Roubos EW et al. (2008), Brain distribution and evidence for both centra...

XB-ART-37162

J Comp Neurol 2008 Apr 01;5074:1622-38. doi: 10.1002/cne.21641.

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Brain distribution and evidence for both central and neurohormonal actions of cocaine- and amphetamine-regulated transcript peptide in Xenopus laevis.

Roubos EW , Lázár G , Calle M , Barendregt HP , Gaszner B , Kozicz T .

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We tested the hypothesis that, in the amphibian Xenopus laevis, cocaine- and amphetamine-regulated transcript peptide (CARTp) not only has widespread actions in the brain but also acts as a local factor in endocrine pituitary cells and/or is neurohemally secreted into the circulation to control peripheral targets. CARTp-immunoreactive cells occur in the olfactory bulb, nucleus accumbens, amygdala, septum, striatum, nucleus of Bellonci, ventrolateral nucleus, central thalamic nucleus, preoptic nuclei, and suprachiasmatic nucleus, and particularly in the medial pallium, ventromedial nucleus, hypothalamus, Edinger-Westphal nucleus, optic tectum, raphe nuclei, central gray, nucleus of the solitary tract, and spinal cord. From the hypothalamic magnocellular nucleus, CARTp-containing axons run to the neurohemal median eminence, and to the neural pituitary lobe to form neurohemal terminals, as shown by immunoelectron microscopy. Starvation increases the number of CARTp-cells in the optic tectum by 46% but has no effect on such cells in the torus semicircularis. CARTp does not affect in vitro release of alpha-melanophore-stimulating hormone from pituitary melanotrope cells. Our results support the hypothesis that in X. laevis, CARTp not only has multiple and not exclusively feeding-related actions in the brain but is also secreted as a neurohormone 1) into the portal system to control endocrine targets in the pituitary distal lobe and 2) from neurohemal axon terminals in the neural pituitary lobe to act peripherally. The differences in CARTp distribution between X. laevis and Rana esculenta may be related to different environmental and physiological conditions such as feeding, sensory information processing, and locomotion.

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Species referenced: Xenopus laevis
Genes referenced: bdnf cartpt cer1 crh pomc trh ucn3
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	Fig. 1. A–L: Distribution of CARTp-immunoreactivity in schematic drawings of the brain of Xenopus laevis. The left upper panel shows the transverse planes chosen for the illustrations. Planes A–C are from the telencephalon, D and E are from the diencephalon, F–I are from the mesencephalon, J and K are from the rhombencephalon, and L shows the spinal cord. The locations of immunoreactive perikarya and of nerve fibers (black small dots) are shown in the right half of each drawing. Neuroanatomical abbreviations (see list) are designated at the left.
	Fig. 2. Distribution of CARTp-immunoreactive cells and fibers in the telencephalon and the preoptic area. A: Immunostaining in the main olfactory bulb. Only a few cells are stained in the internal granular layer (igl). Sagittal section. B: Immunoreactive perikarya in the medial pallium (mp) and the septum (s). Sagittal section. C: Immunoreactive neurons and fiber terminals in the nucleus accumbens (Acc) and the striatum (Str). D: Three groups of immunoreactive neurons in the preoptic area (POa). Sagittal section. E: Intensely stained neurons in the magnocellular preoptic nucleus (Mg). Note caudal extent of the nucleus over the optic chiasm (oc). Sagittal section. gl, glomerular layer; lv, lateral ventricle; ml, mitral cell layer; olfn, olfactory nerve. Scale bar 100 m in A; 200 m in b–E.
	Fig. 3. CARTp-immunoreactive perikarya in the diencephalon and the mesencephalon. A: Immunoreactivity in the rostral diencephalon. Note stained cells in the nucleus of Bellonci (B; arrows). B: Immunoreactive cells and fibers in the infundibulum (inf) and the dorsal hypothalamic nucleus (dHy). C: Stained cells of the magnocellular preoptic nucleus (Mg) in the wall of the infundibulum (inf). Note that their long dendrites are oriented laterally, toward the surface of the brain. D: Immunoreactive cells in the hypothalamus and the Edinger- Westphal (EW) nucleus and intensely stained axon terminals in the neural lobe of the pituitary gland (pn). Sagittal section. Rostral is to the left. E: Immunostained infundibular neurons with long dendrites at the level of plane E in Figure 1. Note stained perikarya in the ventromedial (VM) and the posterior entopeduncular nuclei (epp). F: Immunostained fibers in the pars nervosa (pn) of the pituitary gland and median eminence of the hypothalamus. The intermediate pituitary lobe (pi) is immunonegative, whereas some cells in the distal lobe (pd) reveal weak immunoreactivity. G: Detail of the median eminence, with small and less numerous stained dots in the internal zone (iz) and large stained dots and short varicose fibers in the external zone (ez). Hd, dorsal habenula; Hv, ventral habenula. 3rd, third ventricle. Scale bar 200 m in A,B; 100 m in C,E; 300 m in D; 150 m in F; 15 m in G.
	Fig. 4. CARTp-immunoreactivity in the mesencephalon and rhombencephalon. A: Pretectal region. Note heavy staining of neurons in the central thalamic nucleus (C). B: Optic tectum. Note stained cells in layer 9 and intense fiber staining in the periventricular layers (pvl). C: Immunostained fibers and cells in the optic tectum (tect) and the magnocellular nucleus (mt) of the torus semicircularis. D: Sagittal section of the mesencephalon and the rhombencephalon. Note intense fiber staining in the tectum and fasciculus retroflexus (arrow), and heavily stained neurons in the central thalamic nucleus (C). E: Intense fiber staining in the isthmic region. Note that the isthmic (Is) and the interpeduncular (Ip) nuclei are free of immunostaining. F: Stained cells and fibers in the superior raphe nucleus (r). aq, cerebral aqueduct; cer, cerebellum; cg, central gray; Lpv, lateral thalamic nucleus, posteroventral division; P, posterior thalamic nucleus; Pn, pretectal neuropil; Pv, posteroventral tegmental nucleus; 4th, fourth ventricle. For other abbreviations, see list. Scale bar 300 m in A; 100 m in B,F; 200 m in C,E; 500 m in D.
	Fig. 5. CARTp-immunoreactivity in the rhombencephalon and the spinal cord. A: Neurons in the nucleus of the solitary tract (nst, arrow). B: Intense staining of fiber terminals in the ambiguous nucleus (IX–Xm, arrow). Note stained cells dorsomedially. C: Neurons in the gracile nucleus (Ng) of the dorsal column nuclei. D: Fiber terminals and neurons in the intermediate zone of the spinal cord. Horizontal section. E: Stained cells (arrows) medial and dorsal to the lateral motoneurons in the spinal cord in a lumbar segment. Note fiber staining in the dorsal horn (dh), and among unstained motoneurons (vh). The midline is to the left. af, anterior funiculus; cc, central canal; cg, central gray; lf, lateral funiculus; pf, posterior funiculus; sol, nucleus of the solitary tract; 4th, fourth ventricle. Scale bar 100 m in A–D; 200 m in E.
	Fig. 6. Immunoelectron microscopy of CARTp immunoreactivity in the neural lobe of the pituitary gland of X. laevis. A: Conventional fixation. Low-power micrograph with neurohemal axon terminals (a) and a capillary (c) with endothelial cell (e) and blood cell (b). B: Conventional fixation. Round secretory granules (g) in neurohemal axon terminal, with some immunogold particles (arrowheads) indicating the presence of CARTp. C: High-pressure freezing and cryosubstitution followed by silver intensification of postembedding immunogold labeling, showing strongly immunopositive neurohemal terminals (a). D: As in C but the axon terminal is shown in detail. E: As in C but immunonegative, as sections were processed with antiserum preadsorbed to CARTp. s, intercellular space. Scale bar 1 m in A,C; 100 nm in B; 250 nm in D; 300 nm in E.
	Fig. 7. CARTp-immunoreactivity in the X. laevis optic tectum, showing more stained perikarya (arrows) in starved animals (B) than in fed controls (A). Scale bar 25 m in A,B.
	Fig. 8. Effect of starvation on the number of CART-immunoreactive neurons in the optic tectum (expressed as average number per section); asterisk indicates statistically significant difference (P 0.01) between control and starved X. laevis. No effect is seen on the number of CART-immunoreactive neurons in the torus semicircularis. Data are expressed as mean standard error of the mean (n 5).
	Fig. 9. Absence of effect of increasing concentrations of CARTp (10 10 to 10 6 M) on the release of 3H-lysine-labeled peptides (mainly MSH; Scheenen et al., 1995) by dissociated melanotrope cells superfused in vitro. Viability and capability of release of melanotropes appears from stimulation by sauvagine (SA) and inhibition by dopamine (DA). Vertical gray bars indicate 10-minute periods of CARTp application. Each data point represents amount of radioactivity in a 2-minute sample. Secretion is expressed as percentage of control (unstimulated) secretion level, which was set at 100% (n 4).