Stress responses are fundamental for the survival of an organism and constitute an adaptive reaction to a real or perceived threat. Neuronal, endocrine, and behavioral responses are activated after the perception of a stressor and are aimed to re-establish the organism's homeostasis. A complex network of neuronal circuits involving several brain regions orchestrates the activation of the major stress system, the Hypothalamic Pituitary Adrenal (HPA) axis, and most of the neuronal populations that control its activity reside in hypothalamic nuclei. Activation of the HPA axis starts within the paraventricular nucleus of the hypothalamus, in which a population of peptidergic neurons secretes corticotropin-releasing hormone (CRH) into the circulation, which in turn initiates a signaling cascade culminating with the production of cortisol. The activity of the HPA axis and CRH-producing neurons must be tightly controlled and rapidly modulated to ensure proper responses to threats but also to avoid overactivation of the stress system, which is deleterious for an animal’s wellbeing. However, the hypothalamic circuits responsible for the activation and termination of the stress response and the underlying neuromodulatory mechanisms are still largely unknown. In this study, I used the zebrafish larva as a model to elucidate the hypothalamic neuronal circuits modulating the behavioral and neuroendocrine responses to acute stress. The transparent and small larval zebrafish brain provides the unique opportunity to study neuronal responses in vivo, facilitating the identification of the neuronal populations modulating stress. Importantly the main brain regions and stress axes are conserved between mammals and teleosts. The Hypothalamic Pituitary Interrenal (HPI) axis is homologous to the mammalian HPA axis and its activation is controlled by Crh-producing neurons located in the preoptic area (PoA), homologous to the mammalian paraventricular nucleus. Among the peptidergic neurons of the hypothalamus that might be involved in the modulation of acute stress, I chose to focus on a population of cells secreting the neuropeptide Galanin (Galn). I identified in the PoA of zebrafish a subpopulation of Galanin-producing neurons (Galn+) highly responsive to different types of stressful stimuli. Ablation of Galn+ neurons led to exacerbated stress responses, elevated cortisol levels, and caused increased activation of Crh-producing neurons, suggesting an inhibitory effect of Galn+ neurons over the HPI axis. I also found that Galn+ neurons in the PoA of zebrafish larvae are GABAergic, suggesting the possibility that GABA is the neurotransmitter released by Galn+ neurons to inhibit downstream stress-promoting circuits. I further investigated the molecular mechanisms by which Galn+ neurons negatively modulate stress responses by manipulating the peptide Galn. Lack of Galn elicited a diminished response to stress and increased the activity of Galn+ neurons in the PoA. Conversely, overexpression of Galn exacerbated stress- related responses and decreased the activity of Galn+ neurons, suggesting a self-inhibitory action of Galn peptide on Galn+ neurons. Taken together, the results reported in this study indicate that Galn+ neurons in the PoA negatively modulate Crh+ neurons, likely through GABAergic transmission, to prevent overactivation of the HPI axis. In parallel, the neuropeptide Galn mediates an additional modulatory control within this hypothalamic circuit, reducing the activity of Galn+ neurons through an autocrine mechanism. This dual system likely regulates a balance of activation and inhibition over Crh+ neurons, which allows fine-tuning of the HPI axis activity and mediate behavioral responses to stress.
