural circuitry mechanisms.
Cooperation, in which multiple participants act together for mutual benefits , is critical to the survival and evolution of many species, including humans [1, 2]. In the laboratory, animals can also learn to accomplish cooperative tasks. Monkeys can learn to cooperatively control the movement of a cursor on a screen , and rodents can learn tasks such as coordinated shuttling and nose-poking [4,5,6]. Although mice demonstrate relatively less cooperation , they do exhibit various prosocial behaviors [7,8,9]. Considering the abundance of transgenic mice available for recording and manipulating the activity of specific neuronal populations , it is worthwhile to explore the cooperation capability in mice. In this study, we trained mice to learn coordinated lever-pressing and then examined their social interactions during this cooperative behavior. We further evaluated potential brain circuits involved in cooperation with whole-brain imaging of c-Fos expression.
For training the mice to learn cooperation, we designed a training box that was divided into two chambers by a transparent windowed partition wall, with a lever and a lickometer on the two ends of each chamber (Fig. 1A). The mice were first trained individually to obtain water rewards after lever-pressing (Additional file 1: Fig. S1A–C). Then they were divided into cooperative and non-cooperative groups and trained differently (Additional file 1: Fig. S1A). Mice in the non-cooperative group continued to be trained individually for lever pressing, whereas mice in the cooperative group were trained in pairs and needed to press the levers synchronously within a 1 s or 0.5 s window to receive rewards (Fig. 1B, C and Additional file 1: Fig. S1A). Because mice could also show synchronous lever-pressing by chance, we shuffled the timing of lever-pressing and licking behaviors of each pair of mice, and computed the ratios of synchronous pressing and total pressing under 1000 shuffled conditions. We then define the cooperation index (CI) as the synchronous pressing ratio (the number of measured synchronous pressing over total pressing), subtracting the top 95th percentile of the “synchronous pressing” ratios from the shuffled data. The mean CI of the cooperative group was found to increase with training and was significantly higher than that of the non-cooperative group on the last training day (Fig. 1D, Additional file 2: Video S1 and Additional file 3: Video S2). To evaluate the stability of this learned cooperative behavior, we performed three different tests including (1) partner swapping test, (2) obstacle test, and (3) long-term memory test (see “Methods”). None of the manipulations significantly affected the resulting CI (Additional file 1: Fig. S1D–F). Thus, the mice were able to learn the task, resist interference and stably express cooperative behavior.
Social contact is known to be necessary for cooperation [4, 6, 11]. To test the importance of social contact in the present task, we used DeepLabCut  to identify the neck and body coordinates of the mice and found that the mice indeed tended to shuttle synchronously during cooperation (Additional file 1: Fig. S2A, B). The body locations of the two paired mice in the cooperative group exhibited more overlap than the non-cooperative group (Additional file 1: Fig. S2C), and were positively correlated with CI (Additional file 1: Fig. S2D). To further analyze social behaviors, we extracted social contact events based on neck distance and body angle (Fig. 1E, Additional file 1: Fig. S2E and Additional file 4: Video S3). We found that the number of social contact events in the cooperative group was greater than that in the non-cooperative group (Fig. 1F). Furthermore, the number of social contact events was positively correlated with the CI in the cooperative group (Fig. 1G). In addition to social contact, mice also showed voluntary waiting behavior, i.e., a mouse did not press the lever until his partner had also reached the press area (Fig. 1H, Additional file 1: S2A and Additional file 5: Video S4). The number of waiting events in the cooperative group was significantly greater than that in the non-cooperative group (Fig. 1I) and was positively correlated with CI (Fig. 1J).
To evaluate the necessity of communication between partners during this task, we paired cooperatively trained mice with non-trained partners. It is interesting to note that the trained mouse apparently waited for the non-trained mouse to come close to the lever, and then pressed its lever and shuttled to the water nozzle (Additional file 6: Video S5). This suggests that the trained mouse could use the partner’s location information to direct its action. However, in such tests where the trained mice did not have effective communication with their non-trained partners, the CI of the trained animals decreased significantly to almost chance level (Additional file 1: Fig. S3A). This indicates the importance of effective communication for the animals to achieve high-level performance in coordination tasks, consistent with previous studies using robotic rat partners for similar tests . In parallel experiments, we turned off the illumination light during cooperation tests for pairs of trained mice. The CI decreased significantly compared to the normal light-on condition but was still significantly higher than that of non-trained animals (Additional file 1: Fig. S3B), suggesting that other sensory modalities such as auditory or olfactory systems could also contribute to the performance. Finally, when we used an unwindowed partition wall in the test box and turned off light to block all sensory information needed for social interaction during the cooperation test, the CI for the tested pairs decreased to near the chance level (Fig. 1K, L). These results suggest that the coordinated lever-pressing of the mice is likely a form of cooperation that requires social interaction and communication.
Cooperation requires individuals to coordinate actions with their partners, and could involve concerted activity of various brain circuits [3, 5, 6]. To identify such circuits involved in cooperation, we used a volumetric imaging with synchronized on-the-fly-scan and readout (VISoR) system for whole-brain imaging  to obtain brain-wide c-Fos expression  in mice performing cooperative or non-cooperative tasks (Fig. 1M–P and Additional file 1: Fig. S4). Of all 61 brain regions evaluated, 5 were found to exhibit significantly elevated c-Fos expression in the cooperative group (Fig. 1M–Q). These regions include the frontal pole (FRP), somatomotor areas (MO), anterior cingulate area (ACA), prelimbic area (PL) and lateral amygdala nucleus (LA), suggesting possible involvement of various functions, such as decision making, motor planning, socializing and emotions, in cooperative behavior .
It is noted that the c-Fos expression across different brain areas showed strong individual variability in the cooperation group. This may be due to the variability of spontaneous behaviors and mental states during the period when the mice performed the test. It could also reflect different strategies of cooperation. In the future, refined behavior analysis and real-time neural activity recording are needed for further in-depth investigation. In the current study, we established an efficient paradigm of cooperative behavior in mice based on synchronized lever-pressing and revealed the relationship between characteristic social behaviors and cooperation. Together with brain-wide activity trace mapping, our work provides useful tools and clues for further studies of the neural mechanisms underlying cooperative behavior and its development through learning.
Availability of data and materials
The datasets obtained and/or analyzed in the current study are available from the corresponding author on reasonable request.
High-speed volumetric imaging with synchronized on-the-fly-scan and readout
Anterior cingulate area
Agranular insular area
Posterior parietal association areas
Temporal association areas
Lateral amygdalar nucleus
Basolateral amygdalar nucleus
Basomedial amygdalar nucleus
Posterior amygdalar nucleus
Striatum dorsal region
Striatum ventral region
Lateral septal complex
Striatum-like amygdalar nuclei
Pallidum dorsal region
Pallidum ventral region
Pallidum medial region
Pallidum caudal region
Thalamus sensory-motor cortex related
Thalamus polymodal association cortex related
Hypothalamic medial zone
Hypothalamic lateral zone
Superior colliculus sensory related
Substantia nigra reticular part
Ventral tegmental area
Midbrain reticular nucleus retrorubral area
Midbrain reticular nucleus
Superior colliculus motor related
Substantia nigra compact part
Midbrain raphe nuclei
Pons sensory related
Pons motor related
Pons behavioral state related
Medulla sensory related
Medulla motor related
Medulla behavioral state related
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We thank Lulu Qiu and other members of the LNP for helpful discussions and assistance.
This study was supported by grants from NSFC‑Guangdong Joint Fund (U20A6005), the Strategic Priority Research Program of Chinese Academy of Science (XDB32030200) and National Natural Science Foundation of China (32100896).
Ethics approval and consent to participate
All procedures involving the use of animals were approved by the Animal Care and Use Committee of the University of Science and Technology of China.
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The authors declare no competing interests.
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Mice were able to learn the cooperative lever-pressing task. A Schematic diagram of the cooperative training procedure. B, C Mice were able to learn the individual lever-pressing task. B The total press number during pretraining increased. There was no significant difference between the coop and non-coop groups across the 3 pretraining days. One-way ANOVA test. C The total lick number during pretraining increased. There was no significant difference between the coop and non-coop groups across the 3 pretraining days. One-way ANOVA test. D Swapping partners did not affect the cooperation index. The CI of mice cooperating with familiar partners or strangers are shown. Paired t test or Mann−Whitney test. E Introducing obstacles did not affect the cooperation index. The CI of mice cooperating without obstacles or with obstacles are shown. Paired t test or Mann−Whitney test. F Cooperative memory last long. The CI of mice on day 21and day 37 are shown. Error bars represent the SEM. Paired t test or Mann−Whitney test. *p < 0.05, ***p < 0.001, n.s. = not significant. Fig. S2. Shuttling synchronization was higher in the coop group. A Photo of the cooperation box illustrating video-based analysis. Colorful dots on the mice indicate the neck and body coordinates. Tags on the left indicate area segmentation of the training box. Axis and tags on the right indicate body locations. B Example of synchronous and asynchronous shuttle behaviors. The red and blue lines represent the projection of body centers of the two mice on the axis in A. C Shuttling synchronization of the coop group was higher than that of the non-coop group. One-way ANOVA test. Error bars indicate the SEM. ***p < 0.001. D Shuttling synchronization was positively correlated with the cooperation index. Dots with different shapes indicate the data of six training pairs on various training days. The heat level indicates the number of training days. E Schematic diagram of the definition of social contact. Fig. S3. Communications between partners are important. A Cooperation index decreased when mice cooperated with non-trained partners. Paired t test. B Cooperation index decreased during the light-off test. Paired t test or Mann-Whitney test. Error bars represent SEM. *p < 0.05, **p < 0.01. Fig. S4. Cooperation test results of animals to be sacrificed for whole-brain imaging of c-Fos antibody expression and activity trace mapping. Results of both the coop and control groups are shown. Error bars represent SEM. Mann−Whitney test. ***p < 0.001.
Additional file 2: Video S1. Example of lever-pressing of mice in the coop group, related to Fig. 1D. Video playback speed is 1 × .
Additional file 3: Video S2. Example of lever-pressing of mice in the non-coop group, related to Fig. 1D. Video playback speed is 1 × .
Additional file 4: Video S3. Example of social contact behavior, related to Fig. 1E and F. Video playback speed is 0.5 × .
Additional file 5: Video S4. Example of waiting behavior, related to Fig. 1H and I. Video playback speed is 0.5 × .
Additional file 6: Video S5. Example of cooperative behavior when mice cooperated with trained and non-trained partners, related to Figure S3A. Video playback speed is 1 × .
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Zhang, KM., Shen, Y., Jia, CH. et al. A new paradigm of learned cooperation reveals extensive social coordination and specific cortical activation in mice. Mol Brain 16, 40 (2023). https://doi.org/10.1186/s13041-023-01032-y