<i>Bdnf</i> mRNA distribution in cross-sections through the adult zebrafish mid- and hindbrain.

<p>In the ventral hypothalamus (<b>A, A’, F and G</b>), the dorsal thalamic region (<b>B, B’</b>), the optic tectum (<b>C, C’ and G</b>), the posterior tuberal nucleus (<b>D, D’ and G</b>) and medulla oblongata (<b>E, E’ and H</b>). In A', B', C', D' and E’, cell nuclei are labeled in blue with DAPI. F, G and H are representative sections taken from the zebrafish atlas (Wullimann et al., 1996). <i>Bdnf</i>-expressing cells are represented by red dots. DiV: diencephalic ventricle, DP: dorsal thalamic nucleus; Hd: dorsal zone of the periventricular hypothalamus; Hv: ventral zone of the periventricular hypothalamus; IRF: inferior reticular formation; OT: optic tectum; PGZ: periventricular gray zone of the optic tectum. PTN: posterior tuberal nucleus. Scale bar: 120 μm.</p

Immunohistochemical characterization of <i>bdnf</i>-expressing cells in adult zebrafish brain.

<p>Double staining for <i>bdnf</i> mRNA (red) and PCNA protein (green) on cross-sections through the telencephalon (<b>A to A”‘</b>), the thalamus (<b>B and B’</b>) and the ventral hypothalamus (<b>B and B”</b>). A and B are representative sections taken from the zebrafish atlas (Wullimann et al., 1996). <i>Bdnf</i>-expressing cells are represented by red dots and PCNA-labeled cells are green dots. Dl: lateral zone of the dorsal telencephalon; Dm: medial zone of the dorsal telencephalon; Hyp: hypothalamus; TelV: telencephalic ventricle; Thal: thalamus; Vv: ventral zone of the ventral telencephalon. Scale bar = 200 μm in A’; 100 μm in A”; 50 μm in A”‘.</p

Expression of <i>bdnf</i> in the brain of adult zebrafish.

<p>+ = few cells; ++ = moderate number of cells; +++ = numerous cells.</p

<i>Bdnf</i> mRNA are expressed in the brain of 7 days old zebrafish.

<p>Olfactory rosettes (<b>A-B</b>), telencephalon (<b>A-F</b>), preoptic area (<b>E-H</b>), dorsal thalamus, (<b>I-J</b>), optic tectum (<b>G-H and K-L</b>). Figures B, D, F, H, J and L show cell nuclei labeled with DAPI. OR: olfactory rosettes; POA: preoptic area; Tel: telencephalon; Thal: thalamus; OT: optic tectum. Scale bar: 120 μm.</p

Transverse sections of adult zebrafish brain showing co-expression of <i>bdnf</i> mRNA with neuronal markers.

<p>Orthogonal projections of Z-stacks from telencephalon (11 sections of 0.5 μm) evidencing the expression of <i>bdnf</i> (red) in neurons identified by MAP2 protein (green) (<b>A and B</b>). Double staining for <i>bdnf</i> mRNA (red) and acetylated-tubuline (green) in the habenula (<b>C</b>), the thalamus (<b>D</b>) and the optic tectum (<b>E-E’</b>). In E’, cell nulei are labeled in blue with DAPI. Dl: lateral zone of the dorsal telencephalon; Had: dorsal habenular nucleus; OT: optic tectum; PGZ: periventricular gray zone of the optic tectum; TelV: telencephalic ventricle; Thal: thalamus; TL: torus longitudinalis. A, B, C and D were obtained with the confocal microscope. E and E’ were obtained with the Apotome. Scale bar: 50 μm in E and E’; 40 μm in A and C; 25 μm in B and D.</p

Immunohistochemical characterization of <i>bdnf</i>-expressing cells in adult zebrafish brain.

<p>A, B, C, D and E are representative sections taken from the zebrafish atlas (Wullimann et al., 1996). <i>Bdnf</i>-expressing cells are represented by red dots and Aromatase B (<b>A-D</b>) or BLBP-labeled cells (<b>E</b>) are represented by black dots with thin lines indicating radial glia cytoplasmic processes. Double staining for <i>bdnf</i> mRNA (red) and Aromatase B protein (green) on cross-sections through the telencephalon (<b>A-A’</b>), the preoptic area (<b>B-B’</b>), the entopedoncular nucleus (<b>B-B”</b>), the thalamus (<b>C-C’</b>) and the ventral hypothalamus (<b>D-D”</b>). Double staining for <i>bdnf</i> mRNA (red) and BLBP protein (green) on cross-sections through the telencephalon (<b>E-E”</b>). DiV: diencephalic ventricle; Dl: lateral zone of the dorsal telencephalon; Dm: medial zone of the dorsal telencephalon; ENv: endopedoncular nucleus; Hd: dorsal zone of the periventricular hypothalamus; Hv: ventral zone of the periventricular hypothalamus; POA: preoptic area; TelV: telencephalic ventricle; Vv: ventral zone of the ventral telencephalon. D’ and D” are obtained with an Apotome-equipped Zeiss. Scale bar = 60 μm in A’, C’, D’, D”, E’ and E”; 30 μm in B’ and B”.</p

Immunohistochemical characterization of <i>bdnf</i>-expressing cells in the brain of 7 days old zebrafish larvae.

<p>Double staining for <i>bdnf</i> mRNA (red) and PCNA protein (green) on cross-sections through the telencephalon (<b>A-B</b>) and the thalamus (<b>C-D</b>). Double staining for <i>bdnf</i> mRNA (red) and the neuronal marker Hu (green) in the thalamus (<b>E,F and G</b>), the optic tectum (<b>H, I and J</b>) and at the level of the superior reticular formation (<b>H, I and J</b>). In F and I cells nuclei are counterstained with DAPI. The dotted lines indicate the ventricles. OT: optic tectum; SRF: superior reticular formation; Tel: telencephalon; Thal: thalamus. Scale bar: 150 μm in H, I and J. 120 μm in A, B, C. 60 μm in D, E, F and G.</p

<i>Bdnf</i> mRNAs in juvenile zebrafish.

<p>+ = few cells; ++ = moderate number of cells; +++ = numerous cells.</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