Long-term behavioral effects of social separation during early life in a social mammal, Octodon degus

doi:10.21203/rs.3.rs-1988682/v1

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Long-term behavioral effects of social separation during early life in a social mammal, Octodon degus

https://doi.org/10.21203/rs.3.rs-1988682/v1

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published 12 Jun, 2023

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Social separation is thought to induce a strong stress response in social juvenile mammals, but little is known about how this response might vary throughout the development. The present study examines the long-term effects of early-life stress (ELS) induced by social separation on individual behaviors later in life using the social and precocious species Octodon degus. Four experimental groups were established a positive control group of mothers and siblings from three litters comprised the socially housed (SH) group, while pups from six litters were randomly assigned to three treatments: pups experiencing no separation (NS) treatment while their siblings did; repeated bouts of consecutive separation (CS); intermittent separation (IS). We analyzed the effects of separation treatment on the frequency and duration of freezing, rearing and grooming behaviors. ELS was correlated with higher hyperactivity later in life, especially in developed males, and hyperactivity increased with more frequent separation. The SH group containing siblings that did not experience parental separation gained more weight than the other groups. The findings suggest that the NS group was indirectly affected by ELS, especially females. In summary, the long-term effects of ELS are more direct in males while those are more indirect in females.

It is considered that the effect of early-life environmental stresses raises the risk of later onset of mental illness. In the process of brain development, synapse formation occurs from birth to several months postnatal, and during this experience, efficient neural networks will be made [1]. Thus, animals have high sensitivity to their environment, especially at the time of birth, and need a stable environment [2]. It has been observed that stress on animal pups after birth blocks normal behavioral and physiological development in both humans and animals. It has been shown that stress during this period is an important risk factor for mental health postdevelopment [3]–[5]. Reportedly, animals that experience stresses repeatedly or chronically will change their level of endocrines in the system [6]. Neuroscience and neuroanatomy studies have documented how negative mental health experiences negatively affect the development of limbic circuit functions and structures [7], [8]. The effects of stress also vary by sex because, during an animal’s development, sex differences induced by gonadal hormones influence brain development and hormonal dynamics [9]–[12].

The greatest early-life stress (ELS) occurs during social separation which is the cutting off of direct communication and contact with a caregiver of the same group, such as a mother, regardless of the animal's will. Social animals are affected by social separation and have significant stress and detrimental psychological effects [13]. Thus, social separation during this sensitive period has impact on juvenile development. However, few studies have long-term observations. Hence, the sex dependent effects of separation and indirect effects from separations of siblings are understudied because these effects are expected to become apparent after a long time.

The effects of social separation may cause problems in various animal breeding situations. For example, on farms, livestock production frequently has young juveniles separated, and in zoos, social separation of offspring is often unavoidable due to a limited number of animals in captivity, abandonment, or inter-individual conflict. These ELS events induced by separation are possible causes of serious problems in the juvenile’s developmental process. However, to research the effects of separation, tracking an individual’s development in a controlled situation, including social environmental factors in livestock or zoo animals, is difficult because of problems in production, the long experimental periods, ensuring large sample sizes, space constraints, and high costs. Hence, research that focuses on separation experiments using model mammals is urgently needed.

Octodon degus (Fig. 1) are highly intelligent, diurnal, herbivorous, small, social mammals [14]–[16]. They live in groups [17] and have complex vocal communication [8], [18]. The group comprised 1–3 males and 1–10 females and is predicted to be a promiscuous mating system [15]. Degus nest and raise their young communally with females nursing not only their own young but also other females’ young born at the same time [19]. The gestation period is about 90 days and natural weaning occurs at 4–6 weeks. Furthermore, they are precocious, meaning they are considered advanced at birth because they can move independently and vocalize within 3–4 h of birth, displayed functional grooming (face washing, hind-paw scratching, and rapid headshake) by the 1st or 2nd day, and communicate with their parents and siblings [20]. Thus, as a model species, we can analyze behavioral reactions at an earlier developmental level compared with experimental mice or rats. In addition, this species demonstrates strong social attachment and strong stress responses [21]. Because of these characteristics, degus are considered to be a useful model animal in the fields of psychopathology, psychology, neuroscience, and animal behavior [22].

In previous research, the focus was on the development of the brain, hormones, and endocrine stimulators such as neurotransmitters. Rising stress hormones in plasma after separation was reported, and a daily 1-hour separation increases individual activity [23]. Moreover, ELS precipitates widespread reduction of brain activity in the infant’s brain and changes in interregional functional coupling and results in attention-deficit and brain metabolic hypoactivity in degus [24], [25]. However, these results were observed around weaning time (Postnatal day 35, PND35), and how this response varies through the developmental process has only been reported by Rivera [26]. In addition, to our knowledge, there is no study to illustrate sex dependent responses to the direct ELS (DELS) and to examine directly the behavioral effects of indirect ELS (IELS) in social animals.

Thus, to reveal the long-lasting effects of ELS experienced from PND3 to PND20 on individual behaviors exhibited after development, we used the open-field test (OFT) and compared behavior on PND21, PND50, and PND245 among the four following groups: (i) social housing (SH) group (control group): the whole litter did not experience any separation treatment; (ii) no separation (NS) group: the pups did not experience separation from their mother, but their siblings did; (iii) consecutive separation (CS) group: the pups were removed from their mother and home cage for 1-hour every day; and (iv) intermittent separation (IS) group: the pups were removed from their mother and home cage for 1-hour on PND3, PND8, and PND14 (see Table S1 and Fig. 2). We defined that the NS group as the IELS group and the CS and IS groups as the DELS groups. Furthermore, the sex differences were analyzed for these groups.

Bodyweight

There were no obvious effects of ELS on the bodyweight gain from PND21 to PND50. From PND21 to PND64 and PND245, the siblings in the control group with no separation (SH) gained more weight than the other groups (Table S2; Separation group × PND). Consequently, the overall bodyweight of the SH group was higher than the other three groups on PND245 at the end of the experiment (multiple comparisons: NS < SH; estimate ± SE = 20.64 ± 5.85, t = 3.53, p = 0.02, CS < SH; estimate ± SE = 18.58 ± 5.45, t = 3.41, p = 0.030, IS < SH; estimate ± SE = 24.34 ± 5.25, t = 4.64, p < 0.001). In males, from PND21 to PND245, the SH group gained more weight than other groups (Figure 3a, Table S3; Separation group × PND). The bodyweight of the SH group was heavier than that of the IS group on PND245 (Figure 3a, multiple comparisons: IS < SH; estimate ± SE = 25.95 ± 7.22, t = 3.60, p = 0.02). In females, from PND21 to PND64 and PND245, the SH group gained more weight than the other groups (Figure 3b, Table S4; Separation group × PND). The bodyweight of the SH group tended to be heavier than that of the NS group on PND64 (Figure 3b, multiple comparisons: NS < SH; estimate ± SE = 30.88 ± 9.35, t = 3.30, p = 0.08).

Behavior

Effects of separation and PND on freezing, rearing, and grooming behaviors in all individuals

Overall, the time spent freezing for all individuals was not different between groups (Table S5a; Separation group). By contrast, the time spent freezing was shorter in this order: PND50 < PND21 < PND245 (Table S5a; PND). From PND21 to PND50, the time spent freezing decreased less in the NS group than in the SH group, whereas it decreased more in the CS group than in the SH group (Table S5a; Separation group × PND). From PND21 to PND245, the time spent freezing decreased in the NS and CS groups while it increased in the SH group (Table S5a; Separation group × PND). On PND21, the time spent freezing tended to be shorter in the IS group than in the NS group (Figure 4a, multiple comparisons: IS < NS; estimate ± SE = 0.96 ± 0.33, z = 2.92, p = 0.06). On PND50, the time spent freezing was shorter in the CS and IS groups than in the NS group (Figure 4a, multiple comparisons: CS < NS; estimate ± SE = 1.70 ± 0.36, z = 4.74, p < 0.001, IS < NS; estimate ± SE = 1.09 ± 0.34, z = 3.25, p = 0.02), and the time spent freezing in the CS group tended to be shorter than that in the SH group (Figure 4a, CS < SH; estimate ± SE = 0.97 ± 0.34, z = 2.82, p = 0.09). On PND245, the time spent freezing was shorter in the CS group than in the SH and NS groups (Figure 4a, multiple comparisons: CS < SH; estimate ± SE = 1.30 ± 0.33, z = 3.90, p = 0.002, CS < NS; estimate ± SE = 1.16 ± 0.35, z = 3.28, p = 0.02). Especially, the time spent freezing in the SH group was long, with a maximum value of 117 s for PND245.

The total number of rearing behaviors was higher in the CS and IS groups than in the SH group (Table S5b; Separation group). The total number of rearing behaviors was higher on PND50 than on PND21 (Table S5b; PND). From PND21 to PND50, the total number of rearing increased less in the CS and IS groups than in the SH group, whereas from PND21 to PND245, it increased larger in the NS and CS groups than in the SH group (Table S5b; Separation group × PND). On PND21, the total number of rearing behaviors was higher in the IS group than in the SH and NS groups (Figure 4b, multiple comparisons: IS > SH; estimate ± SE = -0.69 ± 0.22, z = -3.11, p = 0.03, IS > NS; estimate ± SE = -0.96 ± 0.25, z = -3.84, p = 0.002), and tended to be higher in the CS group than in the NS group (Figure 4b, multiple comparisons: CS > NS ; estimate ± SE = -0.75 ± 0.26, z = -2.84, p = 0.08). On PND50, the total number of rearing behaviors did not differ between these separation groups (Figure 4b, multiple comparisons: NS < SH; estimate ± SE = 0.49 ± 0.23, z = 2.16, p =0.55, CS > SH; estimate ± SE = -0.07 ± 0.20, z = -0.33, p = 1.0, IS < SH; estimate ± SE = 0.08 ± 0.19, z = 0.41, p = 1.0, CS > NS; estimate ± SE = -0.56 ± 0.24, z = -2.39, p = 0.31, IS > NS; estimate ± SE = -0.41 ± 0.23, z = -1.81, p = 1.0, IS < CS; estimate ± SE = 0.15 ± 0.20, z = 0.73, p = 1.0). On PND245, it was higher in the CS group than in the SH and NS groups (Figure 4b, multiple comparisons: CS > SH; estimate ± SE = -0.91 ± 0.23, z = -3.92, p = 0.002, CS > NS; estimate ± SE = -0.70 ± 0.25, z = -2.80, p = 0.09). Moreover, the total number of rearing behaviors tended to be higher in IS group than in SH group (Figure 4b, multiple comparisons: IS > SH; estimate ± SE = -0.67 ± 0.23, z = -2.94, p = 0.06).
Overall, the total number of grooming behaviors tended to be higher in the IS group than in the SH group and most evidently on PND50 than on PND21 (Table S5c; Separation group, PND). By contrast, the total number of grooming behaviors were not different between separation groups on each observation day (Figure 4c).

Sex differences in the effects of separation and PND on behaviors

Regarding fixed effects, males showed similar patterns to the case of all individuals pooled as they showed no differences in the time spent freezing between the separation groups, whereas females did not. Here the time females spent freezing tended to be longer in the NS group than in the SH group (Table S7a; Separation group). Similar to the case of all individuals pooled, the time males spent freezing showed the order: PND50 < PND21 < PND245, whereas that of females did not differ between PND21 and PND245 (Table S6a, Table S7a; PND). Conversely, the effects of separation group × PND on the time spent freezing males and females followed a similar pattern to the case of all individuals. On PND21, the time spent freezing of males did not differ between the groups. On PND50, the time spent freezing was shorter in the CS group than in the SH and NS groups and tended to be shorter in IS group than in NS group (Figure 5a, multiple comparisons: CS < SH; estimate ± SE = 1.48 ± 0.43, z = 3.44, p = 0.01, CS < NS; estimate ± SE = 1.99 ± 0.46, z = 4.37, p < 0.001). On PND245, the time spent freezing was shorter in the CS group than in the SH and NS groups (Figure 5a, multiple comparisons: CS < SH; estimate ± SE = 1.60 ± 0.41, z = 3.91, p = 0.002, CS < NS; estimate ± SE = 1.41 ± 0.44, z = 3.21, p = 0.02). By contrast, the time females spent freezing was not different between separation groups on each observation day (Figure 5b).

The total number of rearing of males was higher in the CS and IS groups than in the SH group, like the case of all individuals pooled, whereas those of females were lower in the NS group than in the SH group (Table S6b, Table S7b; Separation group). In males and females, the total number of rearing was higher on PND50 than on PND21 (Table S6b, Table S7b; PND). The effects of separation group × PND on the total number of rearing of males and females had a similar pattern to the case of all individuals pooled. The differences between groups according to age were more distinct in males than in females. On PND21, the total number of rearing of males was not different between the groups (Figure 5c), whereas that of females was higher in the IS group than in the NS group (Figure 5d, multiple comparisons: IS > NS; estimate ± SE = -1.49 ± 0.40, z = -3.72, p =0.004). On PND50, the total number of rearing of both males and females was not different between the groups. On PND245, the total number of rearing in males was higher in the CS and IS groups than in the SH group (Figure 5c, multiple comparisons: CS > SH; estimate ± SE = -1.50 ± 0.34, z = -4.47, p < 0.001, IS > SH; estimate ± SE = -1.27 ± 0.33, z = -3.81, p = 0.003).

The total number of grooming in males tended to be higher on PND50 than on PND21 like the case of all individuals pooled, whereas that of females did not (Table S6c). From PND21 to PND 50, the total number of grooming of males increased in the SH group but decreased in the IS group, whereas such a tendency was not observed in females (Table S6c). Also, in males and females, there were no differences between groups on each observation day (Figure 5e, Figure 5f).

Short-term effects of ELS

Our results indicate that ELS does not affect bodyweight gain. The previous studies in O. degus reported that 1-hour of daily parental separation immediately affected the plasma levels of glucocorticoids, a key indicator of stress, during the first 3 weeks of life [23], whereas the effects of stress do not manifest in the bodyweight [31]. Uekita [29] also reported that ELS did not affect the bodyweight for 6 weeks postbirth ELS degus and no significant effects of ELS on mother-infant interactions, which indicates that no malnutrition occurred, or the infant did not experience neglect by the mother. Similar to these previous studies [29], [31], our results suggest that ELS does not cause malnutrition or result in neglect by their mother during the early development period.

Generally, the groups that experienced DELS were more active. On PND21, the time spent freezing tended to be shorter in the IS group than in the NS (IELS) group, and the total number of rearing behaviors was higher in the IS group than in the SH and NS (IELS) groups and tended to be higher in the CS group than in the NS (IELS) group. On PND50, there was no difference in the total number of rearing behaviors among all groups. This period is the most active in this study since the pups are fully weaned on PND35. Nevertheless, we can focus on the total time spent performing freezing behavior. On PND50, the DELS groups spent less time performing freezing behaviors than the NS (IELS) group, and the CS group tended to spend less time freezing than the SH group. From these results, we can assume that the DELS groups pups tend to be more active and move around constantly. The increase in activity observed in this study has also been observed in previous studies. Uekita [29] reported that when the not separated degus were put in the elevated zero maze test area, they stayed in the closed area for a while before starting to explore the other quadrants, whereas the ELS degus frequently moved from closed to open areas from the start of the trial. Bock [25] reported that repeated ELS induced locomotor hyperactivity. The present study confirmed that the DELS groups especially the CS group displayed hyperactivity. This finding suggests that DELS induces hyperactivity in animals; however, it is difficult to illustrate this hyperactivity in DELS groups when the activity of pups is high.

The number of grooming behaviors was not high overall; nevertheless, the number of grooming behaviors in the IS group tended to be higher than that in the SH group, and the number of grooming behaviors on PND50 tended to be higher than that on PND21. Bock [25] reported that the number of grooming behaviors during the standard OFT did not differ between ELS and control animals. However, in a modified OFT when degu vocalizations were presented from one corner of the arena, grooming behavior increased in DELS animals. Hence, these results indicate that self-grooming behaviors may reduce anxiety when exposed to stronger conflict situations.

Long-term effects of ELS

In the animal life cycle, the period after sexual maturation is longer than the early development period, and there are many events to preserve the next generation. Whether the effects of ELS manifest after sexual maturation or not should be examined. However, the reports on the long-term effects of ELS are lacking; thus, this research reflects a pioneering effect on the long-term effects of ELS.

The trend of gaining bodyweight from PND21 to PND245 in the SH group was higher than in the other groups. Here, we focus on the two points that the difference between the SH group and the DELS groups, and between the SH group and the NS group. There is a difference between the SH group and the DELS groups that whether the animals experienced the DELS or not, thus the ELS induced by separation could have affected the trend of gaining bodyweight in the DELS groups. Noteworthy, we observed differences in bodyweight gain between the SH group and the NS (IELS) group in which neither group experienced separation stress. In short, as expected, the NS (IELS) group may also be indirectly feeling the ELS. In this study, we suggest three possible stressors. First is communications with the DELS animals. The difference between the SH and NS (IELS) groups is whether the siblings who have been reared in the same cage experienced parental separation or not. The NS (IELS) group, which has been reared in the same cage with the siblings of the CS group and/or the IS (DELS) group, may have experienced stress through interactions with siblings who had experienced separation. Since degus are social animals, interaction with other individuals is an important process [22]. We did not separate pups chronically in this study, so the degus have social interactions with their siblings outside of the 1-hour social separation. However, it is not clear whether this interaction is comfortable for the animals. Endo [32] reported that in group-housed and socially isolated adult mice that were co-housed, the isolated mice group was slowest to exhibit huddling behavior with other individuals. Moreover, they reported that the number of times that isolated mice approached group-housed mice was greater than the reverse approach situation. Hence, we should continue to observe whether individuals who have experienced DELS could make appropriate relationships and interactions with cohabitant relatives or not. Second, the experiences of temporary losing siblings, and third, having heard the distressed vocalizations of the separated animals. Degus live in groups and communicate through vocalization [17], [18]. Thus, the NS (IELS) group might have been stressed by the group that experienced separation and by the distressed vocalizations emitted by these animals during the period when only the CS group was separated (PND4–7, 9–13, 15–20). If the NS (IELS) animals felt stressed by these, the NS (IELS) group may feel empathy for the stress of the DELS animals and the IS group could have been similarly stressed when only the CS group was separated. In future, a more focused study on the effects of NS (IELS) is needed and it may uncover new findings to better understand social interactions. Also, since the bodyweight of degus will continue to increase in captivity even after the period of this experiment, it will be necessary to continue to observe the animals until the weight gain reaches a plateau and to investigate whether this will affect the degree of development or whether the differences will only slow down and eventually disappear if enough time is given.

On PND245, the total number of rearing in the DELS animals tended to be higher than that in the SH group. Therefore, the DELS will induce the individual behavioral pattern to be hyperactive after becoming an adult. In terms of changes in behavioral trends over time, the CS group experiencing the greatest number of separations showed a distinctly different pattern compared with the behavioral changes in the SH groups. Conversely, the IS group showed fewer differences from the SH group, but their behavioral tendencies did not change much with time. These data suggest that DELS acts to converge an individual’s behavioral tendencies in a certain direction. The results of the NS (IELS) group were difficult to interpret although they seemed to have few differences from the SH group; however, we should more closely examine the differences between the NS (IELS) group which spent time with the DELS groups, and the SH group which did not. Rivera [26] reported that on the long-lasting effects of ELS. There were no differences among the groups at the age of 31 months and it is contrary to the results of the present study. This may be related to the different observation parameters and the older age of the animals. Alternatively, they reported that learning ability and memory were the lowest in the group that had been kept alone after separation, and these performances fully recovered at the age of 31 months when the rearing method of the single-reared animals was changed to group rearing after the behavioral test at the age of 25 months. The long-term effects on learning ability and memory are similar to the long-term effects on the behavioral parameters in this study.

Stress is a strong modulator of brain structure and function [33], and it is considered to affect the learning and memory process [34]. The short-term effects of ELS on individual behaviors and neurophysiology have been well researched [21], [29]. Similar results to those were observed in this study. The negative effects on the hippocampus activity of PND21 pups experiencing ELS were not observed [25]. Conversely, Rivera [26] reported that ELS animals reared in solitary conditions resulted in lower learning and shorter memory. The degus are known to spontaneously develop Alzheimer’s-like symptoms similar to those of humans [35]–[37] and it has been suggested that stress is associated with the pathogenesis and progression of Alzheimer’s disease [38], representing a unique tool to study age-associated morbidity [22]. The ELS may promote the development of age-related diseases, but these relations have not been argued for. Hence, new insights will be obtained by examining the learning and memory of the animals that experienced the ELS in this study.

Sex differences in the effects of ELS

Inter-group sex differences in bodyweight gain appeared after development and occurred faster in females than males after the first inter-group differences appeared, although the difference between the SH and other groups on PND245 was more obvious in males than in females. Similar to the changes in bodyweight gain, male pups showed a more pronounced effect of DELS on behavioral tendency than female pups, and this trend became clearer in observations after sexual maturation. Thus, it was generally suggested that the long-term effects of DELS were larger in males than in females. Additionally, ELS affects the development of catecholaminergic systems, and ELS-induced differences in tyrosine hydroxylase-immunoreactive fibers were more pronounced in male brains than in female brains [39]. In females, the time spent freezing in the NS (IELS) group was longer than in the SH group, and the total number of rearing in the NS (IELS) group was lower than in the SH group. This pattern was not observed in males. Since the difference between the NS (IELS) group and the SH group is the experience of separation from siblings, females from the NS (IELS) group might be more stressed indirectly than males when housed with siblings who have experienced separation. We predict that female degus are more sensitive to interactions with separated individuals because they nurse each other’s pups [19]. As such, the effects of stress may differ by sex. Nevertheless, since the sample size of females in the SH and NS (IELS) groups was small, more female samples are needed to unequivocally reveal the sex differences of the degu’s susceptibility to ELS.

Summary

We illustrated there were the "direct" effects and the "indirect" effects of ELS in the degus. DELS was correlated to higher hyperactivity later in life, especially in fully developed males, and the hyperactivity increased by more frequent separation. The SH group containing all siblings who did not experience parental separations gained more weight than the other groups. While the group in which the individual did not experience separation themselves, their siblings experienced parental separations (NS (IELS)), did not differ in bodyweight compared with individuals who experienced separation (DELS). Moreover, females tended to be lower activity than the SH group. These findings suggest that the “direct” effects of ELS were more obvious in males than in females, whereas the “indirect” effects of ELS were more obvious in females. Future studies focusing on the effects of IELS will provide insights on the underlying stressors or factors behind the development of anxiety or stress-related behaviors in social animals, which in turn will contribute to the animal welfare in domestic animals, livestock, zoo animals, and laboratory animals.

Animals and Housing Conditions

The degus were bred at the Environmental Control room 1 (room 1), Faculty of Agriculture, University of Miyazaki. Room 1 was maintained 22 ± 2℃, and on a 14:10 h light/dark cycle from January 26, 2017, to May 26, 2019, and a 12:12 h light/dark cycle from May 27, 2019, to November 07, 2019, with the light on starting at 08:00 JST (Japan Standard Time: UTC + 9). A female and a male were paired in a plastic cage (34.5 cm l × 40.3 cm w × 17.7 cm h) with wood chips for bedding, which is defined as a home cage. Commercial rodent diets (Labo MR Stock and Labo G Standard, Nosan Corporation, Kanagawa, Japan) and water were provided ad libitum. Each breeding couple was checked for a new litter daily, and the male was removed from the home cage after the presence of a litter was confirmed. Litter size was not standardized (three to seven pups per litter), and the degus were maintained as a family group composed of one mother and her pups before pup weaning on PND35. Their siblings were reared in the same cage until PND50, after which up to three siblings of the same sex were reared in the same cage. Four experimental groups (Fig. 2) were considered in this study: (i) the social housing (SH) group (a control group); a whole litter did not experience any separation treatment. Conversely, the litters were randomly assigned to three groups within the siblings to avoid the influence of bias in temperament and characteristics of each dam among the families and to the following treatments (for the number of animals and litters used at each age, see Table S1):

(ii) no separation (NS) group; the pups did not experience separation from their mother, but their siblings did, (iii) consecutive separation (CS) group; the pups were removed from their mother and home cage for 1-hour every day, (iv) intermittent separation (IS) group; the pups were removed from their mother and home cage for 1-hour on PND3, PND8, and PND14. To minimize the stresses from direct handling, the pups were identified by ear punching when they experienced the first handling treatment; the animals in the CS and IS groups were ear-punched after the separation on PND3, and the animals in the SH and NS groups were ear-punched after the OFT on PND21.

All procedures adhered to national and institutional guidelines and were approved by the Institutional Animal Experimentation Committee at the University of Miyazaki (Permission Nos. 2017-025-1 to 3 and 2020-012-1).

Separation Treatments and Open-field tests (Fig. 2)

Separation was defined as being when a subject was housed alone for 1-hour in a novel white opaque plastic rodent cage (34.5 cm l × 40.3 cm w × 17.7 cm h). On PND3, PND8, and PND14, the CS and IS pups were separated in the Environmental Control room 2 (room 2) maintained at 22 ± 2℃, and under light conditions. Thus, during separation, the separated pups could communicate with separated siblings each other using acoustic and olfactory cues; however, no visual or social contact (touching) could be made with separated siblings in room 2, and any contact could not be made with their siblings and parents in their home cages in room 1. On PND4–7, PND9–13, and PND15–20, the CS group pups were kept individually in a novel white opaque plastic cage for 1-hour daily in room 1. Thus, during separation, the pups had acoustic and olfactory, but no visual or social contact with their siblings and parents. After 1-hour separation, the pups were given back to their home cages and left undisturbed until the next separation. All groups were performed by the OFT using a novel cage (34.5 cm l × 40.3 cm w × 60.0 cm h) in room 2 for 1-hour on PND21 and PND50, and for 20 min on PND245. The behaviors of the animals were recorded with a digital video camera (Panasonic HX-A1H). All experimental procedures were carried out between 10:00 and 16:00 during the peak activity phase of this day-active species. All subjects were selected randomly at different times to control for any possible sequence effects of sex and treatment.

Bodyweight Measuring

Bodyweights, recorded to the nearest 0.1 g, were used as a general indicator of stress responsivity after the animals of CS and IS were separated on PND3, PND8, and PND14. All animals were separated and weighed on PND21, PND50, and PND245 for behavioral observations. No separation was performed on PND35 and PND64, but their weights were measured.

Observation of Behavior (PND21, PND50, and PND245)

In OFT, to observe the stress responses of animals when introduced into a novel environment, behavioral analysis was conducted during the first 2 min after the animal was introduced into a novel cage. The recorded videos of the animal behaviors were visually observed, and using the previously described method [40], the following three parameters that may indicate a stress response were recorded: 1) freezing: time the animals spent immobile ≥ 2 s; 2) rearing: number of instances where the animals stood on their hind legs, with or without contacting the sides of the box; and 3) grooming: number of self-grooming behaviors.

Statistical Analyses

All statistical analyses were performed using R version 4.1.0 (Core Team, 2021). Factors affecting freezing behavior and rearing behavior and bodyweight were analyzed with a generalized linear mixed model (GLMM) using the GLMM function of the lme4 package 1.1–27–1 [41].

In the analysis of bodyweight, separation treatments (SH, NS, CS, and IS), PND (from PND21–PND245), and the interaction between the separation treatments and PND were treated as fixed effects. In the analysis of behavior, the pooled data of all individuals was analyzed first, and each sex was then analyzed separately. In both cases, separation treatments (SH, NS, CS, and IS) and PND (PND21, PND50, and PND245), and the interaction between the separation treatments and PND were treated as fixed effects. In all statistical analyses, “individual” was regarded as a random effect to control for repeated observations of the same individuals.

The results of the analysis of the models were analyzed using the multiple comparison method with the least squares averaging to compare the values between the separated conditions at each age [42]. All pairwise comparisons were Bonferroni corrected. For all statistical tests, the level of significance was set at p < 0.05.

Acknowledgements

We wish to thank the editors for useful comments and Enago (www.enago.jp) for English language editing. This work is supported by JST SPRING, Grant Number JPMJSP210 and by JSPS KAKENHI, Grant Number JP20K15865.

Author contributions statement

R.U. conceived the project, contributed to experimental designs, performed experiments, interpreted the results, generated figures, and wrote the manuscript; S.H.S. conceived the project, contributed to experimental designs, interpreted the results, and wrote the manuscript. A.S., C.K., G.A.K., S.I., and Y.T. interpreted the results and wrote the manuscript. All authors approved the manuscript.

Competing interests

The authors declare no competing interests.

Spear, L. P. The adolescent brain and age-related behavioral manifestations. Neurosci. Biobehav. Rev. 24, 417–463 (2000). 10.1016/s0149-7634(00)00014 – 2, 10817843
Andersen, S. L. Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci. Biobehav. 27, 3–18 (2003). 10.1016/S0149-7634(03)00005–8
Lehmann, J. & Feldon, J. Long-term biobehavioral effects of maternal separation in the rat: consistent or confusing? Rev. Neurosci. 11, 383–408 (2000). 10.1515/revneuro.2000.11.4.383, 11065281
Sánchez, M. M., Ladd, C. O. & Plotsky, P. M. Early adverse experience as a developmental risk factor for later psychopathology: evidence from rodent and primate models. Dev. Psychopathol. 13, 419–449 (2001). 10.1017/s0954579401003029, 11523842
Gutman, D. A. & Nemeroff, C. B. Neurobiology of early life stress: rodent studies. Semin. Clin. Neuropsychiatry 7, 89–95 (2002). 10.1053/scnp.2002.31781, 11953932
McCormick, C. M., Kehoe, P. & Kovacs, S. Corticosterone release in response to repeated, short episodes of neonatal isolation: evidence of sensitization. Int. J. Dev. Neurosci. 16, 175–185 (1998). 10.1016/s0736-5748(98)00026 – 4, 9785114
Braun, K., Lange, E., Metzger, M. & Poeggel, G. Maternal separation followed by early social deprivation affects the development of monoaminergic fiber systems in the medial prefrontal cortex of Octodon degus. Neuroscience 95, 309–318 (1999). 10.1016/s0306-4522(99)00420-0, 10619487
Ziabreva, I., Poeggel, G., Schnabel, R. & Braun, K. Separation-induced receptor changes in the hippocampus and amygdala of Octodon degus: influence of maternal vocalizations. J. Neurosci. 23, 5329–5336 (2003). 10.1523/JNEUROSCI.23-12-05329.2003, 12832558
Bale, T. L., & Epperson, C. N. Sex differences and stress across the lifespan. Nat. Neurosci <background-color:#FFCC66;bu>18</background-color:#FFCC66;bu>, 1413–1420 (2015). 10.1038/nn.4112
Heck, A. L. & Handa, R. J. Sex differences in the hypothalamic–pituitary–adrenal axis’ response to stress: an important role for gonadal hormones. Neuropsychopharmacology 44, 45–58 (2019). 10.1038/s41386-018-0167-9
Oyola, M. G. & Handa R. J. Hypothalamic–pituitary–adrenal and hypothalamic–pituitary– gonadal axes: sex differences in regulation of stress responsivity. Stress 20, 476–494 (2017). 10.1080/10253890.2017.1369523
Sandi, C. & Haller, J. Stress and the social brain: behavioural effects and neurobiological mechanisms. Nat. Rev. Neurosci. 16, 290–304 (2015). 10.1038/nrn3918
Benner, S., Endo, T., Endo, N., Kakeyama, M. & Tohyama, C. Early deprivation induces competitive subordinance in C57BL/6 male mice. Physiol. Behav. 137, 42–52 (2014). 10.1016/j.physbeh.2014.06.018, 25089814
Okanoya, K., Tokimoto, N., Kumazawa, N., Hihara, S. & Iriki, A. Tool-use training in a species of rodent: the emergence of an optimal motor strategy and functional understanding. PLOS ONE 3, e1860 (2008). 10.1371/journal.pone.0001860, 18365015
Quirici, V., Faugeron, S., Hayes, L. D. & Ebensperger, L. A. Absence of kin structure in a population of the group-living rodent Octodon degus. Behav. Ecol. 22, 248–254 (2011). 10.1093/beheco/arq196
Ebensperger, L. A. et al. Ecological drivers of group living in two populations of the communally rearing rodent, Octodon degus. Behav. Ecol. Sociobiol. 66, 261–274 (2012). 10.1007/s00265-011-1274-3, 22344477
Ebensperger, L. A., Hurtado, M. J., Soto-Gamboa, M., Lacey, E. A. & Chang, A. T. Communal nesting and kinship in degus (Octodon degus). Naturwissenschaften. 91, 391–395 (2004). 10.1007/s00114-004-0545-5
Long, C. V. Vocalisations of the Degu Octodon degus, a social caviomorph rodent. Bioacoustics. 16, 223–244 (2007). 10.1080/09524622.2007.9753579
Ebensperger, L. A., Veloso, C. & Wallem, P. K. Do female degus communally nest and nurse their pups? J. Ethol. 20, 143–146 (2002). 10.1007/s10164-002-0063-x
Reynolds, T. J., & Wright, J. W. Early postnatal physical and behavioural development of degus (Octodon degus). Lab. Anim. 13, 93–99 (1979). 10.1258/002367779780943576
Colonnello, V., Iacobucci, P., Fuchs, T., Newberry, R. C. & Panksepp, J. Octodon degus. A useful animal model for social-affective neuroscience research: basic description of separation distress, social attachments and play. Neurosci. Biobehav. Rev. 35, 1854–1863 (2011). 10.1016/j.neubiorev.2011.03.014, 21477615
Cuenca-Bermejo, L. et al. Octodon degus: a natural model of multimorbidity for ageing research. Ageing Res. Rev. 64, 101204 (2020). 10.1016/j.arr.2020.101204
Gruss, M., Westphal, S., Luley, C. & Braun, K. Endocrine and behavioural plasticity in response to juvenile stress in the semi-precocial rodent Octodon degus. Psychoneuroendocrinology 31, 361–372 (2006). 10.1016/j.psyneuen.2005.08.017, 16274934
Bock, J., Riedel, A. & Braun, K. Differential changes of metabolic brain activity and interregional functional coupling in prefronto-limbic pathways during different stress conditions: functional imaging in freely behaving rodent pups. Front. Cell. Neurosci. 6, 19 (2012). 10.3389/fncel.2012.00019, 22590453
Bock, J., Breuer, S., Poeggel, G. & Braun, K. Early life stress induces attention-deficit hyperactivity disorder (ADHD)-like behavioral and brain metabolic dysfunctions: functional imaging of methylphenidate treatment in a novel rodent model. Brain Struct. Funct. 222, 765–780 (2017). 10.1007/s00429-016-1244-7, 27306789
Rivera, D. S. et al. Effects of long-lasting social isolation and re-socialization on cognitive performance and brain activity: a longitudinal study in Octodon degus. Sci. Rep. 10, 18315 (2020). 10.1038/s41598-020-75026-4, 33110163
Colonnello, V., Iacobucci, P. & Panksepp, J. Analysis of the disruption of maternal social bonds in Octodon degus: separation distress in restricted reunion tests. Dev. Psychobiol. 53, 657–669 (2011). 10.1002/dev.20539
Uekita, T. & Kawakami, S. The effects of pre-weaning social isolation and mother’s presence on the object exploration behavior of infant Octodon degus. Psychologia. 59, 136–147 (2016). 10.2117/psysoc.2016.136
Uekita, T., Ishibashi, A. & Sakamoto, T. Influence of pre-weaning social isolation on post-weaning emotion tendency and mother–infant interactions in infant Octodon degus. Int. J. Environ. Res. Public Health 16, 31126012 (2019). 10.3390/ijerph16101824, 31126012
Malcangi, S. et al. Post-natal maternal stress decreases locomotor play behaviors in Octodon degus pups. J. Ethol. 38, 207–213 (2020). 10.1007/s10164-020-00642-6
Becker, K., Abraham, A., Kindler, J., Helmeke, C. & Braun, K. Exposure to neonatal separation stress alters exploratory behavior and corticotropin releasing factor expression in neurons in the amygdala and hippocampus. Dev. Neurobiol. 67, 617–629 (2007). 10.1002/dneu.20372, 17443812
Endo, N. et al. Multiple animal positioning system shows that socially-reared mice influence the social proximity of isolation-reared cagemates. Commun. Biol. 1, 225 (2018). 10.1038/s42003-018-0213-5, 30564746
Sandi, C. & Haller, J. Stress and the social brain: behavioural effects and neurobiological mechanisms. Nat. Rev. Neurosci. 16, 290–304 (2015). 10.1038/nrn3918, 25891510
Schwabe, L., Joëls, M., Roozendaal, B., Wolf, O. T. & Oitzl, M. S. Stress effects on memory: an update and integration. Neurosci. Biobehav. Rev. 36, 1740–1749 (2012). 10.1016/j.neubiorev.2011.07.002, 21771612
Inestrosa, N. C. et al. Human-like rodent amyloid-β-peptide determines Alzheimer pathology in aged wild-type Octodon degu. Neurobiol. Aging 26, 1023–1028 (2005). 10.1016/j.neurobiolaging.2004.09.016, 15748782
Steffen, J. et al. Revisiting rodent models: Octodon degus as Alzheimer’s disease model? Acta Neuropathol. Commun. 4, 91 (2016). 10.1186/s40478-016-0363-y, 27566602
van Groen, T. et al. Age-related brain pathology in Octodon degu: blood vessel, white matter and Alzheimer-like pathology. Neurobiol. Aging 32, 1651–1661 (2011). 10.1016/j.neurobiolaging.2009.10.008, 19910078
Sotiropoulos, I. et al. Stress and glucocorticoid footprints in the brain-the path from depression to Alzheimer’s disease. Neurosci. Biobehav. Rev. 32, 1161–1173 (2008). 10.1016/j.neubiorev.2008.05.007, 18573532
Kunzler, J., Braun, K. & Bock, J. Early life stress and sex-specific sensitivity of the catecholaminergic systems in prefrontal and limbic regions of Octodon degus. Brain Struct. Funct. 220, 861–868 (2015). 10.1007/s00429-013-0688-2, 24343570
Popović, N. et al. Aging and time-of-day effects on anxiety in female Octodon degus. Behav. Brain Res. 200, 117–121 (2009). 10.1016/j.bbr.2009.01.001, 19162080
Bates, D., Maechler, M., Bolker, B. & Walker, S. Fitting linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015)
Lenth, R. V. (2021). Emmeans: Estimated Marginal Means, Aka Least-Squares Means. R Package Version 1.6. 2–1

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Long-term behavioral effects of social separation during early life in a social mammal, Octodon degus

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Discussion

Short-term effects of ELS

Long-term effects of ELS

Sex differences in the effects of ELS

Summary

Methods

Animals and Housing Conditions

Separation Treatments and Open-field tests (Fig. 2)

Bodyweight Measuring

Observation of Behavior (PND21, PND50, and PND245)

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