Investigating the molecular and neurobehavioral effects of monoamine antidepressants and ketamine in a rodent model of ACTH-induced HPA axis dysfunction

Abstract

Major depressive disorder (MDD) is a debilitating mental illness and is among the leading causes of disability worldwide, severely impacting patients' quality of life. The pathophysiology of MDD is multifactorial. Several theories have been proposed, including neuroinflammation, monoamine and neurotrophic dysregulation, hippocampal atrophy, and excitatory/inhibitory imbalances. Of these theories, hypothalamic-pituitary-adrenal (HPA) axis dysregulation is suggested as a frequently pathophysiological mechanism of MDD. HPA axis dysregulation is attributed to chronic stressors, both physiological and emotional, which disrupt glucocorticoid signalling and impair negative feedback mechanisms. Studying the pathophysiology of MDD in humans is limited by confounding factors and access to post-mortem tissues. Therefore, in rodents, administration of exogenous adrenocorticotropic hormone (ACTH) has been used to induce HPA axis dysregulation and produce depressive-like responses. However, there is a lack of comprehensive neurobehavioral and mechanistic evidence to support the validity of this model. In addition, the investigation of the molecular effects of different classes of antidepressants is important to understand the clinical translation of this model. In Chapter 2, I developed and validated the ACTH model using a battery of neurobehavioural tests. Previous studies demonstrated the inefficacy of tricyclic antidepressants (TCAs) in this model suggesting that ACTH-induced HPA axis dysregulation may be associated with treatment resistant depression (TRD), therefore, I also investigated the effect of impramine, a TCA, in this chapter. Male and female Sprague-Dawley rats were randomly assigned to the control or ACTH groups that received saline or ACTH, respectively, for two weeks. Thereafter, rats in the ACTH group were subdivided to receive ACTH plus saline or ACTH plus imipramine for a further four weeks. Neurobehavioral changes were assessed using the forced swim test (FST), open field test (OFT), and sucrose preference test (SPT). The brain regional mRNA expression of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB), neuroplasticity molecules implicated in depression pathology, was determined using real-time polymerase chain reaction (RT-PCR). ACTH administration resulted in a depressive-like phenotype, significantly increasing immobility in the FST, decreasing interaction with the centre of the OFT, and increasing sucrose consumption in male, but not female rats. ACTH administration significantly increased the expression of BDNF, a neurotrophin important for synaptic plasticity, in the hippocampus and CREB, a transcription factor that regulates neuronal development, in all brain regions in males, but not in female rats. Co-treatment with imipramine did not ameliorate these ACTH-induced neurobehavioral or molecular changes. This chapter showed that ACTH administration resulted in a sex-specific onset of depressive-like symptoms and changes in brain regional expression of neuroplasticity markers, suggesting sex-specific mechanisms in a model of ACTH-induced HPA axis dysregulation. Furthermore, molecular analyses indicate that chronic ACTH alters BDNF and CREB signalling, suggesting that dysregulated neurotrophic signalling is associated iv with HPA axis dysregulation. Although these agents primarily act to regulate neurotransmitters, their effects on neurotrophic signalling, particularly in the ACTH model, remain poorly understood. Following the development and neurobehavioural evaluation of the ACTH model, in Chapter 3, I further validated the model by comparing the neuroplasticity effects of imipramine, a TCA, to citalopram, a selective serotonin reuptake inhibitor (SSRI) and first-line therapy for MDD. The ACTH model was repeated with experimental animals being treated with either citalopram or imipramine for four weeks, respectively. I determined the brain regional mRNA expression of intracellular markers of neurotrophic signalling, including BDNF, CREB, tropomyosin receptor kinase B (TrkB), protein kinase B (Akt), mechanistic target of rapamycin (mTOR), and eukaryotic elongation factor 2 (eEF2) using RT- PCR. In addition, the brain distribution of citalopram and imipramine was determined using atmospheric pressure matrix-assisted laser desorption/ionisation mass spectrometry imaging (AP- MALDI-MSI). ACTH administration increased hippocampal BDNF mRNA expression, coupled with decreased mRNA expression of intermediate neurogenesis signalling factors, TrkB, Akt, mTOR, and eEF2. Citalopram showed a greater capacity to ameliorate ACTH-induced neurotrophic dysregulation compared to imipramine, particularly by enhancing BDNF-TrkB-mTOR signalling, independent of brain drug distribution. These findings highlight the distinct pharmacodynamic effects of different classes of antidepressants in targeting dysregulated neurotrophic signalling. Despite the widespread use of monoamine-based antidepressants, approximately one-third of individuals diagnosed with MDD have TRD, where patients do not respond to two or more classes of conventional antidepressants. Ketamine has emerged as a novel, rapid-acting therapeutic agent for the management of TRD. Unlike monoamine antidepressants that require months to reach their full therapeutic effect, ketamine provides antidepressant relief following a single dose. However, the respective effects of acute and chronic ketamine treatment on neurobehavior, as well as on neurotrophic and monoamine neurotransmitter signalling in an ACTH model of depressive-like symptoms, remain unclear. In Chapter 4, I investigated the behavioural, molecular, and neurochemical effects of acute and chronic ketamine treatment in ACTH-treated rats. In the acute group, following two weeks of ACTH administration, rats received a single dose of ketamine. In the chronic group, after two weeks of ACTH administration, rats received a ketamine injection once a week for four weeks. Neurobehavioural changes were assessed using the FST, OFT, and SPT. Following termination, brain regional mRNA expression of BDNF, TrkB, Akt, mTOR, eEF2, and CREB was measured using RT-PCR. Brain regional protein expression of phosphorylated-CREB (p-CREB) and corticosterone were assessed using an enzyme-linked immunosorbent assay (ELISA). In addition, serotonin, dopamine, and norepinephrine neurotransmitter abundance were determined at various bregma levels using MALDI-MSI. ACTH administration increased behavioural despair in the FST and altered corticosterone and p-CREB protein v expression, mainly in the prefrontal cortex. However, by week six, neurobehaviour and gene and protein expression remained largely unchanged, suggesting a state of homeostatic adaptation. At week six, ACTH-treated rats showed changes in serotonin and norepinephrine levels in the hippocampus and midbrain. Acute ketamine treatment effectively reversed ACTH-induced behavioural despair in the FST and modulated the region-specific expression of monoamines (serotonin) and increased CREB mRNA expression. With chronic ketamine treatment, besides increased striatal mTOR mRNA expression, there were limited effects on the expression of neuroplasticity markers. Our findings indicate that when administered acutely, ketamine modulates neurotrophic pathways and monoaminergic neurotransmission, which impacts behavioural outcomes. In contrast, the minimal behavioural and molecular effects of repeated ketamine treatment suggest that prolonged exposure attenuates ketamine’s neuroplastic and antidepressant actions in this model. These findings highlight the differential mechanism of action of ketamine following acute and chronic dosing, in a model of HPA axis dysregulation. In conclusion, the findings from this thesis contribute to our understanding of the molecular mechanisms underlying the neurobehavioural effects of chronic exogenous ACTH administration in rodents as well as the model-specific effects of imipramine, citalopram, and ketamine. I provide evidence that chronic ACTH exposure induces depressive-like behaviours as assessed using the FST, OFT, and SPT. These effects were sex-dependent, with male rats exhibiting both behavioural changes and elevated hippocampal BDNF and CREB mRNA expression, while females were largely unaffected. I further demonstrate that citalopram was more effective than imipramine at ameliorating ACTH- induced disruptions in the mRNA expression of neurogenesis-related signalling factors across brain regions. I also show that acute ketamine treatment has more pronounced effects on ACTH-induced neurobehaviour and molecular changes, than chronic ketamine. Overall, this thesis provides evidence that chronic ACTH administration models HPA-dysregulation induced depressive-like symptoms, resulting in disturbances in neurotrophic and monoaminergic signalling. Moreover, neurogenesis- enhancing agents such as SSRIs and acute ketamine may offer greater therapeutic efficacy than classic antidepressants in depressive disorders where HPA axis dysfunction is a core aetiological feature