Social hierarchies form within virtually all animal groups and are generally characterized by higher-ranking animals maintaining priority to resources and/or exhibiting more aggressive behaviors than lower-ranking individuals (Drews, 1993; Fulenwider et al., 2022). Not surprisingly, the differing ranks within these hierarchies can significantly affect an individual's physical and emotional health, most often at the greatest expense of the lowest-ranking subordinates, or omegas, resulting in a phenomenon referred to as subordination stress (Blanchard et al., 1993a, Blanchard et al., 1993b; Sapolsky, 2004). For example, it has been shown that subordinates have shorter lifespans and exhibit immune and hypothalamic-pituitary adrenal (HPA) axis dysregulation, increased amygdalar corticotropin-releasing factor (CRF) levels, and even increased levels of drug intake compared to dominants (Ellison et al., 1983; Blanchard et al., 1987; Blanchard et al., 1993b; Albeck et al., 1997; Stefanski et al., 2001; Melhorn et al., 2017; Williamson et al., 2017; Razzoli et al., 2018; Lee et al., 2022).

Notably, the processes outlined above have been primarily examined using male rats and more aggressive mouse strains, such as CD1s, and in experiments designed to incorporate overt agonistic interactions and physical injury. In other words, many of these findings were products of severe aggression and stress exposure, and they might not reflect animals' behaviors and physiological processes in the more common moderately stressful conditions. Therefore, additional studies investigating the consequences of social rank in less overtly aggressive species and strains, such as C57BL/6 J mice, are still needed. Further, as this strain is so frequently used for transgenic manipulations, examining the effects of social rank in C57s has the potential to contribute to more complex studies in this area.

Social rank can be assessed without the exhibition of overt agonistic behaviors using the tube test. This assay involves competition for sole occupation of a clear tube, in which two mice are simply placed at opposite ends of the tube and allowed to approach one another in the center. The dominant subject will then force the subordinate subject out of the tube. The tube test is therefore well-suited to determining social rank in female rodents and males in less aggressive species/strains, such as C57BL/6Js (Fan et al., 2019).

OXT and AVP are two closely related nonapeptides primarily synthesized in the paraventricular and supraoptic nuclei of the hypothalamus (PVN, SON), and to a lesser extent in the suprachiasmatic nucleus of the hypothalamus (SCN), accessory nucleus of the hypothalamus, amygdala and bed nucleus of the stria terminalis (BNST) (Caldwell et al., 2008; Lee et al., 2009). These peptides are then released into the circulation and various brain regions to exert a diverse array of endocrine and synaptic effects, including social behavior and stress responsivity (Benarroch, 2013; Terranova et al., 2017; Jurek and Neumann, 2018; Takayanagi and Onaka, 2021). As such, these two systems are likely candidates for regulating social hierarchy formation, maintenance and subordination stress.

While involved in a variety of complex behaviors and processes, OXT is generally associated with social memory, bond formation, and sexual and parental behavior (Jurek and Neumann, 2018). Additionally, OXT typically suppresses HPA axis activation, reducing cortisol/corticosterone levels and anxiety/anxiety-like behaviors following stress exposure (Heinrichs et al., 2003; Smith and Wang, 2014; Takayanagi and Onaka, 2021). Particularly, PVN OXT levels have been shown to increase in male rats in response to acute, chronic homotypic, and chronic heterotypic stress exposure, though this increase is greatest when a stressor is chronic and homotypic (Zheng et al., 2010). These data suggest that while the PVN OXT system may contribute to stress adaptation, its functionality in this regard varies as a product of the type and duration of stressor presented.

In contrast, AVP has been classically associated with offensive agonistic behaviors in males and with maternal aggression in females (Terranova et al., 2017). AVP is also associated with HPA axis activation and is thought to be involved in maintaining HPA axis hyperactivity in certain individuals with major depressive disorder, essentially serving an opposing role to OXT's generally anxiolytic effects (Dinan and Scott, 2005). In fact, while CRF and its receptor's activity are negatively modulated by elevated CORT levels, AVP and the V1b receptor's activity are positively modulated in these conditions, serving as at least one of the mechanisms through which the AVP system can induce sustained HPA axis activation during chronic stress (Chaki, 2021). It has also been shown in males that the total number of AVP-expressing neurons and AVP- and CRF-co-expressing neurons in the PVN increases following exposure to certain chronic stressors (de Goeij et al., 1992; Ma and Lightman, 1998).

However, it must also be noted that other studies have reported unchanged PVN OXT and/or AVP levels following social stress exposure (Wotjak et al., 1996; Albeck et al., 1997; Reber and Neumann, 2008). Collectively, these data suggest that both AVP and OXT may serve regulatory roles in both social hierarchy and social rank determination and/or resulting subordination stress, but the consistency of these effects across different experiments remains ambiguous.

Lastly, many behaviors, including social dominance/hierarchy formation, can depend on the level of environmental enrichment (EE). Differences between species, strains, age, sex, type of enrichment provided, and duration of enrichment exposure can all influence EE outcomes (Kentner et al., 2021). Generally, EE has been shown to attenuate stress-induced depressive- and anxiety-like behaviors in male C57BL6/J mice exposed to social defeat stress (Schloesser et al., 2010; Lehmann and Herkenham, 2011). For within-group interactions, however, EE can actually potentiate aggressive behaviors, as demonstrated in group-housed male CD1 and CS mice (Marashi et al., 2003; Howerton et al., 2008; McQuaid et al., 2012). In male and female C57BL/6 J mice, though, the effects of EE level on social rank, subordination stress, and associated neuroendocrine measures remain unknown.

As such, the aims of the current study were to: 1) characterize the formation and maintenance of social hierarchies in group-housed male and female C57BL/6 J mice using the tube test, 2) compare tube test-determined social ranks to those determined by palatable liquid competition, 3) elucidate the relationship between tube test-determined social rank and hypothalamic and plasma OXT and AVP levels, 4) assess the roles of EE level and length of testing in these processes. These aims were addressed in 3 separate experiments in group-housed male and female mice housed at 4 same-sex animals per cage. Experiment 1 (Exp 1) examined the dynamics of social hierarchies using the tube test, how social ranks from these tests related to palatable liquid competition performance, and differences in OXT and AVP levels within the PVN. Because animals in Exp 1 were housed in moderately enriched environments to measure individual liquid intake, we next sought to then assess whether the observed differences between dominant and subordinate animals depended on EE. Therefore, in Experiment 2 (Exp 2), mice were housed in high or low EE conditions and exposed to the same number of tube tests as in Exp 1, as well as measurements of PVN and plasma OXT and AVP levels, but were not tested for palatable liquid competition. Lastly, given the inconsistent results in our hypothalamic data between Exps 1 and 2, in Experiment 3 (Exp 3) we aimed to characterize how length of testing and time between last test and tissue collection affected PVN OXT and AVP levels.