Anxiety disorders are among the most prevalent psychiatric disorders globally, representing approximately 3.6 % of the global population according to World Health Organization (WHO, 2017). A study on the Global Burden of Diseases in 2019 further underscored their significance, ranking anxiety disorders among the top 25 leading causes of global health burden (GBD, 2020). Moreover, the year 2020 witnessed a significant surge in the prevalence of both anxiety and major depressive disorders, primarily attributable to the far-reaching impact of the COVID-19 pandemic. Data reveals a striking uptick of 26 % in anxiety disorders and a substantial 28 % increase in major depressive disorders within the span of just one year (WHO, 2022).

Chronic repeated stressful situations are known risk factors triggering anxiety in rodents and humans (Hammels et al., 2015) leading to the development of neuropsychiatric disorders (see review by Marcolongo-Pereira et al., 2022). A common model used in rodents to simulate the variability of stressors encountered in daily life (construct validity) is the unpredictable chronic stress (CUS) model (Papp et al., 1996; Santos-Rocha et al., 2018; Malta et al., 2021), which has been used to study behavioral and physiological stress responses, such as anxiety and corticosterone/cortisol (CORT) levels (McEwen, 2007; Santos-Rocha et al., 2018). Usually, the CUS model continuously raises the activity of the hypothalamic-pituitary-adrenal (HPA) axis, making it challenging for animals to adapt to aversive conditions (Willner et al., 1987; Franco et al., 2016). While most researchers employing the CUS model adhere to fundamental principles with regards to protocols, such as exposing animals to a variety of unpredictable stressors over a prolonged period, the exact nature and sequence of stressors employed can differ between research groups. In general, CUS is a well-accepted experimental model of stress-induced mood disorders (Monteiro et al., 2015), however, it does not always necessarily elicit anxiety-like behavior (Malta et al. 2021), which depends on multiple variables (Monteiro et al., 2015; Malta et al. 2021), such as genetic factors, duration, intensity, among others (Franklin et al., 2012; Monteiro et al., 2015).

The HPA axis comprises important components involved in the stress response. These include corticotropin-releasing hormone (CRH) neurons located in the paraventricular nucleus, which play a crucial role in initiating the stress response by triggering the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary. ACTH circulates in the bloodstream and acts on the adrenal cortex, stimulating the secretion of glucocorticoids. The primary glucocorticoid in rodents is corticosterone, which exerts feedback control by signaling back to the brain and pituitary, effectively shutting off the stress response (Rivier and Vale, 1983). Metyrapone, an inhibitor of glucocorticoid synthesis, is a valuable tool that can modulate the HPA axis activity by blocking the production of glucocorticoids and is frequently used to assess the integrity of the HPA axis (Fiad et al., 1994; Schimmer and Parker, 1996).

Several rodent studies have demonstrated a relationship between elevated plasma corticosterone levels and the development of anxiety-like behaviors (Adams et al., 2003; Aguillar-Valles et al., 2005; Aisa et al., 2007; Heim et al., 2008). For example, Mitra and Sapolsky (2008) observed an anxiogenic effect in rats treated with corticosterone. These findings align with clinical studies that have established dysregulated hyperactivity of the HPA axis in patients diagnosed with anxiety disorders (Buchanan et al., 2004; Kukolja et al., 2008). Thus, both preclinical and human investigations suggest a positive correlation between stress and high plasma glucocorticoid levels (Alfarez et al., 2003; Choy et al., 2008). However, it is important to note that changes in anxiety phenotype and in glucocorticoid levels can vary depending on several factors such as type and duration of stressors, as well as the species under investigation (Monteiro et al., 2015).

On the other hand, environmental enrichment (EE) is an experimental protocol employed in rodents that acts as a protective factor against stressful stimuli. EE entails a stimulating environment that exposes animals to inanimate objects with various colors and textures, such as exercise wheels, tunnels, and stairs besides social enrichment (Nithianantharajah and Hannan, 2006; Rae et al., 2018). EE can confer a resilient phenotype on rodents subjected to chronic stress, such as social defeat (Lehmann and Herkenham, 2011), maternal separation (Francis et al., 2002), or acute restraint stress (Novaes et al., 2017).

Most studies tend to emphasize the impact of CUS either before, in-between or after EE (Zeeni et al., 2015; Shen et al., 2019; Costa et al., 2021). Our objective in this study was to assess the effects stemming from the combination of two distinct stimuli: EE and CUS - an ethological model for investigating the consequences of psychological stressors (Willner, 2017a) - in Swiss mice, an outbred strain. We evaluated the impact of CUS in male mice reared in EE on anxiety-like behavior, corticosterone, and ACTH levels. The plus-maze test served as a behavioral measure to examine anxiety-like responses in this context. To gain a deeper understanding of the underlying mechanisms, we used metyrapone as a pretreatment, which acts as an inhibitor of corticosterone, to assess the functionality of the HPA axis.