Factors Modulating the Effects of Juvenile Social Isolation on Adult Social Behavior in Rodents

Abstract

For decades, rodent social isolation models have been used to explore how social experience influences brain maturation and adult behavioral outcomes, but studies employing these models have produced seemingly inconsistent experimental results. Here, we provide a comprehensive overview of the effects of social isolation during development on adult social behavior, highlighting how differences in experimental paradigms (including the duration and timing of isolation, the sex of subjects, and whether subjects are returned to social housing prior to testing) can help explain disparate experimental results across the four most-studied types of social behaviors in rodent social isolation research (social preference, social investigation, agonistic behavior, and social recognition). Our analysis shows that experimental results are not as inconsistent as they may seem at first glance and that understanding the nuances of rodent social isolation paradigms is necessary for the field to be able to leverage these models to discover the neural mechanisms underlying experience-dependent shaping of social behavior.

1 |. Introduction: Rodent Juvenile Social Isolation Revisited

Social experiences during development shape human brain maturation and have a profound influence on social functioning and neuropsychiatric health in adulthood (Atzil et al. 2018; Champagne and Curley 2005; Cushing and Kramer 2005; Howe 2005; McCrory et al. 2011; Sachser et al. 2013). Deficits in social functioning are also associated with a wide variety of neuropsychiatric disorders and are especially pronounced in neurodevelopmental disorders such as autism and schizophrenia (Bicks et al. 2015; Chevallier et al. 2012; Couture et al. 2006; Segrin 2000). Human clinical and neuroimaging studies have identified important links between early social experience and adult outcomes (Chiang et al. 2015; Cushing and Kramer 2005; Sachser et al. 2013; Yehuda et al. 2010), but the specific neural mechanisms by which social experience affects social functioning are poorly understood. Understanding the consequences of social isolation on a mechanistic level is more important than ever, as loneliness is on the rise around the world (Buecker et al. 2021; Twenge et al. 2021), especially in the wake of the COVID-19 pandemic (Lampraki et al. 2022; C. M. Lee et al. 2020), and loneliness is strongly associated with adverse health outcomes (C. Park et al. 2020). The investigation of the effects of social deprivation on brain and behavior has been ongoing since the 1930s (Bayroff 1936; Duffy and Hendricks 1973; Einon and Morgan 1977; Hatch et al. 1965; King and Gurney 1954; King et al. 1955; Valzelli 1973), and the impacts of social isolation have been demonstrated across a broad range of behavioral abilities, such as locomotor activity, anxiety, learning and memory, attention-related behavior, addiction-related behavior, and social behavior (Arakawa 2018; Walker et al. 2019). Among these, cross-species studies collectively show that social isolation during development is associated with a variety of social behavior abnormalities in adulthood (Freedman et al. 1961; Harlow and Novak 1973; King and Gurney 1954). However, our mechanistic understanding remained historically limited due to the complexity of social experiences and technical challenges, such as the difficulty of experimentally manipulating specific neural circuits or other factors. In recent years, there has been rapid advancement in technologies that enable causal experiments on the effects of social isolation on specific circuits and behavior, thus promoting deeper understanding, particularly regarding its impact on social processing. This review aims to revisit the growing body of findings on the influence of social isolation on social behavior outcomes, as demonstrated from the early stages of research, and to provide a comprehensive overview of them to facilitate future mechanistic studies.

In rodent models of social isolation, mice or rats are singly housed for a number of weeks during development and are typically compared to mice that are group-housed (GH) throughout their lifespan. A great body of work in this area employs a specific isolation paradigm that we refer to here as “chronic social isolation” (CSI) but is also commonly referred to as post-weaning social isolation or isolation rearing. In this paradigm, rodents are usually housed in isolation upon weaning at postnatal day (p) 21, which is near the onset of adolescence and is considered the start of the pre-pubertal “juvenile”adolescent period (Brust et al. 2015). CSI rodents then remain housed in isolation throughout later development into adulthood. In addition to the CSI paradigm, several studies have employed isolation paradigms where subjects are isolated for a number of weeks during development (usually 2–3 weeks, starting on p21) and then returned to social housing prior to adult data collection. Herein, we refer to this model of temporary social isolation during development as “juvenile social isolation” (JSI). JSI subjects are typically rehoused with other JSI conspecifics, but JSI subjects were rehoused with GH conspecifics in a handful of studies.

Despite the general findings on the influence of prolonged social isolation during development on social behavior in rodents, literature also reveals several inconsistencies. This is partly because previous reviews, while presenting high-level summaries that social isolation during development disrupts adult social behavior, often lack a critical analysis of how differences in experimental paradigms and the type of social behavior in question can affect experimental outcomes. Here, we discuss isolation-induced changes in specific adult social behaviors following the structure outlined in Figure 1. Adult social behavior outcomes induced by social isolation are influenced by various experimental factors. These experimental factors include isolation parameters (e.g., timing and duration), behavior assay permutations (e.g., the age and sex of conspecifics used as social stimuli), and subject identity (e.g., the age and sex of subjects), all of which vary across studies. The outcome factors include social preference, social investigation, agonistic behavior, and social recognition. We provide a systematic evaluation of the influence of experimental factors on social behavior outcomes. We close by suggesting priorities for future research to resolve discrepant findings and allow the field to move toward a fuller understanding of the neurodevelopmental effects of social isolation and the brain mechanisms underlying social behavior. The effects of social isolation during development on nonsocial behaviors have been reviewed in detail elsewhere (Arakawa 2018; Burke et al. 2017; Fone and Porkess 2008; D. C. Li et al. 2021; Noschang et al. 2021; Walker et al. 2019), but it should be noted that social behavior deficits resulting from developmental social isolation are highly likely to be influenced by changes in cognition that are not strictly related to social functioning. As mentioned above, anxiety-like behaviors, cognitive and decision-making functions, learning and memory, and reward-associated behaviors are also altered by social isolation during development, all of which are necessary for normal social interaction. This suggests that the social behavioral changes observed after developmental social isolation may reflect a broader disruption in cognitive integration and behavioral flexibility.

FIGURE 1 |.

Experimental factors and social behavior outcomes associated with social isolation studies. Schematic showing the complexity of the various experimental factors that can influence results in social behavior studies and the most commonly studied social behavior constructs and assays.

2 |. Factors Modulating the Effects of Social Isolation on Adult Social Preference and Investigation

The three-chamber social preference test, or reciprocal interaction paradigm (see Glossary) with a novel conspecific used as a social stimulus is frequently used to assess social preference and social investigation, respectively (Brodkin et al. 2004; Moy et al. 2004; Pasciuto et al. 2015). The reciprocal interaction paradigm’s strength lies in its ability to mimic ethologically natural interactions through free interaction between two mice. However, standardized assessment of contributing factors is challenging due to its complexity (Pasciuto et al. 2015). In contrast, the three-chamber social preference test confines an unfamiliar stimulus mouse to a restricted area, enabling the evaluation of a testing mouse’s non-agonistic (affiliative) social approach tendencies as well as comparison to non-social approach tendencies. Preference to engage in a social approach over a non-social approach is often referred to as sociability (Rein et al. 2020). This controlled setup permits visual, tactile, auditory, and olfactory interaction while minimizing potential confounds from aggressive interactions (Moy et al. 2004). Thus, the assay provides a standardized method to assess non-agonistic (affiliative) social approach/avoidance behavior. Its strength lies in assessing social preference deficits, particularly the tendency to avoid unfamiliar social partners. Indeed, it serves as a fundamental tool with broad applications and strong face validity for assessing social deficits, including characterizing social deficits in transgenic and environmental mouse models of autism, studying the development of social impairments, and evaluating the effects of pharmacological treatments and other interventions on social deficits (Jabarin et al. 2022; Moy et al. 2004; Pasciuto et al. 2015). Therefore, this review paper will summarize the findings derived from the three-chamber social preference test to discuss social preference deficits. However, it should be noted that this assay alone cannot capture the full complexity of social behavior or its deficits, due to limitations such as the limited number of monitored variables and a lack of assessment for potential confounding factors or underlying affective states. To address these shortcomings, additional independent control experiments and multidimen-sional approaches that target different aspects and contexts of social behavior are necessary. Other critical factors that must be carefully considered when interpreting the results of each assay include the duration of behavioral assessments, the magnitude of effect sizes, and the reproducibility of findings across experiments. For instance, the duration of observation in reciprocal interaction tests can vary considerably—from just a few minutes to > 15 min. Although this review does not explore this issue in detail, it is important to carefully evaluate how such methodological differences may impact the interpretation of results. In this section, we discuss the effects of JSI and CSI on social behavior with a focus on how different experimental factors can influence social preference and investigation outcomes (Figure 2; Table S1). While our analysis did not uncover apparent major differences between mice and rats related to the effects of social isolation, future studies are warranted to fully dissect possible differences in how these species differentially respond to experimental variables associated with social isolation experiments given the differences in their social motivation (Netser et al. 2020) and play behavior (Pellis et al. 2023).

FIGURE 2 |.

Streamlined summary of the effects of JSI and CSI on social behavior in male subjects relative to GH controls. Results presented are for JSI models where subjects are isolated from p21 to 35 and rehoused with other JSI subjects and for CSI models where subjects are isolated starting from p19 to 23 without rehousing. Experiments where subjects are tested before p56 or where opposite-sex conspecifics are used as social stimuli are not considered in this table. JSI typically reduces adult social preference in the three-chamber social preference tests (Komori et al. 2024; Makinodan et al. 2012; Bicks et al. 2020; Yamamuro et al. 2020; Makinodan et al. 2017; but see Park et al. 2021) and reduces adult social investigation in free reciprocal interaction assays (Hol et al. 1999; Bicks et al. 2020; van den Berg et al. 1999). On the other hand, the effects of CSI on social preference and investigation are inconsistent and depend on the specific behavioral assay that is used. While CSI often reduced social preference in three-chamber test (Zheng et al. 2023; Makinodan et al. 2012; Kuniishi et al. 2022; Huang et al. 2021; Makinodan et al. 2017; Lopez et al. 2024, but see Kim et al. 2021; Manojlović et al. 2024 reporting increased sociability), CSI often increases social investigation in free reciprocal interaction assays (Wongwitdecha and Marsden 1996; Ferdman et al. 2007; Han et al. 2011; but see Huang et al. 2021). In the resident intruder test, several studies show that CSI increases agonistic behavior (Tóth et al. 2008; Chang et al. 2020; Biro et al. 2023; Biro et al. 2017; Oliveira et al. 2019; Toth et al. 2011; Tan et al. 2021; Toth et al. 2012), but there are no studies using the p21–35 JSI model. In social recognition paradigms, a single study suggests that JSI does not affect social recognition impairment (Park et al. 2021), while there is limited evidence that CSI disrupts social novelty preference or recognition (Huang et al. 2021; Oliveira et al. 2019).

2.1 |. Timing of Social Isolation and Evidence for a Developmental Sensitive Period

Rodent isolation studies generally support the hypothesis that adult sociability depends on juvenile social experience. However, the specific effects of isolation during development on adult social preference and investigation are nuanced and sensitive to variations in the duration and timing of social isolation, the behavioral assays used to measure social preference and investigation, the identity of conspecifics used as social stimuli, and the sex of experimental subjects.

P21–35 in rodents spans early adolescence to mid-adolescence. From p21 onward, later developmental events, such as synapse production and elimination, and myelination, are still in progress (Chini and Hanganu-Opatz 2021). In particular, the prefrontal cortex (PFC), which is heavily implicated in social behavior, develops in a protracted manner and is the last brain region to fully mature (Chini and Hanganu-Opatz 2021; Kolk and Rakic 2022). Synaptic density peaks between p21 and 28 in the PFC of rodents and declines through the remainder of adolescence, and PFC myelination reaches its peak rate around p21 (Chini and Hanganu-Opatz 2021; Semple et al. 2013), supporting the hypothesis that JSI impairs social behavior by disrupting this circuit maturation within the PFC during development (Bicks et al. 2020; Makinodan et al. 2012; G. Park et al. 2021; Yamamuro et al. 2020).

In male mice and rats, both CSI from p21 and JSI from p21 to 35 typically reduce adult social preference in the three-chamber social preference test (Bicks et al. 2020; Huang et al. 2021; Komori et al. 2024; Kuniishi et al. 2022; Lopez et al. 2024; Makinodan et al. 2017; Makinodan et al. 2012; Yamamuro et al. 2020; Zheng et al. 2023); but see (G. Park et al. 2021), while CSI starting from p28 or later (Makinodan et al. 2012; Zhang et al. 2021), or 2 weeks of isolation in adulthood followed by rehousing (Bicks et al. 2020) fails to significantly reduce three-chamber test social preference. These findings support the hypothesis that there is a sensitive period during development where social isolation has a more profound impact on adult social preference (Makinodan et al. 2012). Importantly, male GH mice that meet with new cagemates on p35 do not display social preference deficits in the three-chamber test in adulthood (Bicks et al. 2020), suggesting that the observed social preference deficits in p21–35 male JSI mice cannot be explained by meeting with new cagemates on p35. However, one study found that p21–35 male JSI mice that were rehoused on p35 with GH mice rather than other JSI mice had normal levels of social preference in the three-chamber test (Makinodan et al. 2017), and a separate study showed modest evidence that p22–29 male JSI rats rehoused with GH rats had milder social investigation deficits than standard JSI rats (Hol et al. 1999). These findings suggest that, although isolation from p35 or later may fail to induce deficits in social preference and investigation, the post-p35 social environment is still important for determining adult social preference and investigation, and there may be some level of social circuit plasticity remaining (Leventhal and Morishita 2024).

Contrary to this hypothesis, a recent study reports that 2 weeks of adult social isolation without rehousing is sufficient to reduce social preference in the three-chamber test with a juvenile stimulus mouse and that this deficit is not rescued by rehousing subjects with another mouse that is separated by a perforated barrier (Guo et al. 2024). It remains unclear whether the social preference deficits observed in rehoused mice in this study are caused by an acute lack of social touch or if they are resistant to full social rehousing. Notably, there are discrepancies in the reported effects of social isolation during adulthood. One study found that 2 weeks of adult social isolation without rehousing in male mice decreased the amount of time spent in the social chamber in the three-chamber test but also reduced the latency to enter the social chamber (Zelikowsky et al. 2018). In contrast, an early study in male NIH Swiss mice systematically tested various durations of adult isolation (0, 1, 2, 5, 10, or 20 days) and found that increasing duration of isolation was associated with increasing time spent interacting with a novel mouse during reciprocal interaction (Lister and Hilakivi 1988). Similarly, it was reported that 4, 7, or 28 days of adult isolation without rehousing in male rats significantly increased social investigation during reciprocal interaction (Niesink and van Ree 1982). This finding was corroborated by a more recent study showing that 5 weeks of social isolation without rehousing starting from p56 in male mice increases social preference in the three-chamber test (Lv et al. 2022). Other studies report that 3 weeks (Lander et al. 2017) or 6 weeks (Rivera-Irizarry et al. 2020) of adult social isolation without rehousing in male mice had no effect on social preference in the three-chamber test. These studies did not assess the effects of resocialization, making it unclear whether any observed deficits are reversible. Taken as a whole, there is modest evidence that there is some form of social preference deficit induced by prolonged isolation in adulthood, but inconsistent findings suggest that adult isolation causes social preference deficits that are milder or more circumstantial compared to social isolation starting from p21. In our view, the sensitive period hypothesis is supported by the available literature.

2.2 |. Effects of JSI Versus CSI on Adult Social Preference and Investigation

CSI leads to a variety of robust changes in adult behavior, such as increasedanxiety-like behavior and decreased cognitive flexibility (Arakawa 2018; D. C. Li et al. 2021; Walker et al. 2019). However, as CSI rodents are housed in isolation at the timepoints when experimental data are collected, it is difficult to parse the acute effects of isolation during adulthood from the developmental effects of isolation. In addition, the interpretation of anxiety-like behavior tests should be viewed with caution. These tests, including the open field test and the elevated plus maze test, assess anxiety-like responses based on exploratory behavior. Typically, an animal is considered less anxious if it spends more time in the open arms of the elevated plus maze or in the center of the open field. However, such changes may also be influenced by alterations in locomotor activity (Fernandes et al. 1999), and therefore require careful analysis and interpretation. The CSI model produces reliable behavioral changes and is thus useful for exploring associations between contemporaneous changes in brain function and behavior, but its utility for exploring the effects of social isolation on development is limited, as chronic isolation that occurs only in adulthood is known to cause meaningful changes in brain function and behavior, such as increased agonistic behavior (C. R. Lee et al. 2021). JSI models, unlike CSI models, rule out the possibility that changes in brain and behavior are the result of adult isolation and make it possible to pinpoint which specific windows of development are sensitive to the effects of social isolation. However, the interpretation of the effects of JSI is also complicated, as JSI may induce changes to the post-rehousing homecage social environment, which, in turn, may influence adult social behavior outcomes (Leventhal and Morishita 2024). To ensure that any observed differences between JSI and GH animals are attributable to isolation-rearing rather than the resocialization procedure, some studies controlled for the effect of regrouping by randomly reassigning GH animals to new groups of GH animals at the same time that JSI animals were regroup-housed. These studies demonstrated that the re-socialization procedure itself cannot account for the reduced social preference or investigation induced by JSI (Lukkes et al. 2009 and Bicks et al. 2020).

The effects of CSI on adult social behavior are inconsistent in the literature. Although CSI from p21 in male mice or rats usually decreases adult social preference in the three-chamber test (Huang et al. 2021; Kuniishi et al. 2022; Lopez et al. 2024; Makinodan et al. 2012; Zheng et al. 2023), some groups have reported that CSI starting from p21 in males increases social preference in the three-chamber test (Kim et al. 2021; Manojlović et al. 2024). Increased sociability in the three-chamber test was also reported in CSI mice isolated from p38 (Lander et al. 2017). Conversely, there are relatively consistent reports that CSI rodents have increased adult social investigation during free-moving reciprocal interaction (Ferdman et al. 2007; Han et al. 2011; Wall et al. 2012; Wongwitdecha and Marsden 1996); but see (Huang et al. 2021). Further, CSI mice of both sexes (pooled together) isolated from p22 showed dramatically increased social investigation of an adult female stimulus mouse during reciprocal interaction on p48 (Arakawa et al. 2024), and CSI rats isolated from p21 showed an increased number of social interactions during reciprocal interaction at p28 (Niesink and van Ree 1982). No changes in social investigation were observed in male CSI rats isolated from p21 in a sequential social preference test where subjects interact with an empty corral followed by a corral containing a novel conspecific (Oliveira et al. 2019).

In contrast to CSI, JSI has been more consistently reported to reduce adult social preference and investigation. During free reciprocal interaction, reduced social investigation has been reported in adult JSI mice (Bicks et al. 2020) and rats (Hol et al. 1999; van den Berg et al. 1999) isolated from p21 to 35, and in JSI rats isolated from p21 to 42 (Lukkes et al. 2009). CSI rodents are known to have increased agonistic behavior (see next section), and a possible explanation for the discrepancy in social behavior between CSI and JSI rodents is that increased agonistic behavior in CSI rodents can complicate measurement of social investigation, especially during free reciprocal interaction. Rodents commonly signal their intent before initiating agonistic behavior (Haller 2017), and this signaling often manifests as social approach and following behavior that can be difficult to discern from non-agonistic social investigation. The theory that measurement of social preference and investigation is complicated by the difficulty of distinguishing between prosocial versus agonistic forms of social behavior is supported by findings that both CSI mice (Zhang et al. 2021) and rats (Han et al. 2011; Oliveira et al. 2019; Wall et al. 2012; Wongwitdecha and Marsden 1996; Zhao et al. 2009) display increased agonistic behavior in the reciprocal interaction context. CSI subjects may exhibit somewhat more consistently reduced social preference in the three-chamber test because the stimulus conspecific is protected by a barrier; thus, stimulus mice are less likely to elicit a territorial response from CSI subjects, and there is no opportunity for CSI subjects to exert aggression. Additionally, CSI subjects may sometimes display increased social investigation due to the general novelty of social stimuli rather than a specific motivation for social investigation.

2.3 |. Duration of Social Isolation

A study by Musardo et al. (2022) found that male JSI mice isolated for only 1 week from p28 to 35 show increased social investigation during reciprocal interaction with a novel juvenile mouse on p60, despite that multiple studies show reduced social preference and investigation resulting from p21 to 35 JSI (Bicks et al. 2020; Komori et al. 2024; Makinodan et al. 2017; Makinodan et al. 2012; Yamamuro et al. 2020). In this study (Musardo et al. 2022), 24 h of social isolation without rehousing was not sufficient to increase social investigation on p35. It has been proposed that short-term social isolation will trigger homeostatic mechanisms that promote social investigation, but once isolation becomes prolonged enough, there is a shift in the animal’s homeostatic set point and level of desire for social interaction (Matthews and Tye 2019). Indeed, a previous study found that 24 h of social isolation (without rehousing) in adult male mice caused a trending increase in social preference in the three-chamber test (Matthews et al. 2016). However, other studies found that 24 h of adult social isolation in male rats had no significant effect on social investigation in the reciprocal interaction test, but that 4, 7, or 28 days of isolation increased sociability (Niesink and van Ree 1982), and that 24 h of adult social isolation in male mice had no significant effect on social investigation, while longer durations of isolation increased social investigation (Lister and Hilakivi 1988). Further, 3 days of CSI in rats of both sexes from p18 markedly increased social interaction, including play behavior, on p21 (Panksepp and Beatty 1980). Overall, it appears that short-term (i.e., 24-h) isolation has little to no effect on investigation, but that longer-term social isolation without rehousing often increases social investigation in reciprocal interaction. However, there is a need for additional studies that directly compare different durations of social isolation while controlling for factors such as rehousing and the developmental timing of isolation. Since chronic isolation in adulthood seems to increase social investigation, the developmental effects of CSI are likely confounded by adult social isolation, and there is an especially great need for additional JSI studies.

2.4 |. Age and Sex of Conspecifics Used as Social Stimuli

The presence or absence of social preference and investigation deficits induced by social isolation may also depend on the conspecific used as a social stimulus. Most studies use a social stimulus that is the same sex, species, and strain as the subject, but experimental paradigms commonly vary as to whether the social stimulus is age-matched to the subject or is a juvenile. Our literature review did not reveal any clear association between the age of stimulus conspecifics and adult social preference and investigation because the effects of stimulus conspecific age were often confounded by other experimental variables (such as JSI vs. CSI or the timing/duration of isolation). One study found that male and female (pooled together) CSI mice isolated from p22 show reduced social preference in the three-chamber test on p47 when the stimulus mouse is male but display no social preference deficit if the stimulus mouse is female (Arakawa et al. 2024). Overall, there is a strong need for future studies that determine the extent to which isolation-induced social preference deficits are generalizable to different types of stimulus conspecifics.

2.5 |. Sex of Experimental Subjects

In female subjects, the effects of social isolation on adult social preference and investigation are less consistent and less well-characterized than in males. One study reported that female CSI rats isolated from p21 or from p30 had no social investigation deficit during adult reciprocal interaction (Ferdman et al. 2007), and another study reports that female CSI rats isolated from p21 have no adult social preference deficits in the three-chamber test (Zheng et al. 2023). Other investigators found that female CSI rats isolated from p21 had increased social investigation during reciprocal interaction (Wall et al. 2012) and increased social preference in the three-chamber test (Manojlović et al. 2024). In a simple social approach test where subjects were exposed to a corralled conspecific in an open field arena, adult female rats that underwent JSI from p21 to 42 showed no significant social investigation deficit (Kinley et al. 2021). However, another group using a similar behavior test found that female CSI mice isolated from p21 had reduced social investigation (Tan et al. 2021), and it was reported that pairs of female CSI rats isolated from p21 displayed reduced social investigation toward each other during reciprocal interaction compared to pairs of female GH control rats (Hermes et al. 2011). Given that these studies reporting reduced social investigation did not include a comparison between social and nonsocial investigation, it cannot be ruled out that the observed reductions in social investigation in females were a result of general investigative deficit (such as those caused by general anxiety) rather than a specific reduction in social investigation. Increased anxiety-like behavior as a result of social isolation is commonly reported, especially in females (Harvey et al. 2019; Kumari et al. 2016; Lukkes et al. 2012; Wu et al. 2022). Overall, the evidence for reduced adult social investigation resulting from JSI or CSI in females is relatively weak, but further investigation is warranted. To our knowledge, there is only one published study reporting the effects of JSI on adult social investigation in female subjects (Kinley et al. 2021). It should be noted that male and female rodents are typically housed only with conspecifics of the same sex, and there are considerable differences in the homecage social environment of all-male and all-female homecages. It is likely that sex differences in how JSI affects adult social behavior are influenced by both genetics and gene-environment interactions, such as the effects of sex on the homecage social environment.

2.6 |. Summary of Social Preference and Investigation Deficits Caused by JSI and CSI

Taken together, these studies show that the effects of social isolation on social preference and investigation are highly influenced by variations in experimental paradigms. There are important differences in adult social preference and investigation phenotypes between male JSI rodents and CSI rodents, and between male CSI rodents and female CSI rodents (Figure 2). The literature is generally consistent with the hypothesis that there is a p21–35 sensitive period where isolation reduces adult social preference and investigation in males, but lack of a consistent adult phenotype in CSI males isolated from p21 suggests that developmental events and/or experiences occurring after p35 may be important for determining adult social preference and investigation. There also appears to be a sex difference in the effects of social isolation on adult social preference and investigation, where females are less affected than males, but differences between males and females are poorly characterized, and the mechanisms underlying any potential sex difference are largely unknown.

3 |. Differential Impact of Isolation Paradigms on Agonistic and Social Novelty-Related Behaviors

This section examines the effects of JSI and CSI on agonistic and social novelty-related behaviors (Figure 2; Table S1). We assess how different experimental factors affect social behavior outcomes wherever there is enough available data to make a reasonable comparison. The resident intruder test (see Glossary) has been used extensively to test isolation-induced changes in agonistic behaviors. Isolation-induced changes in social recognition-related behaviors have been tested with the three-chamber social novelty test and with social habituation-dishabituation paradigms (see Glossary).

3.1 |. Agonistic Behavior

Agonistic behavior is a common and inherited trait across the animal kingdom. It was originally defined from an ethological perspective as adaptations for situations involving physical conflict or contests between members of the same species (Scott 1966). In rodent models, agonistic behavior is classified as offensive versus defensive by considering preceding circumstances, the specific action observed, and its consequences (Takahashi and Miczek 2014). In a natural setting, offense adaptively increases access to breeding females and often increases access to other important natural resources such as food and territories or safe nest sites (Blanchard and Blanchard 1988). The resident intruder test is a paradigm to measure offensive (territorial) and defensive agonistic behavior in a semi-natural laboratory setting (Blanchard and Blanchard 1988; Koolhaas et al. 2013; Lin et al. 2011; Miczek and O’Donnell 1978). The underlying principle is to partially reflect natural situations by promoting adult male rodents to establish an asymmetry, which to some extent reflects territory when provided with sufficient living space, and attack an adult male that intrudes into the territory (Koolhaas et al. 2013).

Male CSI rodents consistently display increased agonistic behavior in the resident intruder test in adulthood (Biro et al. 2023; Biro et al. 2017; Chang et al. 2020; Kim et al. 2021; Oliveira et al. 2019; Tan et al. 2021; Tóth et al. 2008; Toth et al. 2011; Toth et al. 2012) compared to GH subjects, which were usually isolated for only 1–3 days prior to testing to induce some level of agonistic behavior and to control for the acute effects of social isolation. Additionally, one study reports that CSI mice exhibit a higher degree of territorial urine-marking behavior in the urine-marking assay (see Glossary) (Hyun et al. 2021). However, prolonged social isolation that occurs only in adulthood is also known to robustly increase agonistic behavior in males (Matsumoto et al. 2005; Zelikowsky et al. 2018), and, to our knowledge, previous studies have not compared CSI subjects to “GH” subjects that were isolated during adulthood for an equivalent duration to CSI subjects. As a result, the extent to which developmental dysfunction plays a role in increased agonistic behavior induced by CSI is questionable. An informative study found that CSI mice isolated from p28 showed increased agonistic behavior in the resident intruder test on p63 relative to GH controls who were never isolated, but no significant differences in agonistic behavior levels were detected between CSI mice isolated from p63 and GH controls on p119 (X. Li et al. 2022). However, this discrepancy appears to be driven by both increased agonistic behavior in adolescent CSI mice compared to adult-isolated mice and increased agonistic behavior in p119 GH mice compared to p63 GH mice, suggesting that there is a developmental increase in agonistic behavior across early adulthood in GH mice that can complicate the interpretation of how social isolation affects agonistic behavior. Another study provides insight into whether there is a sensitive period of development for the effects of social isolation on agonistic behavior by performing a resident intruder test with a juvenile intruder mouse in both CSI males isolated from p21 and JSI males isolated from roughly p21–49 (Kim et al. 2021). This study revealed that adult male JSI mice display low levels of agonistic behavior that were significantly lower than CSI mice and non-significantly reduced compared to GH controls, suggesting that increased agonistic behavior in male CSI mice may be primarily driven by the effects of prolonged social isolation or isolation during adulthood rather than developmental dysfunction.

Previous studies have failed to detect increased agonistic behavior in female CSI mice in the resident intruder test (Ago et al. 2013; Tan et al. 2021). During reciprocal interaction, it was reported that female CSI rats spent more time exhibiting agonistic grooming, but no significant differences in duration of chasing behavior or number of pins were detected (Wall et al. 2012). These findings may reflect a genuine sex difference, which would not be surprising given female rodents are generally considered to have lower levels of agonistic behavior compared to males. Alternatively, faithful comparisons in aggression between males and females could be complicated by sex differences in the social contexts where rodents exhibit agonistic behavior (Been et al. 2019). The lack of isolation-induced effects on agonistic behavior in females could be due to the specific social context associated with the resident intruder test, which may not be ideal for measuring agonistic behavior in females.

3.2 |. Isolation-Induced Changes in Social Recognition and Habituation

Both the three-chamber social novelty test and social habituation-dishabituation paradigms measure the duration of social investigation of a novel conspecific versus a familiar conspecific and operate under the assumption that subjects prefer to investigate a novel social stimulus over a familiar social stimulus. The choice of assay is important, as loss of social novelty preference can be the result of multiple factors, including failure to recognize a familiar conspecific, lack of motivation to investigate a novel conspecific, or slowed social habituation (making the “familiar” conspecific effectively more novel). For both assays, isolation-induced social preference deficits may decrease the subject’s amount of social exposure during the habituation phase, and it is not possible to distinguish social recognition deficits from lack of motivation to investigate novel social stimuli.

Adult deficits in social novelty preference in the three-chamber social novelty test have been reported in male CSI mice isolated from p21 or later (Huang et al. 2021; Zhang et al. 2021) and in male mice that underwent long-term JSI from p21 to 77 (G. Park et al. 2021), but JSI mice isolated from p21 to 35 did not display social recognition deficits (G. Park et al. 2021). These results raise the possibility that there is a sensitive period between p35 and adulthood where isolation disrupts adult social recognition. Another possible interpretation is that isolation must be sufficiently prolonged to measurably impact adult social novelty preference. Lack of social novelty preference was reported in CSI mice that were isolated from p28 and tested on p35, but this effect was also found in CSI mice that were isolated for only a single day, starting on p34, and tested on p35 (Musardo et al. 2022). This finding suggests that even relatively short experiences of social isolation can acutely impair social novelty preference, calling into question whether any observed deficits in social novelty preference in CSI mice are due to any developmental phenomenon.

Studies employing social habituation-dishabituation paradigms show that both social habituation and social novelty preference in adulthood was markedly impaired in male CSI mice isolated from p28 (Zhang et al. 2021) and in both male and female CSI mice isolated from p30 (Kercmar et al. 2011), raising the possibility that social novelty preference deficits in the three-chamber test can be explained by reduced rates of habituation to social stimuli rather than a social recognition deficit. Impaired social habituation was also shown in CSI mice isolated from p28 and tested on p35 (Musardo et al. 2022). A study by Oliveira et al. (2019) also showed that CSI rats isolated from p21 of both sexes (pooled together) show a social novelty preference deficit in a social discrimination test with a single habituation session. G. Park et al. (2021) found social novelty preference deficits alongside a normal rate of habituation in male mice that were isolated from p21 to 77 but were returned to social housing several weeks before behavioral tests, suggesting that adult social novelty preference (rather than just rate of social habituation) is indeed sensitive to prolonged JSI. Together, the findings that slowed social habituation is exhibited by male CSI subjects (who are isolated at the time of testing) and not by male subjects isolated from p21 to 77 suggest that the rate of social habituation is strongly influenced by the subject’s current social environment. Whether or not there is a sensitive period for isolation-induced effects on social habituation remains unclear, and there is a need for studies that directly compare the effects of JSI to equivalent periods of adult isolation. One study found that 1 week of adult isolation without rehousing is sufficient to induce social novelty preference deficits in male mice, but this phenotype was rescued after 1 week of resocialization (Liu et al. 2018).

There is also a need for additional studies that investigate the effects of social isolation on social habituation and recognition in females and make direct comparisons between the sexes. In the handful of social recognition studies that included female subjects, there were no significant differences between CSI males and females isolated from p21 (Manojlović et al. 2024; Kercmar et al. 2011; Oliveira et al. 2019), with two studies reporting a social novelty preference deficit and one study failing to detect a significant deficit. A single study found that p30–60 JSI was sufficient to induce significant deficits in females, but not in males (Kercmar et al. 2011).

4 |. Summary, Future Directions, and Conclusion

The social isolation literature generally supports the hypothesis that there is a sensitive period (or multiple sensitive periods) during development where social experience has a more profound impact on adult social behavior. The strength of the effect of isolation on adult behavior is dependent on the developmental timing and duration of isolation, whether subjects are returned to social housing prior to adult data collection, the sex of the experimental subjects, and the specific behavioral construct under investigation. In male rodents, there is relatively strong evidence for the existence of a sensitive period for the establishment of adult social preference and investigation, as there are numerous studies reporting reduced social preference and investigation resulting from JSI (Bicks et al. 2020; Komori et al. 2024; Makinodan et al. 2017; Makinodan et al. 2012; Yamamuro et al. 2020), and adult isolation fails to reproduce social preference and investigation deficits caused by JSI (Bicks et al. 2020). While social isolation does affect adult social novelty preference and agonistic behavior, the evidence for a sensitive period for the establishment of these behaviors is more limited due to a lack of direct comparisons between the effects of adult isolation versus JSI. Further, studies of the effects of social isolation during development on agonistic behavior have focused on CSI rodents, which are likely to display increased agonistic behavior due to concurrent adult isolation. In female rodents, consistent effects of JSI on adult social behavior have not been observed, highlighting the need for more studies that include female subjects and raising questions about the neurobiological bases for apparent sex differences in how JSI affects adult social behavior.

4.1 |. Future Directions

This review reveals a need for more social isolation studies that systematically compare different experimental parameters such as the duration and timing of isolation or the sex of experimental subjects. Currently, our ability to draw conclusions about the effects of isolation is limited by a lack of standardized protocols and the need to perform “apples-to-oranges” comparisons of different isolation paradigms used by different research groups. There are also many aspects of social behavior that remain under-explored in the context of social isolation. Some examples include resiliency to stressors (such as social defeat), social dominance behavior, social play, emotion recognition, sexual motivation and competency, and social transmission of information (such as food safety). It is also unclear how different social behavioral constructs may interact with each other: for example, changes in agonistic behavior could influence social investigation behavior, and vice versa.

Our analysis also showed that the vast majority of existing developmental social isolation studies examine the adult consequences of social isolation. In a separate review article, we argue that JSI-induced brain and behavior deficits may be both directly caused by social deprivation and caused by social isolation by creating a “developmental mismatch” between the late adolescent social environment and adaptations formed from living in social isolation (Leventhal and Morishita 2024). The differences in the adult social behavior of JSI and CSI mice discussed in this article suggest that such a developmental mismatch effect may be important for determining the consequences of social isolation during development. In order to truly understand the developmental mechanisms disrupted by social isolation, it will be necessary to explore the brain and behavior changes that are occurring while development is ongoing. This line of investigation is expected to reveal the specific reason that social isolation disrupts brain and behavior maturation. For example, social isolation could be deleterious because isolated subjects are left without any meaningful source of cognitive stimulation, or it could be due to the loss of social-specific sensory stimuli (such as social touch or smell). In rats, social play peaks during the juvenile stage and is essential for normal social and cognitive development (Pellis et al. 2023). Remarkably, just 1 h of daily interaction with an age-matched peer can mitigate cognitive deficits in juveniles reared in isolation (Einon et al. 1978), suggesting that specific aspects of social experience, particularly social play, are essential for the maturation of the brain and behavior. Future studies, which incorporate machine learning-based 3D tracking of postural dynamics in freely interacting animals (Klibaite et al. 2025), can help discern what aspects of social experience are important for brain development by varying the level of cognitive stimulation provided to isolates (e.g., by providing environmental enrichment) or by varying the social sensory stimuli that are provided to subjects (e.g., isolates could be provided only with social odor stimuli). Gaining this knowledge would be instrumental for developing effective interventions before the closure of the sensitive period, thereby preventing or even reversing isolation-induced social deficits.

4.2 |. Conclusion

In this review, we provided a comprehensive overview of the effects of social isolation during development on adult social behavior, highlighting how differences in experimental paradigms (including the duration and timing of isolation, the sex of subjects, and whether subjects are returned to social housing prior to testing) can help explain disparate experimental results. Future studies that more fully characterize the effects of specific experimental parameters on adult outcomes produced by social isolation during development will help the field drive toward an understanding of the specific developmental mechanisms that are disrupted by social isolation and may inform the development of novel treatments for neuropsychiatric disorders associated with impaired social experience-dependent brain maturation. Such efforts are especially important in present times given the prevalence of social isolation resulting from COVID-19 and increasing digital social interaction at the expense of in-person social interaction.

5 |. Glossary

5.1 |. Three-Chamber Social Preference Test

An assay where subjects are exposed to both a novel social stimulus (conspecific) and a novel object, allowing the experimenter to parse experimentally induced changes in social preference (relative to non-social object) from general changes in investigative behavior that apply to both social and non-social stimuli. However, the three-chamber test requires that social stimuli be corralled, restricting normal social interaction. Three-chamber test paradigms often vary with respect to the novel object that is used, with some investigators using only an empty corral and other investigators using a corral-enclosed object, such as a small figure made of connectable blocks. Further, different investigators employ different dependent variables within the three-chamber test. While some investigators measure the amount of time that a subject spends in the social chamber versus the object chamber, with the advent of automated video tracking, it is common to measure the amount of time that the subject spends with its nose occupying an “interaction zone” surrounding each stimulus.

5.2 |. Reciprocal Interaction

This assay entails simply allowing two mice to freely interact within an open environment. It is commonly conducted in an open field setting but also can be conducted in a clean homecage. Investigators often measure the amount of time that subjects spend sniffing a conspecific stimulus. However, this test produces a multitude of possible dependent variables, including various types of social investigation (distinguished by, e.g., which body part is being investigated or whether the investigation is reciprocated) or the frequency and duration of other social behaviors such as approaches and social withdrawals. An advantage of reciprocal interaction over the three-chamber test is that it allows for the assessment of social investigation in a more naturalistic context, but this comes at the cost of social behavior being more difficult to quantify. Quantification of reciprocal interaction behavior requires either laborious manual scoring or advanced automated tracking and behavior classification.

5.3 |. Resident Intruder Test

This assay is typically performed using adult male subjects and involves socially isolating the subject or housing the resident male and the companion female together in the resident cage, usually for 1–3 weeks, prior to the introduction of an intruder conspecific into the subject’s homecage. Under normal circumstances, an adult male rodent will attack the intruder within minutes. The intruder is often age-matched to the subject but may also be a juvenile to capture “abnormal” heightened agonistic behavior. The fact that this assay typically requires adult social isolation to achieve a baseline level of agonistic behavior complicates the interpretation of how isolation during development influences adult agonistic behavior.

5.4 |. Three-Chamber Social Novelty Test

In this assay, subjects are first habituated to the “familiar”conspecific over the course of the 10-min social preference testing period. During the social novelty test phase, the novel object that is present during the social preference phase is swapped for a novel conspecific, and the subject is allowed to investigate both familiar and novel stimulus conspecifics. Under normal circumstances, a GH mouse or rat will spend more time investigating the novel conspecific compared to the familiar conspecific. The three-chamber social novelty test’s relatively short habituation to the familiar mouse (typically 10 min) makes it difficult to identify deficits in social habituation.

5.5 |. Social Habituation-Dishabituation Paradigms

In this assay, the subject undergoes multiple habituation sessions (sometimes across multiple days, sometimes within the same day), and the familiar conspecific is replaced with a novel conspecific on the final session of testing. A typical GH mouse or rat will increase the amount of time they spend socially investigating during the final session with the novel conspecific. The social habituation-dishabituation paradigm typically involves more extensive habituation compared to the three-chamber social novelty test, making it easier to determine whether social novelty preference deficits are caused by disruption of social habituation versus social recognition deficits or lack of motivation to investigate a novel social stimulus.

5.6 |. Urine-Marking Assay

This assay assesses the subject’s urine-marking behavior, which is often associated with territoriality. The subject is placed alone in a clean cage with a floor that is lined with filter paper. The center of the filter paper is treated with urine from an unfamiliar strain of conspecific to evoke territorial urine counter-marking or is treated with saline as a control. The subject is then left in the cage, typically for 1 h, and allowed to urinate on the filter paper. A fluorescent image is taken of the urine-marked filter paper, and the subject’s urine marking pattern is analyzed. When they encounter urine from an unfamiliar mouse strain, more territorial or dominant rodents tend to disperse their urine marks broadly and countermark near the urine stimulus, while less territorial or submissive rodents tend to urinate in the corners of the cage away from the urine stimulus.

Supplementary Material

Table S1

Supplementary Table 1. Detailed summary of the effects of JSI and CSI on social behavior. Summary of behavioral results for all JSI and CSI studies reviewed in this manuscript. The red cells represent isolation window, the green cells represent group-housing window, and the astarisks represent testing age. Yellow fill is used only to call attention to certain variables that may have an important effect on results. Yellow fill is used when the subjects are female, when JSI subjects are rehoused with GH conspecifics, and when an atypical behavior test is used. Results symbols are marked with an asterisk if the result or the interpretation of the result is unclear for any reason. See specific findings section for details.

Additional supporting information can be found online in the Supporting Information section.

Data Availability Statement

The data that supports the findings of this study are available in the supporting Information of this article.