The role of the stress hormone, corticosterone, in rodent diurnal behavior not only begins at the hypothalamic-pituitary axis (HPA), but also at the binding site of mineralocorticoid (MRs) and glucocorticoid receptors (GRs). Disruption in the function of these corticosterone-responsive transcription factors is implicated in dysregulation of sleep and metabolic processes, which, in turn, affect behavior (Kalman and Spencer, 2002; Keller et al., 2017; Otte et al., 2003; Spencer et al., 1998; Young et al., 2003). MRs have a higher binding affinity for corticosterone than GRs and are thought to be sensitive to even low levels of circulating hormone (Reul and Kloet, 1985; Reul et al., 2015). Accordingly, relative binding saturation for MRs remains more tonic than that of GR whose binding fluctuates with stress and circadian events (De Kloet et al., 2000). Levels of circulating corticosterone fluctuate across day and night (De Boer and Van der Gugten, 1987; Kakihana and Moore, 1976; Kamakura et al., 2016). In rats, corticosterone tends to be elevated in the evening, early in the animals' active period, compared to the morning (Oitzl et al., 1995) with this elevation persisting into the early night phase (Butte et al., 1976). Given this, it is hypothesized that MR and GR play a unique function in the diurnal response to corticosterone.

MRs and GRs are present throughout the brain (Chao et al., 1989; Prager et al., 2010; Van Eekelen et al., 1988) with notable expression within the hippocampus (Herman et al., 1993; Sarabdjitsingh et al., 2009; Van Eekelen et al., 1988). The hippocampus is a limbic structure necessary for learning, memory, emotion, spatial processing, and response to stress. Of interest, within the rodent hippocampus, MR expression tends to be more highly concentrated in area Cornu Ammonis 2 (CA2) compared to GRs, which are mainly expressed within the CA1 and dentate gyrus (Herman et al., 1989; Kalman and Spencer, 2002; McCann et al., 2021). These transcription factors play a crucial role in the control, adaptation, and output of physiological responses to stressful events at the level of the HPA axis, which in turn influences the function of the hippocampal formation through actions of corticosterone at MR and GR receptors (Henry et al., 1994; Phillips et al., 2006; Sapolsky et al., 2000). MRs differentially regulate the diurnal release of adrenocorticotropic hormone (ACTH) (Young et al., 1998). Further, in humans, blockade of MR binding disrupts circulating levels of cortisol, the primary glucocorticoid in humans (Heuser et al., 2000). Finally, both MR and GR follow a diurnal expression pattern within the hippocampus (Herman et al., 1993). Given these effects, along with the high binding affinity of MRs for corticosterone, whether MRs have a downstream effect on the fluctuations of behavior across day and night requires investigation.

Previous work has shown that conditional knockout (KO) of MR in hippocampus and/or forebrain disrupts the acquisition of the CA2 molecular profile and various behaviors, including anxiety, reactivity to novelty, and social memory (Berger et al., 2006; McCann et al., 2021; Ter Horst et al., 2014). Traditional behavioral assays, such as open field or novel object recognition, do not allow for assessment of continual spontaneous behavior as they are usually bound to discrete, 5- to 30-min observations. Given this limitation, a more in-depth and continuous monitoring approach to understanding the effects MRs can have on daily behaviors could supplement the information that can be obtained through traditional behavioral assays.

In order to study the role of MRs in the modulation of spontaneous behavior across day and night, we used automated home-cage monitoring. In comparison to more commonly used paradigms that measure a rodent's spontaneous behavior, such as the open-field test, automated home-cage monitoring offers the benefit of being a high-throughput method of longitudinal recording that minimizes investigator bias and researcher interference (Aarts et al., 2015; Kiryk et al., 2020; Grieco et al., 2021; Ahloy-Dallaire et al., 2019). This study sought to determine whether MR deletion from the whole brain and/or from the hippocampal area CA2 affects rodent spontaneous behavior by measuring activity across three murine conditional MR KO strains generated based on the Cre line bred with flox MR mice. These strains include Amigo2-Cre, EMX-Cre, and Nestin-Cre. We used the Noldus PhenoTyper to measure behavior in these differing MR KO strains across a 60-h observation period which allowed for continuous assessment of animals' locomotor and exploratory behavior.