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The amphibian Xenopus laevis can adapt the color of its skin to the light intensity of the background. A key peptide in this adaptation process is alpha-melanophore-stimulating hormone (alpha-MSH), which is derived from proopiomelanocortin (POMC) and released by the endocrine melanotrope cells in the pituitary pars intermedia. In this study, the presence of alpha-MSH in the brain, cranial placode derivatives, and retina of developing Xenopus laevis was investigated using immunocytochemistry, to test the hypothesis that POMC peptide-producing neurons and endocrine cells have a common embryonic origin and a common function, i.e., controlling each other's activities and/or being involved in the process of physiological adaptation. The presence of alpha-MSH-positive cells in the suprachiasmatic nucleus, ventral hypothalamic nucleus, epiphysis, and endocrine melanotrope and corticotrope cells, which are all involved in regulation of adaptation processes, has been detected from stage 37/38 onward. This is consistent with the presumed common origin of these cells, the anterior neural ridge (ANR) of the neural-plate-stage embryo. The olfactory epithelium and the otic and epibranchial ganglia also contain alpha-MSH, indicating that these placodal derivatives originate from a common placodal domain continuous with the ANR. Furthermore, we demonstrate the presence of alpha-MSH in a subpopulation of retinal ganglion cells (RGCs), which is possibly also derived from the ANR. Immunoreactivity for alpha-MSH in RGCs that are located in the dorsal part of the retina is dependent on the background light intensity, suggesting that these cells are involved in the regulation of background adaptation. Taken together, the results support the hypothesis that POMC peptide-producing cells have a common embryonic origin and are involved in adaptation processes., (Copyright 2002 Wiley-Liss, Inc.)

Adaptation of the skin colour to the background light condition in the amphibian Xenopus laevis is achieved by migration of pigment granules in the skin melanophores, a process regulated by alpha-MSH secretion from melanotrope cells in the pituitary pars intermedia (PI). alpha-MSH secretion in turn, is regulated by various stimulatory and inhibitory messengers synthesized in brain nuclei, especially the hypothalamic suprachiasmatic and magnocellular nuclei and the locus coeruleus in the hindbrain. In the present study, the roles in background adaptation of nitric oxide (NO) and NO synthase (NOS) enzyme activity were evaluated. In situ, using both immunohistochemistry with anti-human brain NOS (bNOS) serum in paraffin-embedded material and using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry in cryo-sections, we showed NOS in neurons in the optic tectum and in the locus coeruleus. NADPH-d reactivity was also found in neurons in the lateral amygdala, the ventral hypothalamic nucleus and in fibers in the median eminence. Using a Western blot stained with an anti-human bNOS serum, we demonstrated a 150 kDa band in Xenopus hindbrain lysates, which is similar to the NOS protein present in the rat anterior pituitary, but which was not detectable in the lysates from both the neurointermediate and distal lobes in Xenopus. No differences in histochemical staining pattern or on Western blotting were observed between animals adapted to a black or a white background. Paraffin sections of the endocrine PI and pars distalis did not reveal bNOS-like immunoreactivity. NADPH-d reactivity was observed in the endothelia of this gland. However, using a new procedure of thin cryo-sections of pituitary neurointermediate lobes, we observed bNOS-immunoreactive fibers as well as cyclic 3',5' guanosine monophosphate (cGMP)-accumulating fibers in the PI. The PI may be regulated by NOergic neurons from higher brain centers. The possibility that NOergic neurons in the locus coeruleus are involved in the innervation of the PI needs further investigation. The latter neurons are probably not noradrenergic because double labeling studies show no co-localization of NADPH-d reactivity and tyrosine hydroxylase immunoreactivity in locus coeruleus neurons.

Our research focusses on the role of brain and hypophysis in the control of background adaptation in the clawed toad, Xenopus laevis. This adaptation is regulated by alpha-melanophorestimulating hormone (alpha-MSH). Previously, it was shown that various neurotransmitters influence alpha-MSH release. Here we report about the origin of these factors. Using retrograde labelling techniques combined with immunocytochemistry, it was found that the inhibitory transmitters dopamine and neuropeptide Y coexist in neurons in the suprachiasmatic nucleus. These neurons project to the pars intermedia and synaptically contact the alpha-MSH-producing melanotrope cells. In the synapses also GABA is present. Tracing of the optic nerve indicated the presence of a direct retinosuprachiasmatic tract. Furthermore, locus coeruleus neurons project to the pars intermedia. They contain the inhibitory transmitter noradrenaline. The stimulatory factors corticotropin-releasing hormone and thyrotropin stimulating hormone originate from the magnocellular nucleus which send its processes to the neural lobe of the hypophysis.

To get more insight into developmental aspects of catecholamine systems in vertebrates, in particular anuran amphibians, these systems were studied immunohistochemically in embryos and larvae of Xenopus laevis and Rana ridibunda. Antisera against tyrosine hydroxylase (TH) and dopamine (DA) revealed that catecholamine systems are already present at early embryonic stages. The first dopamine group to be detected was found ventral to the central canal of the spinal cord of Xenopus, soon followed by DA cell groups in the posterior tubercle, the hypothalamic periventricular organ, the accompanying cell group of the periventricular organ, and the suprachiasmatic nucleus. Although weakly TH-immunoreactive cells were found in the olfactory bulb at about the same embryonic stages, DA immunoreactivity was not detected until premetamorphic stage 49. Dopamine cell groups in the caudal brainstem, midbrain, and pretectum appeared at late premetamorphic and prometamorphic stages, whereas the preoptic group was first observed at the metamorphic climax stage. Rana showed an almost similar timetable of development of catecholamine cell groups, except for the caudal brainstem group which was already present at the end of the embryonic period. When compared with previous studies by means of formaldehyde-induced fluorescence technique, it becomes clear that TH/DA immunohistochemistry enables an earlier detection of catecholamine cell groups and fiber systems in anuran amphibians. The present study also revealed that the DA-immunoreactive cells of the hypothalamic periventricular organ never stained with the TH antiserum during development, thus supporting their putatively DA accumulating nature. Another notable result is the site of origin and rather late appearance of the midbrain dopaminergic cell group. It is suggested that the latter cell group only partly corresponds to the ventral tegmental area and substantia nigra of amniotes.

The distribution of dopamine (DA) and the biosynthetic enzyme tyrosine hydroxylase (TH) has been studied immunohistochemically in the brain of the adult South African clawed frog, Xenopus laevis. The goals of the present study are, firstly, to provide detailed information on the DA system of the brain of a species which is commonly used in laboratories as an experimental model and, secondly, to enhance our insight into primitive and derived characters of this catecholaminergic system in amphibians. Dopamine-immunoreactive cell bodies are present in the olfactory bulb, the preoptic area, the suprachiasmatic nucleus, the nucleus of the periventricular organ and its accompanying cells, the nucleus of the posterior tubercle, the posterior thalamic nucleus, the midbrain tegmentum, around the solitary tract, in the ependymal layer along the midline of the caudal rhombencephalon, and along the central canal of the spinal cord. In contrast to the DA antiserum, the TH antiserum fails to stain the liquor-contacting cells in the periventricular organ. On the contrary, the latter antiserum reveals additional immunoreactive cell bodies in the olfactory bulb, the isthmic region and the caudal brainstem. Both antisera yield an almost identical distribution of fibers. Distinct fiber plexuses are observed in the olfactory bulb, the basal forebrain, the hypothalamus and the intermediate lobe of the hypophysis. Features that Xenopus shares with other anurans are the larger number of DAi cells, which are generally smaller in size than those observed in urodeles, and the lack of DAi fibers in pallial structures. On the other hand, the paired midbrain DA cell group and the innervation of the tectum of Xenopus resemble those found in the newt rather than those in frogs. Despite the existence of these species differences, the brain of Xenopus offers an excellent model for studying general aspects of neurotransmitter interactions and the development of catecholamine systems in this class of vertebrates.

The projections from the subiculum to histaminergic cells in the posterior hypothalamic region of the rat were studied by means of anterograde neuroanatomical tracing with Phaseolus vulgaris-leucoagglutinin (PHA-L) combined with histidine decarboxylase (HDC)-immunohistochemistry. PHA-L was injected at various loci along the dorsoventral and proximodistal axes of the subiculum. This resulted in labeling of the fornix and of terminal plexuses at various locations in the diencephalon and the mammillary body. Following deposition of PHA-L in the proximal part of the dorsal subiculum, labeled fibers in the posterior hypothalamus were confined to the mammillary nuclei, whereas after injections of PHA-L in the distal part of the dorsal subiculum and the entire ventral subiculum, labeled fibers were also present in clusters of histaminergic cells located around the mammillary nuclei. The density of the PHA-L labeled fibers within these clusters increased from low to moderate in association with a shift of the injection sites from dorsal to ventral and from proximal to distal parts of the subiculum, i.e., the highest fiber labeling was seen after injections of PHA-L in the distal part of the ventral subiculum. In the latter experiments, PHA-L labeled fibers reached HDC-immunoreactive neurons in the tuberal magnocellular nucleus, the deepest layer of the caudal magnocellular nucleus, the two bridges of histaminergic cells in the posterior hypothalamus, and the histaminergic neurons scattered in the supramammillary region. A few labeled fibers invaded the postmammillary caudal magnocellular nucleus. The presence of varicosities on the PHA-L labeled fibers in close proximity to the cell bodies and dendrites of the histaminergic neurons suggest the existence of synaptic contacts.