Corticotropin-releasing factor (CRF) and the urocortins (UCN1, UCN2 and UCN3) belong to the same CRF family of neuropeptides, having similar amino acidic structure (Fekete and Zorrilla, 2007), but different anatomical distributions (Reul and Holsboer, 2002), physiological functions (Domin and Smialowska, 2024) and pharmacological profiles (Dedic et al., 2018). CRF is a 41-amino acid peptide that was isolated first from sheep brain, but it was later detected in rats, mice, non-human primates and humans, as well (Vale et al., 1981). Human CRF presents 54 % homology with urotensin from the fish urophysis and 48 % homology with sauvagine from the frog skin, and it is best known for its ability to release pituitary corticotropin, also known as adrenocorticotropic hormone (ACTH) (Vale et al., 1981). CRF is synthesized predominantly in the paraventricular nucleus (PVN) of the hypothalamus, central amygdala (CeA) and locus coeruleus (LC), whereby it regulates the neuroendocrine, autonomic and behavioral responses to stress (Vale et al., 1981). UCN1 is a 40-amino acid peptide that was isolated first from the rat brain (Vaughan et al., 1995). Human UCN1 presents 63 % homology with fish urotensin and 45 % homology with human CRF, its name being derived from these two peptides (Vaughan et al., 1995). UCN1 is expressed predominantly in the non-preganglionic Edinger-Westphal nucleus (EWN), termed the centrally-projecting EWN (Vaughan et al., 1995). Unlike the classical, pre-ganglionic EWN that modulates the oculomotor function, the centrally-projecting EWN regulates the behavioral and biochemical responses to stress through its projections to several brain regions, including the lateral septum (LS), CeA, ventral tegmental area (VTA) and raphe nuclei (RN) (Kozicz, 2007). UCN2 is a 38-amino acid peptide identified first in the mouse brain (Reyes et al., 2001). In humans, it corresponds with stresscopin-related peptide, presenting 34 % homology with human CRF (Reyes et al., 2001). UCN2 is expressed predominantly in the paraventricular, supraoptic, and arcuate nuclei of the hypothalamus, and the LC (Reyes et al., 2001), and based on its mild locomotor suppressive and delayed anxiolytic-like effects it contributes to stress coping (Valdez et al., 2002). UCN3 is another 38-amino acid peptide that was identified together with UCN3 in the mouse brain (Lewis et al., 2001). In humans, its correspondent is stresscopin that presents 36 % homology with human CRF (Lewis et al., 2001). UCN3 is expressed in the hypothalamus, brainstem, LS and the bed nucleus of stria terminalis (BNST) (Lewis et al., 2001), and based on its mild locomotor suppressive and anxiolytic-like effects, it also plays role in stress coping (Valdez et al., 2003). CRF and the urocortins act through two distinct CRF receptors (CRF1 and CRF2). CRF1 is expressed predominantly in the cerebral cortex, cerebellum and anterior pituitary, whereas CRF2 expression is more abundant in the subcortical regions and posterior pituitary (Van Pett et al., 2000). CRF activates preferentially CRF1, binding with 15-fold higher affinity to CRF1, than CRF2 (Vale et al., 1981), while UCN1 activates equipotently both CRF receptors, binding with 7-fold higher affinity to CRF1 and 40-fold higher affinity to CRF2, than CRF itself (Vaughan et al., 1995). UCN2 has a 100-fold higher affinity for CRF2 (Reyes et al., 2001), while UCN3 has a 1000-fold higher affinity for CRF2 (Lewis et al., 2001), than for CRF1.

Serotonin (5-hydroxytryptamine, 5HT) is a neurotransmitter that is synthesized in the brain and the gut from tryptophan and catabolized by monoamine oxidase (MAO) and aldehyde dehydrogenase (ALDH) into its final metabolite, 5-hydroxyindoleacetic acid (5HIAA) (Teleanu et al., 2022). In the brain, the vast majority of 5HT is produced in the RN that are distributed near the midline of the brainstem and can be divided in the rostral and caudal raphe complex (Hornung and Fritschy, 1988). The neurons within the rostral raphe complex (caudal linear, dorsal raphe and median raphe nuclei) project primarily to forebrain, whereas neurons within the caudal raphe complex (raphe magnus, raphe obscurus, raphe pallidus nuclei and parts of the adjacent lateral reticular formation) project to the spinal cord (Hornung and Fritschy, 1988). The 5HT receptors found in these areas of projection of the RN can be divided into 7 families (5HT1–7), comprising totally 15 different receptors that promote various motor, sensory and limbic functions, such as pain, sleep and mood (Hornung and Fritschy, 1988). As regards the distribution of CRF receptors in the RN, CRF1 is expressed usually on glutamatergic and dopaminergic neurons, whereas serotoninergic neurons express more CRF2 (Lovenberg et al., 1995; Refojo et al., 2011). Actually, the RN is the only brain region expressing more abundantly CRF2, than CRF1 (Skorzewska et al., 2011). The dysbalance of serotoninergic neurotransmission was associated with a wide range of psychiatric and neurological disorders (Teleanu et al., 2022). For example, decreased 5HT level in the brain were observed in major depressive disorder, contributing to disturbances of sleep and mood, whereas increased 5HT level in the body can result in serotonin syndrome, consisting of symptoms, such as hyperthermia, tremors and diarrhea (Teleanu et al., 2022).

Stress, anxiety and depression affect the activity of the hypothalamic-pituitary-adrenal (HPA) axis and serotoninergic neurotransmission, both being regulated by CRF and CRF-related peptides (Fox and Lowry, 2013; Kormos and Gaszner, 2013). However, the exact action of CRF and urocortins on the 5HT release was not fully elucidated yet. Therefore, the aim of the present study was to investigate the actions of CRF and urocortins on the 5HT released from the RN, and the participation of CRF receptors in these actions. In order to do so, male Wistar rats were used, their RN were isolated and dissected, and then the RN slices were incubated with tritium-labelled 5HT, superfused and stimulated electrically. During superfusion, the RN slices were treated with 100 nM of CRF, UCN1, UCN2 or UCN3, and, when significant effect was observed, pretreated with 0.1 nM of selective CRF1 antagonist antalarmin or 1 nM selective CRF2 antagonist astressin2B. The release of tritium-labelled 5HT from the RN was determined by liquid scintillation counting. The doses of the materials used were based on our previous in vitro superfusion studies (Bagosi et al., 2006, Bagosi et al., 2008, Bagosi et al., 2012, Bagosi et al., 2015), and the method used was described originally by Gaddum (Gaddum, 1953) and later modified by Harsing (Harsing Jr., 2006).