The Systems Biology of Chronic Stress in Mice: Integrated Neurobiological, Behavioural and Cardiovascular Outcomes

Abstract

The demands of modern life are often challenging and require psychological efforts in order to be effectively met. Conserved through evolution, acute psychological stress (in response to an acute threat) activates physiological systems that are advantageous, exerting appropriately timed responses to promote survival. However, the advantageous outcomes of the acute stress response are reversed under prolonged conditions (chronic stress), detrimentally influencing biological processes and/or behaviours and increasing disease risks. This disease risk is imposed on virtually all organ systems, and ranges from mood disorders (e.g., major depressive disorder; MDD) to cardiovascular (e.g., ischaemic heart disease; IHD) and metabolic (e.g., type 2 diabetes; T2D) diseases. Although the impact of chronic psychological stress on mood disorders has been well documented, its effects on other body systems (including cardiovascular, circulatory, and hepatic systems) are less detailed. Furthermore, chronic psychological stress outcomes vary between females and males, both in disease risk and presentation, yet we lack definitive understanding of the specific mechanistic differences. Our understanding of psychological stress has increased considerably since it’s early conception in 1915 by Walter Bradford Cannon, a view that would later be defined by Hans Selye in 1956. However, our knowledge regarding the systems biology of chronic stress, the neurobiological adaptations and underpinnings of aberrant behaviour, and the mechanistic basis of sex-dependent outcomes, remain relatively limited. The doctoral work presented in this thesis describes the establishment of models of chronic stress in mice and their application in addressing these issues. The first (and fundamental) step in this doctoral project consisted of the development and characterization of murine chronic stress models for further potential study. Three distinct categories of stress were initially trialled: homotypic physical (restraint stress; RS), heterotypic variable (chronic unpredictable mild stress; CUMS), and a novel model of heterotypic social stress (social stress; SS). Differential behavioural outcomes were apparent across these models, with heterotypic variable and social stress paradigms found to induce ‘stress’ behaviours and phenotypes. In contrast, homotypic RS induced hedonia while reducing anxiety behaviours (potentially reflecting habituation). Somewhat unexpectedly, stress-related shifts in circulating mediators were more pronounced with social vs. the other stressors, suggesting particular sensitivity to social forms of stress. Gene expression changes in the frontal cortex, including inflammation, neurotransmitter, and neurotrophic genes, were apparent in CUMS, and less so in SS and RS. In contrast, hippocampal gene transcripts remained largely unchanged under all stress conditions. Results indicate that SS may induce a more stable neurobiological state (associated with anxiety- and depressive-like behaviours) indicative of a later disease state vs. an earlier (unstable) disease state in CUMS. Critically, this study revealed that despite differing modes of stress, anxiety behaviours were consistently linked to CNS expression of inflammatory and monoamine signalling mediators. Thus, independent of stressor type or duration, behavioural outcomes are similarly governed by neuroinflammatory and monoamine signalling changes. Having established that a novel social stressor paradigm appeared to induce the greatest behavioural and endocrine disruption - consistent with the importance of social forms of stress in social mammals (including humans) - studies in Chapter 3 investigated the sex-specific impacts of chronic social stress. It is to be noted that sex differences of chronic stress-induced mood disorders (such as MDD) are well documented in humans. For example, women are twice as likely as men to develop MDD, in addition to presenting with more severe symptoms. However, controversy exists within the field regarding the relative resilience or susceptibility of male and female rodents to chronic stress, and whether this aligns with outcomes in humans. Aligning with clinical observations, female mice did exhibit a heightened ‘biological sensitivity’ to social stress (greater body weight loss, heightened peripheral inflammation) compared with male mice. Conversely, males presented with a more pronounced behavioural phenotype based on traditional markers (presenting with depressive-like behaviours), while females exhibited anxiety without anhedonia. These differing outcomes were matched by sex specific shifts in circulating and CNS mediators, including greater shifts in circulating catecholamines and adipokines in males; and greater stress-related shifts in CNS genes in females. The data support a role for autonomic and adipokine signalling in driving differing outcomes in males and females, yet also highlight the challenges in studying and interpreting sex dependent responses to chronic stress. While most research into chronic psychological stress addresses impacts on neurobiology (with links to mood disorders such as MDD), chronic stress is also strongly linked to cardiovascular disease (CVD). Moreover, when both MDD and CVD interact, they reciprocally increase the risk of the other thereby increasing the risk of worsened outcomes. Chapter 4 sought to examine the mechanisms linking chronic stress to cardiac phenotype (specifically, cardiac function and resistance to ischaemic or infarction), and how these responses are influenced by sex. Worsened myocardial outcomes were evident with heterotypical stressors (variable and social) vs. homotypic restraint stress. Data revealed that myocardial ischaemic tolerance across all experimental models correlated significantly with circulating noradrenaline, and CNS transcript for MAOA and key inflammatory markers. This supports key roles for the ANS in communicating stress response to the heart and confirms roles for monoamine and inflammatory signalling in the CNS. Sexually dimorphic cardiac outcomes were also apparent, with social stress inflicting greater cardiac detriment in male vs. female mice. This dimorphism paralleled shifts in catecholamines, together with transcription of key regulators of myocardial metabolism. Finally, work in Chapter 5 details the transcriptome wide response to chronic social stress in the frontal cortex of male mice. The frontal cortex plays an important role in regulating emotional responses to psychological stress. The 'un-biased' profile revealed by RNA-sequencing supported a dominant role for structural/remodelling adaptations to stress, coupled with involvement of mitochondrial dysfunction, rather than orchestrated shifts in neurotransmitter and inflammatory pathways. This data supports existence of adaptive changes in neurotrophic signalling, together with maladaptive changes in mitochondrial and other relevant paths (e.g., myelination). Coexistence of beneficial adaptive and detrimental maladaptive changes suggests these animals may exist in an unstable pre-disease state. The relevance of this array of observations, and broader issues regarding chronic stress experimentation and interpretation are considered in Chapter 6.