Abstract
Hypocretins/Orexins neuropeptides are known to regulate numerous physiological functions, such as energy homeostasis, food intake, sleep/wake cycle, arousal and wakefulness, in vertebrates. Previous studies on mice have revealed an intriguing orexins/endocannabinoids (ECs) signaling interaction at both structural and functional levels, with OX-A behaving as a strong enhancer of 2-arachydonoyl-glycerol (2-AG) biosynthesis. In this study, we describe, for the first time in the brain of zebrafish, the anatomical distribution and co-expression of orexin (OX-2R) and endocannabinoid (CB1R) receptors, suggesting a functional interaction. The immunohistochemical colocalization of these receptors by confocal imaging in the dorsal and ventral telencephalon, suprachiasmatic nucleus (SC), thalamus, hypothalamus, preoptic area (PO) and cerebellum, is reported. Moreover, biochemical quantification of 2-AG levels by LC-MS supports the occurrence of OX-A-induced 2-AG biosynthesis in the zebrafish brain after 3 h of OX-A intraperitoneal (i.p.; 3 pmol/g) or intracerebroventricular (i.c.v.; 0.3 pmol/g) injection. This effect is likely mediated by OX-2R as it is counteracted by i.p./i.c.v administration of OX-2R antagonist (SB334867, 10 pmol/g). This study provides compelling morphological and functional evidence of an OX-2R/CB1R signaling interaction in the brain of adult zebrafish, suggesting the use of this well-established vertebrate animal model for the study of complex and phylogenetically conserved physiological functions.
Highlights
The high physiological and genetic homology to mammals, as well as the highly conserved anatomical and cytoarchitectural organization have made zebrafish (Danio rerio) a very useful animal model in the neuroscience to analyze both brain functions and dysfunctions (Haffter and Nüsslein-Volhard, 1996; Norton and Bally-Cuif, 2010; Wolman and Granato, 2012; Orexins and Endocannabinoid in Zebrafish BrainLeung et al, 2013; Varshney and Burgess, 2014; D’Angelo et al, 2016b)
The expression of OX-2R has been reported in several brain areas of adult zebrafish, which correspond to the orexinergic neuron projection areas, such as the dorsal and ventral telencephalon, the ventromedial (VM) hypothalamus; the ventral, lateral and caudal hypothalamic nuclei; the caudal zone of the periventricular hypothalamus; the thalamic nuclei; the pretectal nuclei; the periventricular gray zone (PGZ) of optic tectum; the periventricular nucleus of posterior tuberculum; the lateral nuclei in the dorsal tectum and tegmentum, and the locus coeruleus (Kaslin et al, 2004)
In the present study, using immunohistochemistry and confocal imaging, we carried out the neuroanatomical mapping of OX2R/CB1R co-expression in the whole zebrafish brain
Summary
The high physiological and genetic homology to mammals, as well as the highly conserved anatomical and cytoarchitectural organization have made zebrafish (Danio rerio) a very useful animal model in the neuroscience to analyze both brain functions and dysfunctions (Haffter and Nüsslein-Volhard, 1996; Norton and Bally-Cuif, 2010; Wolman and Granato, 2012; Orexins and Endocannabinoid in Zebrafish BrainLeung et al, 2013; Varshney and Burgess, 2014; D’Angelo et al, 2016b). Neuroanatomical evidence indicates that, for example, components of the zebrafish telencephalon are homologous to the mammalian hippocampus and amygdala, while the midbrain tectum shows similar organization to the cortex of amniotes (Kesavan et al, 2017) or to superior colliculus of mammals (Heap et al, 2018). These highly conserved brain areas are involved in the control of emotion, learning, memory and movements as in mammals (Wullimann and Mueller, 2004; Broglio et al, 2005; Mueller and Wullimann, 2009; Kesavan et al, 2017). Long-term food deprivation leads to a significant increase of pre-pro-orexin mRNA levels in zebrafish and goldfish brain, while intracerebroventricular (i.c.v) injection of OX-A induces feeding, as in mammals (Novak et al, 2005; Nakamachi et al, 2006; Yokobori et al, 2011)
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