Development of an early life stress model in larval zebrafish and analysis of stress-induced transcriptomic changes in hypothalamic cells

Abstract

The Hypothalamic-Pituitary-Adrenal (HPA) axis and its final effector, glucocorticoids (GCs), are important players in maintaining homeostasis of an organism upon stress exposure. However, overexposure to GCs during early life is involved in developmental programming of the HPA axis and is linked to detrimental effects in health. The hypothalamus is a key target for developmental programming due to its pivotal role as an integrator of input signals coming from sensory systems and other brain regions and as a translator of neuronal signals into endocrine signals. Yet, little is known about the molecular mechanisms involved in hypothalamic programming mediated by stressful experiences during early life. Elucidation of these mechanisms is essential for understanding the link between early life stress, dysregulation of the stress response, and detrimental health in later stages. Here, I used the zebrafish model to elucidate the molecular correlates of early adverse experience in hypothalamic cells. First, I developed a stimulation protocol using vortex flows to activate the hypothalamic-pituitary-interrenal (HPI) axis, the homolog of the HPA axis in teleost, and characterized the stress response at early stages by measuring cortisol (the main GC in zebrafish) and behavioral correlates. I then identified a critical time window in which HPI axis activity matures. Subsequently, I established an early life stress protocol to induce hypercortisolic states and alter stress response maturation. Endocrinological, behavioral, and cellular characterization of the early life stress paradigm showed an overall downregulation of the stress response with attenuated locomotor and cortisol response to subsequent stressful events as well as reduced calcium activity and expression of stress related peptides (AVP, CRH, and OXT) in hypothalamic cells. To dissect the molecular correlates of early adverse experience, I then performed transcriptomic analysis of hypothalamus-specific cell populations after exposure to the early life stress paradigm. Candidate molecules involved in the adaptive process occurring in hypothalamic cells were identified. Moreover, gene ontology and pathway analysis showed that lipid metabolism and molecular transport pathways were downregulated after zebrafish larvae were subjected to the early life stress protocol. In contrast, cellular movement and inflammatory response pathways were upregulated. Finally, I characterized the cortisol profiles of optogenetic and targeted transgenic tools which have been generated to manipulate the HPI axis activity in freely swimming larvae. Here, I show evidence of altered levels of endogenous cortisol in larvae that were manipulated at any of the three levels of the HPI axis. Altogether, the main contributions of this thesis are: 1) establishment of a novel stress protocol to activate the HPI axis in zebrafish larvae in a highly controlled and strength-dependent manner; 2) characterization of the cortisol response of developing zebrafish and identification of a critical time window of stress response maturation; 3) development of an early life stress paradigm and elucidation of the effects of early adverse experience at the cellular, behavioral, and endocrinological level; 4) identification of candidate molecules and metabolic pathways in hypothalamic cells involved in adaptive processes after early adverse experience, and 5) characterization of the cortisol profiles of optogenetic and genetic tools to manipulate the HPI axis activity at any of its three levels (hypothalamus, pituitary, and interrenal gland).