Traumatic events, occurring early in life, are known to enhance the vulnerability to develop brain diseases in adulthood (Sarkar et al., 2019; Hegde and Mitra, 2020; Palma-Gudiel et al., 2020). To evaluate the vast array of long-lasting alterations after the exposure to various stress paradigms in the early post-natal life, numerous animal models have been developed and validated over the years (Murthy and Gould, 2018). Several changes induced by early-life stress have been described, including impairments in memory formation and cognitive performances (Reincke and Hanganu-Opatz, 2017; Talani et al., 2023), enhanced anxiety level (Brunton, 2015), depression-like symptoms (Vetulani, 2013) and vulnerability to develop drug use disorders (Delavari et al., 2016; de Almeida Magalhães et al., 2017).

In rodents, the neonatal maternal separation is one of the most commonly used models of early-life stress exposure to study the consequent alterations at the endocrine, neurophysiological, and behavioral level (Nishi et al., 2014; Mejía-Chávez et al., 2021). In this model, pups are removed from the dam’s nest for a few hours each day during the first two weeks of postnatal life, although protocols may differ in terms of length of separation or time window in which they are applied (Plotsky and Meaney, 1993; Hall, 1998; Fabricius et al., 2008; Cirulli et al., 2009; Marco et al., 2009; Bailoo et al., 2014). Interestingly, impairments are typically observed in male but not female rodents, which highlights a sex-dependent effect induced by this environmental manipulation (Romeo et al., 2003; Mehta and Schmauss, 2011; Kundakovic et al., 2013; Bailoo et al., 2014; Talani et al., 2023). However, other studies report opposite results (Veenema et al., 2007; Tsuda and Ogawa, 2012; Cui et al., 2020), thereby suggesting that the influence of sex as a factor for vulnerability to the effects of maternal separation needs further investigation and clarification. Age is another factor that may significantly influence the effect of early-life social experiences, as both social isolation and enrichment during the first three postnatal weeks may affect behavior differently in adolescent and adult rats (Macrì et al., 2010; Daoura et al., 2011).

Pre-weaning social isolation provides a further experimental animal model of early-life stress. This model is much less characterized and implies a shorter time window and duration of pups’ separation. Such a pre-weaning social stress significantly enhances locomotor activity in a novel environment in male and female adolescent rats and increases the response to low-to-moderate doses of methamphetamine (Pritchard et al., 2012), suggesting that subjects with a history of early-life social stress may be particularly vulnerable to the effects of psychostimulants. Notably, this latter effect occurred at different doses of methamphetamine for male (3.0 mg/kg) and female (1.0 mg/kg) rats, revealing a sex-dependent effect of early social isolation (ESI) on vulnerability to psychostimulants effects (Pritchard et al., 2012). Other studies demonstrated that early-life social isolation resulted in impulsive behavior (as indexed by faster movements) in adolescent rats and that the effect was more pronounced in males than females, suggesting that adolescent female rats are more resistant to the effects of early social stress on motor impulsive behavior (Pritchard et al., 2012). Yet, early social isolation may also lead to hypoactive behaviors in rats along with greater propensity to show depressive-like responses to stress (Spivey et al., 2009).

In rodents, the behavioral endophenotype most altered by peri-weaning social isolation is the prepulse inhibition (PPI) of the startle reflex (Wilkinson et al., 1994; Varty and Higgins, 1995; Frau et al., 2015; Sun et al., 2021). PPI refers to the inhibition of the startle response by presentation of a weak intensity prestimulus or prepulse (acoustic or tactile) that immediately precedes the pulse (startle) stimulus (Swerdlow et al., 1995). This paradigm provides a well-established operational measure of sensorimotor gating, the process that the brain enacts to attribute the salient aspects of sensory information from the environmental stimuli. As such, this protective mechanism gates out irrelevant and/or redundant sensory stimulation, thereby preventing information overload and preserving cognitive integration with its related neurobehavioral outcomes.

Another behavior affected by neonatal maternal separation is burying behavior, defined as a physiologic process, in laboratory rodents, that consists in burying harmless objects (e.g., glass marbles) using bedding material. When burying behavior become excessive, however, it is considered a valid prediction of perseveration/compulsion related disorders (Taylor et al., 2017). Recent studies have shown that mice exposed to maternal separation showed a significant increase in the number of buried marbles as clear increase of perseverative behavior, providing further empirical support for a link between childhood adversity and development of impulsive/compulsive traits (Ou et al., 2021; Jarrar et al., 2022). Interestingly, sex-dependent differences have been reported in the marble burying test, although with discrepant results (Burke et al., 2016; Mancini et al., 2021; Emtyazi et al., 2022).

If early-life social deprivation has been associated to enhanced risk of altered behavior and vulnerability to mental diseases, a potential positive impact of early social enrichment on behavior and brain development has also been described (Cirulli et al., 2010). An ethological paradigm to create a pre-weaning complex social environment is represented by communal nesting (CN), i.e., the sharing of caregiving behaviors among a group of dams in a single nest (Branchi, 2009). CN mirrors the developing pups’ natural rearing setting, which is typically much richer than the standard laboratory conditions (Branchi et al., 2006). CN has been reported to affect anxiety and depressive-like behaviors in mice (Branchi and Alleva, 2006), stimulate social interaction (D’Andrea et al., 2007), and increase resiliency to social stress in male mice (Branchi et al., 2013). Whether CN condition can attenuate, if not fully prevent, the behavioral alterations induced by an early-life stress in adolescent and/or adult rats remains to be investigated.

Here, we explored, for the first time, the effects of CN on an early social isolation (ESI) protocol, milder than the classical maternal separation paradigm, as it consisted in depriving pups of any social contact from postnatal day (PND) 14 to PND21 for 30 min/day only. Specifically, this study was undertaken to assess whether during adolescence and/or adulthood (i) ESI alters motor activity, obsessive-compulsive behavior and sensorimotor gating in rats, (ii) CN per sé is able to affect animals’ behavioral performances, (iii) CN can reverse the potential negative impact of ESI on motor activity, obsessive-compulsive behavior and sensorimotor gating. Moreover, in light of the sexual dimorphism in the “emotional brain” circuits (Fone and Porkess, 2008; Pallayova et al., 2019) and the sex-dependent differences in the neural response to social behaviors (Zilkha et al., 2021) and stress (Oyola and Handa, 2017), this study also evaluated (iv) whether early-life social rearing conditions affect behaviors of adolescents and adults in a sex-dependent manner. The Wistar rat strain was selected in light of its use in previous studies investigating either the effects of early-life social isolation on behavior and potential nonpharmacological interventions to mitigate its behavioral effects (Daoura et al., 2010; Lundberg et al., 2016; Khalifeh et al., 2019; Mohammadian et al., 2019), including the CN paradigm (Uriarte et al., 2014).

Early-life experiences, including parental care and social interactions, have profound impact on brain development and subsequent behavioral expression. In rodents, pre-weaning social isolation has been associated with behavioral alterations in the offspring that manifest during adolescence and may persist into adulthood (Hall, 1998). This study tested the hypothesis that an early-life socially enriched environment, such as that provided by the communal nesting condition, may attenuate (if not fully prevent) the behavioral effects induced by pre-weaning social isolation in male and female rats. Results revealed that early-life social stress (i.e., pre-weaning isolation) induces a significant deficit in sensorimotor gating in both adolescent males and females and in adult males as well as compulsive burying behavior in adolescent male rats, while no effects were observed on spontaneous motor behavior. Importantly, a protective effect of communal nesting was observed on both sensorimotor gating deficit and compulsive burying behavior, with significant differences between sexes (Figure 8).

Although early-life isolation did not alter the animals’ spontaneous locomotor activity, we observed a significant effect of sex in two out of three parameters investigated during adolescence and adulthood. With respect to males, female Wistar rats spent more time in the center zone of the open field and travelled a greater distance in the arena (Knight et al., 2021), suggestive of decreased anxiety level and increased motor activity, respectively. Further, when compared to corresponding SH groups, a positive (but not significant) trend can be observed in the time spent in the center of the arena by adolescent CN females, suggesting that a pre-weaning social enrichment condition may decrease the level of anxiety. These findings are in line with previous studies showing that when reared in CN conditions, NIH Norway rats were more likely to enter and spend more time in the center of the open field (Martinez et al., 2015), and that Balb/c mice displayed reduced anxiety-like behavior in the same test (Curley et al., 2009). However, other studies suggested the opposite, with CN reducing in Sprague–Dawley rats the time spent in the open arms in the elevated plus maze and the latency to escape from the light side of the chamber in the light/dark test (Connors et al., 2015), and increasing thigmotaxis in the open field in CD1 Swiss-derived (ICR) mice (Branchi and Alleva, 2006). Such discrepancies highlight the need for further characterization of the effect of pre-weaning social enrichment on emotional behavior, with particular attention given to potential underlying sex- and strain-dependent differences.

Deficits in sensorimotor gating are a transdiagnostic marker of several psychiatric disorders, including schizophrenia (Braff and Geyer, 1990), Tourette syndrome (Castellanos et al., 1996), and bipolar disorder (Perry et al., 2001). PPI deficits are typically modeled in rats by individual housing from weaning until adulthood (Roncada et al., 2009), and these deficits can be reversed by the administration of antipsychotic drugs (Varty and Higgins, 1995). Importantly, gentle handling and environmental enrichment reverse PPI deficits in Sprague–Dawley rats (Varty et al., 2000; Krebs-Thomson et al., 2001), as well as other behavioral and endocrine responses related to early-life stress and psychiatric conditions in Lister Hooded rats (Schrijver et al., 2002), suggesting that social enrichment might also reduce PPI deficits induced by early-life social isolation. The impact of social isolation in the early phase of rearing on sensorimotor gating function has been seldom investigated. Isolation protocols have been typically designed to deprive rodents of social contacts soon after weaning, with male and female Sprague–Dawley rats (Weiss et al., 2001), male Lister Hooded rats (Geyer et al., 1993) and male 129 T2 and C57BL/6 J (Varty et al., 2006) socially isolated immediately after weaning (PND 21–23) and tested on PPI after about 8 weeks (PND 77–80), corresponding to yong-adult age. However, the effect of this environmental manipulation on PPI performance has yielded contrasting findings, depending on several methodological factors, such as timing of rearing, strain, caging condition, protocols, and number of PPI sessions (Geyer et al., 1993; Wilkinson et al., 1994; Domeney and Feldon, 1998). To our knowledge, only one study applied a pre-weaning social isolation paradigm to evaluate its consequences on PPI in male and female Sprague–Dawley rats (Weiss et al., 2001), but in this study the social isolation continued after weaning effectively preventing the assessment of the pre-weaning social isolation per sé on PPI. Interestingly, the developmental timing of the onset of social isolation appears to be a key factor in shaping PPI, as it has been observed that male Sprague–Dawley rats deprived of social contacts especially at adulthood do not exhibit PPI disruption (Varty et al., 1999). Here, we focused on isolation rearing only during the pre-weaning period and found that this protocol elicited a robust PPI deficit which was evident during both adolescence and adulthood in males but only during adolescence in females, suggesting a sex-dependent developmental origin of this environmental model with regard to psychiatric disorders featuring sensory gating impairments (Geyer, 2006). Notably, this study shows for the first time that the PPI deficits induced by early-life social stress were fully prevented by the CN condition in both adolescent and adult males but not in the adolescent females. CN is a naturalistic form of social environmental enrichment that begins from birth until weaning, providing pups with enhanced social and physical, and sensorimotor stimulation. As such, the observed rescue effects of CN in our study might align with the beneficial effects of repeated handling in preventing deficits in prepulse inhibition caused by isolation rearing in Sprague–Dawley and Wistar rats (Krebs-Thomson et al., 2001; Rosa et al., 2005). This suggests that, like repeated handling, CN can also have beneficial effects in mitigating the negative consequences of isolation rearing on sensorimotor integrity during development (Bakshi and Geyer, 1999).

Digging (i.e., the displacement of a substrate using mostly the forepaws), burrowing (i.e., the construction of tunnels for habitation), and burying (i.e., the displacement of either aversive or non-aversive objects underneath any available substrate) are core components of the normal behavioral repertoire of rodents (de Brouwer et al., 2019). The marble burying test is commonly used in preclinical research for the identification of anxiety- or compulsive-like features (Albelda and Joel, 2012). Maternal conditions occurring before and during pregnancy have been linked to the risk of developing obsessive-compulsive disorder (OCD) (Arnold et al., 2018; Mahjani et al., 2020), supporting the evidence that patients with anxiety, OCD and other mental disorders present a history of childhood trauma more often than the healthy population. Further, maternal separation has been reported to increase compulsive burying activity in adult male BALB/c mice, without affecting locomotor activity (Jarrar et al., 2022), and in adolescent but not adult Sprague–Dawley rats, with larger effects in males than females (Abraham et al., 2023). Notably, individual housing soon after weaning did not alter burying activity in young adult Long Evans rats, with isolated males burying a slightly (not significant) higher number of marbles than isolated females (Kinley et al., 2021). This study examined for the first time the effect of the exposure to pre-weaning social stress on burying behavior in rats reared in standard or communal nesting condition, revealing a significant effect of both ESI and housing condition during adolescence but not adulthood. Specifically, while ESI significantly increased burying activity in SH adolescent males, CN fully reverted this effect, without showing an intrinsic effect per. Again, a sex-dependent effect of ESI was detected also in this test, with SH-ESI (but not CN-ESI) adolescent females burying a significantly lower number of marbles than corresponding male group.

Due to the numerous variables involved in this study, i.e., housing condition (SH vs. CN), early-life stress (CTRL vs. ESI), sex (M vs. F) and age (adolescence vs. adulthood), to reduce complexity in the analysis of data we evaluated the effect of the first 3 variables separately in adolescent and adult animals. Yet, a simple comparison of adolescent vs. adult animals in each test shows that (i) the effect of sex in the locomotor activity test observed during adolescence is long-lasting, since it is still evident during adulthood, (ii) the CN condition protects against ESI-induced PPI deficits in males at both ages, (iii) the effect of ESI, CN and sex on burying behavior during adolescence does not persist into adulthood.

Overall, our findings show for the first time that early social experiences and housing condition interact in modulating motor, cognitive and emotional functions in a sex- and age-dependent manner. Further studies are needed to assess the potential benefits of a socially enriched rearing environment in males and females. Questions remain regarding whether being exposed to a socially stimulating environment from birth may effectively protect, or at least attenuate, against other behavioral alterations induced by social isolation early in life.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Organismo preposto al benessere degli animali (OPBA), Cagliari. The study was conducted in accordance with the local legislation and institutional requirements.

JB: Data curation, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. MC: Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. AP: Data curation, Investigation, Methodology, Resources, Visualization, Writing – original draft. GT: Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. RF: Data curation, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing. PP: Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing. MD: Supervision, Validation, Writing – review & editing, Conceptualization, Data curation. FF: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. PR: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. LR: Data curation, Formal analysis, Software, Visualization, Writing – review & editing. VT: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. RC: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. FS: Formal analysis, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. LF: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.