The results of this study are significant for understanding the advanced sociality of mammals, such as living in groups and cooperative parenting. In the future, they are also expected to contribute to the understanding of the mechanisms of human loneliness and sociality.

Collaborative research group discovered that when female mice were isolated from their living group by a barrier, the expression of the neuropeptide "amylin" in the central medial preoptic area (cMPOA), a brain region important for parenting, decreased and nearly depleted within a week. When these isolated mice were reunited with their mates, the cMPOA was activated, and the expression of amylin returned to normal in two weeks. Artificially reducing the amount of amylin and its receptor, "calcitonin receptor (Calcr)," in the cMPOA resulted in decreased social contact behavior. Conversely, artificially activating amylin cells using pharmacogenetic techniques increased social contact behavior. These results indicate that the affiliative social behavior of female mice, which sense isolation and seek companionship, is regulated by the binding of amylin and Calcr in the cMPOA.

This study was published online in the scientific journal 'Nature Communications' on February 8 (Japan time).

Discovery of molecules and neural circuits that are important for socialization among adults, which are solitary and mate-seeking

Background

Under the COVID-19 pandemic, many people have likely felt the importance of direct and physical social interactions, rather than just virtual ones. Social animals, including humans and mice, form groups to protect themselves from cold and external threats. They collaborate in nest-building, maintaining nest cleanliness, nursing, and efficient childcare. When social animals are unable to meet their group members, they feel stressed and make efforts to reunite with their companions. If they still cannot reunite, they may exhibit responses similar to depressive states.

In humans, "loneliness" (subjective perception of social isolation) is known to adversely affect not only mental health, such as depression, but also physical health outcomes like cardiovascular diseases and cancer prognosis. Similarly, in mice, loneliness results in increased aggression in males and exacerbates depressive-like behavior and ovarian cancer in females.The importance of alleviating loneliness through social contact is well-known, yet the brain regions and mechanisms responsible for sensing and prompting social interactions have been poorly understood until recently.

Team led by Kumi Kuroda has been studying the brain mechanisms essential for parent-child relationships. In mice, not only mothers but also fathers and even virgin females participate in parenting. They identified neurons in the "medial preoptic area central part (cMPOA)" that express "calcitonin receptor (Calcr)," crucial for parenting behaviors (1, 2). Especially in mothers, it is believed that the amount of Calcr increases alongside the action of female reproductive hormones, activating cMPOA neurons necessary for parenting, demonstrating parental motivation to protect their children even at personal risk.

Calcr is activated by a neuropeptide called "amylin," produced in other cells in the cMPOA. During their research, the team serendipitously discovered that housing solitary female mice drastically reduced amylin expression in the cMPOA. Therefore, they investigated the role of the cMPOA and amylin in adult social behavior.

notes

• (1) Press release on September 30, 2015: "Parenting in the animal world: turning off the infanticide instinct"
• (2) Press release on June 2, 2021: "Why moms take risks to protect their infants"

Methods and results

Amylin expression in the cMPOA reflects the amount of affiliative social contact

First, to investigate the conditions under which amylin expression in the cMPOA varies, collaborative research group placed a single female mouse from a group cage of 4-5 mice into a separate cage, or left a single mouse in the cage while removing the other mice (Figure 1a, top). In both cases, the number of amylin-expressing neurons (amylin cells) in the isolated mice decreased by about half within two days and almost to zero within six days. However, when the isolated mice were returned to group housing, the number of amylin cells recovered to normal levels within approximately two weeks (Figure 1b).

Moreover, in both cases where the mice were completely isolated (Figure 1a, top) and where they were isolated but could see other mice through a partitioned window (Figure 1a, bottom), the number of amylin cells decreased to less than 3% within six days. These results indicate that maintaining the number of amylin cells in female mice requires not only olfactory and visual stimuli but also physical contact with their mates.

In terms of behavior, compared to group-housed mice, isolated mice spent less time remaining still and were observed digging under the partitioned window or exploring around it. Furthermore, the time spent gnawing at the partition by isolated mice, when they could see other mice through the window, was 5.2 times greater than when they were completely isolated (Figure 1c). It is thought that seeing other mice increases their motivation to gnaw and break through the barrier.

Two days after isolation, the window barrier was replaced with a partition that allowed free movement of the mice, and the isolated mouse was reintroduced to the group. The previously isolated mouse actively sniffed and made contact with the other mice. After about an hour, the mice were observed sleeping together in a group.

図1 マウスを用いた社会的条件とアミリン発現に関する実験

• a) Diagrams showing complete isolation of mice (top) and isolation where other mice are visible through a partitioned window (bottom).
• b) When solitary housing was imposed on female mice previously group-housed in groups of five, the number of amylin cells in the cMPOA dropped to nearly zero within six days. Upon returning to group housing, recovery occurred within approximately two weeks. Different alphabets indicate significant differences, P < 0.05.
• c) Mice housed in groups of four with only the partition exchanged, or completely isolated, showed minimal gnawing behavior on the partition. However, mice isolated with visibility of other mice through the partitioned window exhibited significantly increased gnawing behavior on the partition. Different alphabets indicate significant differences, P < 0.05.

The activity of amylin cells in the cMPOA decreased when mice were isolated from the group and increased upon returning to group housing. Therefore, it is believed that social contact information activates amylin cells, maintains amylin expression levels. While other substances may fluctuate in expression due to social isolation or contact, none have been identified that vary in expression levels as rapidly and dramatically in response to social situations as amylin.

The transmission of information in the amylin-Calcr neural circuit is necessary for behaviors seeking social contact

Neuropeptides such as amylin bind to specific receptors on the cell membrane, regulating neuronal activity. The amylin receptor forms a complex with another protein known as calcitonin receptor (Calcr). In the cMPOA, amylin and Calcr are expressed in separate neuronal cells. Activation of Calcr-expressing neurons (Calcr cells) in the cMPOA was observed when amylin was administered to the cMPOA or when isolated female mice were returned to their mates (Figure 2a)

To investigate whether amylin cells and Calcr cells in the cMPOA are activated simply by tactile stimuli such as physical contact, we created situations where mice came into contact due to anxiety or fear. Mice are nocturnal and feel anxiety and fear in bright environments. During their active night period, sudden exposure to bright light causes mice to huddle together defensively, attempting to protect themselves. Brain regions responsive to anxiety and fear were activated in this defensive huddling scenario, but the medial preoptic area (MPOA)^[1], including the cMPOA, did not show activation. Therefore, it is likely that cMPOA is not simply activated by physical contact stimuli, but requires physical contact in affinitive sociality (friendly engagement).

To further explore the role of amylin and Calcr in affiliative social behavior, we generated amylin-Cre transgenic mice ^[5] and artificially activated amylin cells in the cMPOA using pharmacogenetic DREADD methods ^[6]. This activation led to a 4.7-fold increase in isolated mice gnawing at the partition, indicating increased attempts to seek social contact compared to wild-type mice (Figure 2b). Conversely, knockout mice lacking the amylin gene showed a significant 26% reduction in partition gnawing behavior under isolation conditions (Figure 2c). Finally, using RNA interference ^[7] to reduce Calcr expression in the cMPOA knockdown mice by approximately 30%, partition gnawing behavior decreased by more than half under isolation conditions (Figure 2d)

These results demonstrate that signaling through the amylin-Calcr neural circuit plays a crucial role in sensing loneliness and driving behaviors aimed at reuniting with mates.

図2 アミリン-Calcr神経回路と社会的接触を求める行動の関係を調べる実験

• a) When isolated female mice were reunited with their 仲間, neurons expressing calcitonin receptor (Calcr)were significantly activated. The red arrowheads indicate activated Calcr cells. ***P<0.001.
• b) When adeno-associated virus incorporating DREADD, which is expressed in a Cre protein-dependent manner, was injected into the cMPOA of amylin-Cre transgenic mice (left-hand schematic) and activated by the pharmacogenetic method DREADD specifically in amylin cells, isolated mice increased the partition biting behavior. *P<0.05.
• c) Mice deficient in amylin (-/-) showed 26% less partition biting behavior under isolation than wild-type (+/+). In mice heterozygous deficient in amylin (+/-), partition biting behavior under isolation was reduced to half of wild-type. This suggested that the behavioral changes occurred in an amylin dose-dependent manner. Different alphabets indicate significant differences; P<0.05.
• d) When RNA interference limited to the cMPOA (left-hand schematic) was used to reduce the amount of Calcr in Calcr cells, the partition biting behavior under isolation was reduced to less than half of that in wild-type. *P<0.05.

Future expectations

With the recent low birthrate, an aging population and digitalization, people's social interactions are undergoing a number of changes. Furthermore, people's social contact has been severely restricted since the outbreak of the COVID-19 pandemic in 2020. Under these circumstances, measures to address social loneliness are urgently needed both domestically and internationally, with the UK establishing a 'Minister for Loneliness' in 2018 and Japan establishing an 'Office for Loneliness and Isolation Countermeasures' in the Cabinet Secretariat in December 2021. This research results report one part of the mechanism by which the brains of social animals feel loneliness and behave to seek mates, and can be expected to contribute to the clarification of this problem.

Last year, the collaborative research group showed that Calcr cells of the same cMPOA are essential for parenting behavior. One advantage of the same neural basis for the affinitive social and parenting behaviors between adults reported in this study could be the promotion of co-parenting. Mother mice can raise their pups alone, but when mothers gather together, they build large nests and raise their pups by 'communal nursing', where everyone feeds their pups. Communal nursing promotes the growth of the pup, so it makes sense to assume that the expression of amylin increases during parental care, increasing social contact between adults. Furthermore, phylogenetics and comparative behavioral observations have led many researchers, including Darwin, to speculate that advanced adult affinitive sociality, including empathy and altruism, may have originally evolved from parenting. The present results provide material evidence for this age-old hypothesis.

In summary, the amylin-Calcr neural circuit in the cMPOA may be involved in human co-parenting and adult sociality. However, the results of studies in rodents cannot be immediately applied to humans. Therefore, research in non-human primates is necessary. The common marmoset, a primate that uses vocal communication for family parenting, serves as an excellent model for human sociability. The collaborative research group is currently investigating whether amylin-Calcr neural circuits are also present in the common marmoset in brain regions corresponding to the cMPOA, and whether it is involved in parenting and adult sociality.  They are also trying to understand the nature of human sociality and its flip side, loneliness.

https://www.riken.jp/press/2022/20220208_1/index.html