Specimens and samples used for immunohistrochemistry and western blotting analysis.

<p>Specimens and samples used for immunohistrochemistry and western blotting analysis.</p

OXA and OXB single immunohistochemical detection.

<p>OXA immunopositive perivascular fibers in the submucosal layer of main stomach (a); OXA ir neurons and nervous fibers in the myenteric plexus of proximal intestine (b); OXB ir neurons of the submucosal plexus of pyloric stomach (c) and myenteric plexus of proximal intestine (d). Scale bars: 5 µm (a); 20 µm (b); 10 µm (c, d).</p

Orexin system protein expression.

<p>Western blot for orexin 1 and 2 receptors, prepro-orexin and β-actin (used as internal marker) in forestomach, main stomach, pyloric stomach, proximal and distal intestine.</p

OXA plasma levels.

<p>Bottlenose dolphin plasma OXA concentration (mean ± SD) at 10∶00 and 17∶30 hours.</p

Specimens and samples used for immunohistrochemistry and western blotting analysis.

<p>Specimens and samples used for immunohistrochemistry and western blotting analysis.</p

Data_Sheet_2_Overlapping Distribution of Orexin and Endocannabinoid Receptors and Their Functional Interaction in the Brain of Adult Zebrafish.docx

<p>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.</p