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Abstract

Disturbances in the serotonergic neurotransmitter system are known to play a pivotal role in the pathogenesis of many psychiatric diseases, particularly in depression. Further, depressive patients show alterations in the stress response, namely in the hypothalamic-pituitary-adrenal axis. The hypothalamic-pituitary-adrenal axis and the serotonergic neurotransmitter system share close interrelationships; glucocorticoids, as mediators of the body’s stress response, affect serotonergic neurons and vice versa. Understanding the interplay between those systems can help understand the pathogenesis of psychiatric diseases such as depression. Previous research suggested that glucocorticoids cause an immediate increase in extracellular hippocampal serotonin. However, the mechanisms of glucocorticoid receptor-induced serotonin release are unknown. This work examined the molecular process of immediate, glucocorticoid receptor-mediated serotonin release and uptake by employing murine stem cell-derived 5-HT neurons (1C11-5HT). It was investigated if glucocorticoids trigger rapid, vesicular serotonin release in vitro, and if there is spatial proximity between the glucocorticoid receptor and vesicular release sites, which is a prerequisite for a possible interaction. Lastly, it was investigated whether the activated glucocorticoid receptor shows spatial proximity to the membrane-bound serotonin transporter, which is a prerequisite to alter serotonin uptake. Employing life cell imaging with the vesicular dye FFN511 and the styryl dye FM4- 64FX, it was shown that dexamethasone causes immediate vesicular serotonin release and subsequent vesicle recycling and uptake, comparable to the vesicle release and uptake observed after depolarization with potassium chloride. Vesicle release was also observable in the absence of calcium in the extracellular medium and occurred on the level of neurites as well as somas. To further investigate the role of the glucocorticoid receptor in the release process, subcellular fractionation of mice central nervous system and subsequent immunological detection showed that the glucocorticoid receptor has spatial proximity to the vesicular and synaptic markers rab3 and synapsin 1 in vivo. Those results were confirmed in vitro, as it was shown that the glucocorticoid receptor colocalizes with the vesicular protein synaptotagmin 1. To examine whether the activated glucocorticoid receptor could potentially affect serotonin uptake by interacting with the membrane-bound serotonin transporter, colocalization analysis in 1C11-5HT showed an increased spatial proximity between the membrane-bound serotonin transporter and the activated glucocorticoid receptor 15 min after glucocorticoid receptor activation. 30 min after glucocorticoid receptor activation, colocalization decreased below the initial level. Both effects were not observable under the simultaneous application of the glucocorticoid receptor antagonist mifepristone. This work extends the knowledge of the interplay between glucocorticoids and serotonergic neurons. Firstly, it was shown that glucocorticoids cause an immediate release of serotonergic vesicles, which was independent from extracellular calcium and took place at neurites and somas. Secondly, the high proximity between the glucocorticoid receptor and vesicular markers is an indication that the glucocorticoid receptor might directly or via signaling pathways interact with vesicular release sites to trigger serotonin release. Thirdly, an increase of short-term colocalization between the membrane-bound serotonin transporter and the activated glucocorticoid receptor could indicate a possible interaction between the glucocorticoid receptor and the serotonin transporter to modify serotonin re-uptake. Extending the knowledge of the interrelationship between glucocorticoids as mediators of the body’s stress response and the serotonergic neurotransmitter system might ultimately help understand the underlying mechanisms in the pathogenesis of depression and other psychiatric disorders.