Differential distribution and regulation of expression of synaptosomal-associated protein of 25 kDa isoforms in the Xenopus pituitary gland and brain.

Synaptosomal-associated protein of 25 kDa (SNAP-25) regulates various membrane fusion processes including exocytosis by endocrine and neural cells. To increase our understanding of the occurrence and regulation of SNAP-25 isoforms, we identified and characterized SNAP-25a and SNAP-25b mRNAs in the pituitary gland and brain of the amphibian Xenopus laevis. The proteins are strongly conserved and are resistant to botulinum neurotoxin A but not to botulinum neurotoxin E, as shown by Western blotting. The spatial distribution of the two SNAP-25 isoforms was assessed with in situ hybridization. Both SNAP-25a mRNA and SNAP-25b mRNA reside in cells in the pituitary distal lobe and, particularly, in the endocrine melanotrope cells in the pituitary intermediate lobe. The melanotrope cells are involved in the background adaptation process of the skin by releasing alpha-melanophore-stimulating hormone. Quantitation of the respective in situ hybridization signals in the Xenopus pars intermedia indicated a differential response, SNAP-25b mRNA being more highly expressed in black-adapted animals than SNAP-25a mRNA, and more than in white-adapted toads. This differential upregulation was also studied by real-time reverse transcriptase polymerase chain reaction, showing that in the intermediate pituitary lobe, both isoforms are physiologically controlled by the background light intensity stimulus, but with different intensities; in black-adapted animals SNAP-25b mRNA is upregulated by 3.33 times compared with white-adapted animals, but SNAP-25a only by 1.96 times. As to neural tissue, in situ hybridization showed that both isoforms coexist throughout the brain, sometimes with similar strengths, but in various areas either SNAP-25a mRNA or SNAP-25b mRNA expression is prevalent. It is speculated that each of the SNAP-25 isoforms in the Xenopus pituitary and brain has a distinct function in cellular fusion processes including secretion, and that their occurrence and regulation depend on the type of secreted neurotransmitter/hormone and/or the activity state of the cell.

Dynamics and plasticity of peptidergic control centres in the retino-brain-pituitary system of Xenopus laevis.

This review deals particularly with the recent literature on the structural and functional aspects of the retino-brain-pituitary system that controls the physiological process of background adaptation in the aquatic toad Xenopus laevis. Taking together the large amount of multidisciplinary data, a consistent picture emerges of a highly plastic system that efficiently responds to changes in the environmental light condition by releasing POMC-derived peptides, such as the peptide alpha-melanophore-stimulating hormone (alpha-MSH), into the circulation. This plasticity is exhibited by both the central nervous system and the pituitary pars intermedia, at the level of molecules, subcellular structures, synapses, and cells. Signal transduction in the pars intermedia of the pituitary gland of Xenopus laevis appears to be a complex event, involving various environmental factors (e.g., light and temperature) that act via distinct brain centres and neuronal messengers converging on the melanotrope cells. In the melanotropes, these messages are translated by specific receptors and second messenger systems, in particular via Ca(2+) oscillations, controlling main secretory events such as gene transcription, POMC-precursor translation and processing, posttranslational peptide modifications, and release of a bouquet of POMC-derived peptides. In conclusion, the Xenopus hypothalamo-hypophyseal system involved in background adaptation reveals how neuronal plasticity at the molecular, cellular and organismal levels, enable an organism to respond adequately to the continuously changing environmental factors demanding physiological adaptation.

Localization and physiological regulation of the exocytosis protein SNAP-25 in the brain and pituitary gland of Xenopus laevis.

In mammals, the synaptosomal-associated protein of 25 kDa, SNAP-25, is generally thought to play a role in synaptic exocytosis of neuronal messengers. Using a polyclonal antiserum against rat SNAP-25, we have shown the presence of a SNAP-25-like protein in the brain of the South-African clawed toad Xenopus laevis by Western blotting and immunocytochemistry. Xenopus SNAP-25 is ubiquitously present throughout the brain, where its distribution in various identified neuronal perikarya and axon tracts is described. Western blot analysis and immunocytochemistry also demonstrated the presence of SNAP-25 in the neural, intermediate and distal lobes of the pituitary gland. Intensity line plots of confocal laser scanning microscope images of isolated melanotropes indicated that SNAP-25 is produced and processed in the rough endoplasmatic reticulum and Golgi apparatus, and is associated with the plasma membrane. Immunoelectron microscopy substantiated the idea that SNAP-25 is present in the plasma membrane but also showed a close association of SNAP-25 with the bounding membrane of peptide-containing secretory granules in both the neurohemal axon terminals in the neural lobe and the endocrine melanotropes in the intermediate lobe. Quantitative Western blotting revealed that adapting Xenopus to a dark background has a clear stimulatory effect on the expression of SNAP-25 in the neural lobe and in the melanotrope cells. This background light intensity-dependent stimulation of SNAP-25 expression was confirmed by the demonstration of increased immunofluorescence recorded by confocal laser scanning microscopy of individual melanotropes of black background-adapted toads. On the basis of this study on Xenopus laevis, we conclude that SNAP-25 (i) plays a substantial role in the secretion of a wide variety of neuronal messengers; (ii) functions in the central nervous system but also in neurohormonal and endocrine systems; (iii) acts at the plasma membrane but possibly also at the membrane of synaptic vesicles and peptide-containing secretory granules; (iv) acts not only locally (as in synapses), but at various sites of the plasma membrane (as in the endocrine melanotrope cell); and (v) can be upregulated in its expression by physiological stimuli that increase the extent of the molecular machinery involved in exocytosis.

Co-expression in Xenopus neurons and neuroendocrine cells of messenger RNA homologues of exocytosis proteins DOC2 and munc18-1.

The proteins munc18-1 and DOC2 are assumed to play a role in docking of synaptic vesicles in neurotransmitter exocytosis at the presynaptic junction. As the proteins are known to interact, they should co-exist within neurons. We have tested this hypothesis for exocytosis of both classical and peptidergic messengers, by investigating the distribution of the messenger RNAs of munc 18-1 and DOC2 homologues in the brain and pituitary gland of the clawed toad Xenopus laevis, using in situ hybridization. For this purpose we cloned a partial complementary DNA encoding Xenopus unc18 (xunc18) and used a corresponding RNA probe, together with an RNA probe for Xenopus DOC2. At the messenger RNA level DOC2 and xunc18 were found to be expressed throughout the Xenopus brain. All brain nuclei expressing DOC2-messenger RNA showed xunc18-messenger RNA expression as well. Co-expression was shown at the individual cell level in consecutive sections of large-sized neurons. A strong expression was demonstrated in the suprachiasmatic and magnocellular nuclei and in peptidergic endocrine cells in the intermediate and anterior lobes of the pituitary gland, suggesting roles of DOC2 and xunc18 in messenger release from peptidergic secretory systems. Combined in situ hybridization and immunocytochemical analyses show that neuropeptide Y-containing cells in the suprachiasmatic nucleus also express DOC2 and xunc18 messenger RNAs. Since these cells have a high secretory activity, controlling the activity of the pituitary pars intermedia, the levels of expression of DOC2 and xunc18 may be indicators for neuronal secretory activity. The present data represent the first evidence for the co-existence of DOC2 and munc18-1 and suggest co-ordinate action of these proteins at the level of brain nuclei, individual neurons and endocrine cells.

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

Topographical relationship between neuronal nitric oxide synthase immunoreactivity and cyclic 3',5'-guanosine monophosphate accumulation in the brain of the adult Xenopus laevis.

Previous immunohistochemical staining procedures of the brain and pituitary in Xenopus laevis, using an antiserum against neuronal nitric oxide (NO) synthase (nNOS) and nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry, have revealed NOS activity in neurons and fibers in a number of brain areas, as well as in fibers in the pituitary. In the present study we have localized the target structures of the NOergic system in the Xenopus brain by visualizing the sites of NO-sensitive cyclic 3',5'-guanosine monophosphate (cGMP) accumulation, according to a method for cGMP visualization in rat brain slices. Brain slices of unfixed Xenopus are incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine and the NO donor sodium nitroprusside, followed by fixation and cryosectioning. Sections were then processed for immunohistochemistry using rabbit and sheep antisera against cGMP and a sheep antiserum against nNOS. Visualization of single and double labeling of cGMP immunoreactive and/or nNOS immunoreactive structures was performed with combined CY3/fluorescein isothiocyanate fluorescence microscopy. Following this procedure, we provide immunohistochemical evidence for the distribution of cGMP-accumulating neurons in the brain of adult Xenopus. In most brain areas, the distribution of nNOS and cGMP immunoreactive structures (neuron somata and fibers) is distinct and separate, for instance in the dorsal pallium, the lateral thalamic nuclei, the optic tectum, the locus coeruleus and the reticular formation. However, nNOS and cGMP immunoreactive structures are often found in the vicinity of each other, and in the optic tectum even in adjacent neuron fibers and somata. The present observations are in line with the presence of an NO-dependent soluble guanylate cyclase in distinct brain areas of Xenopus laevis, corroborating similar data in the mammalian brain. Further, our observations may add to the understanding of the anatomical connectivity pattern and functional relevance of the NOergic system in the amphibian brain.

Identification of suprachiasmatic melanotrope-inhibiting neurons in Xenopus laevis: a confocal laser-scanning microscopy study.

The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of alpha-melanophore-stimulating hormone (alpha-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of alpha-MSH is inhibited by gamma-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1'dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY+ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis.

Distribution of pro-opiomelanocortin and its peptide end products in the brain and hypophysis of the aquatic toad, Xenopus laevis.

Using in situ hybridization with a pro-opiomelanocortin (POMC)-mRNA probe and immunocytochemistry with antisera to POMC and to various POMC-derived peptides, it is shown that melanotrope cells in the pars intermedia of the hypophysis of the South African aquatic toad Xenopus laevis contain POMC, alpha-melanophore-stimulating hormone (alpha-MSH), gamma-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of gamma-MSH, these peptides are also found in the corticotrope cells in the rostral pars distalis. In the Xenopus brain, neuronal cell bodies in the ventral hypothalamic nucleus express POMC, alpha-MSH, gamma-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, alpha-MSH, gamma-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain alpha-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show alpha-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas alpha-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, alpha-MSH alone is present in the olfactory bulb, the medial septum, the medial and lateral parts of the amygdala, the ventromedial and posterior thalamic nuclei, the optic tectum and the anteroventral tegmental nucleus, and non-acetylated endorphin alone appears in the epiphysis. It is suggested that neurones that form POMC-derived peptides may play a direct or indirect role in the control of POMC-producing hypophyseal cells and/or in the physiological processes these endocrine cells regulate. This idea is supported by the fact that the suprachiasmatic nucleus and the locus coeruleus, both involved in melanotrope cell control, show POMC and POMC-peptide expression. A possible involvement in melanotrope and/or corticotrope control of the anterior preoptic and ventral hypothalamic nuclei, which both express POMC and various POMC-derived peptides, deserves future attention.

Nitric oxide synthase and background adaptation in Xenopus laevis.

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.

Neuropeptide Y in the developing and adult brain of the South African clawed toad Xenopus laevis.

To get more insight into developmental aspects of neuropeptide Y (NPY)-containing neuronal structures in the brain of amphibians and their possible involvement in background adaption, we have studied immunohistochemically the distribution of this neuropeptide in embryos, larvae and adults of Xenopus laevis. Antisera against NPY revealed that already at early embryonic stages NPY immunoreactive cell bodies are present in the ventral thalamus and rhombencephalic tegmentum. Slightly later, cell bodies appear in the olfactory bulb, the basal forebrain including the lateral and medial amygdala, the preoptic area, the ventral and dorsal thalamus, the suprachiasmatic region, the anteroventral tegmental nucleus and the solitary tract area. At late embryonic stages, the NPY cell groups not only show an increase in number of cells, but also stain more intensely. Around the time of hatching, a dramatic decrease in the number of immunodetectable cells occurs, particularly in the basal forebrain and in the rhombencephalic tegmentum. At the same time, however, new cell groups appear in telencephalic pallial regions and in the torus semicircularis. By the end of the premetamorphic stages, the distribution of NPY-immunoreactive cell bodies and fibers resembles closely the pattern observed in adult Xenopus brains. When compared with the development of catecholamine systems, it is clear that the NPY neurotransmitter system develops earlier. However, the expression of NPY- and dopamine-immunoreactivity in the suprachiasmatic nucleus occurs at about the same time (around stage 40) and coincides with several other events related to background adaptation, suggesting that this nucleus plays a key role in this complex neuroendocrine mechanism.

Central control of melanotrope cells of Xenopus laevis.

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.

Ontogeny of catecholamine systems in the central nervous system of anuran amphibians: an immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine.

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.

Involvement of retinohypothalamic input, suprachiasmatic nucleus, magnocellular nucleus and locus coeruleus in control of melanotrope cells of Xenopus laevis: a retrograde and anterograde tracing study.

The amphibian Xenopus laevis is able to adapt the colour of its skin to the light intensity of the background, by releasing alpha-melanophore-stimulating hormone from the pars intermedia of the hypophysis. In this control various inhibitory (dopamine, gamma-aminobutyric acid, neuropeptide Y, noradrenaline) and stimulatory (thyrotropin-releasing hormone and corticotropin-releasing hormone) neural factors are involved. Dopamine, gamma-aminobutyric acid and neuropeptide Y are present in suprachiasmatic neurons and co-exist in synaptic contacts on the melanotrope cells in the pars intermedia, whereas noradrenaline occurs in the locus coeruleus and noradrenaline-containing fibres innervate the pars intermedia. Thyrotropin-releasing hormone and corticotropin-releasing hormone occur in axon terminals in the pars nervosa. In the present study, the neuronal origins of these factors have been identified using axonal tract tracing. Application of the tracers 1,1'dioctadecyl-3,3,3',3' tetramethyl indocarbocyanine and horseradish peroxidase into the pars intermedia resulted in labelled neurons in two brain areas, which were immunocytochemically identified as the suprachiasmatic nucleus and the locus coeruleus, indicating that these areas are involved in neural inhibition of the melanotrope cells. Thyrotropin-releasing hormone and corticotropin-releasing hormone were demonstrated immunocytochemically in the magnocellular nucleus. This area appeared to be labelled upon tracer application into the pars nervosa. This finding is in line with the idea that corticotropin-releasing hormone and thyrotropin-releasing hormone stimulate melanotrope cell activity after diffusion from the neural lobe to the pars intermedia. After anterograde filling of the optic nerve with horseradish peroxidase, labelled axons were traced up to the suprachiasmatic area where they showed to be in contact with suprachiasmatic neurons. These neurons showed a positive reaction with anti-neuropeptide Y and the same held for staining with anti-tyrosine hydroxylase. It is suggested that a retino-suprachiasmatic pathway is involved in the control of the melanotrope cells during the process of background adaptation.

Immunocytochemistry and in situ hybridization of neuropeptide Y in the hypothalamus of Xenopus laevis in relation to background adaptation.

The amphibian Xenopus laevis is able to adapt to a dark background by releasing melanophore-stimulating hormone from the pars intermedia of the pituitary gland. The inhibition of melanophore-stimulating hormone release is accomplished by neuropeptide Y-containing axons innervating the pars intermedia. To determine the production site of neuropeptide Y involved in this inhibitory control, the distribution of neuropeptide Y in the brain has been investigated by immunocytochemistry and in situ hybridization. Immunoreactive cell bodies were visualized in, among others, the ventromedial and posterior thalamic nuclei, and the suprachiasmatic and ventral infundibular hypothalamic nuclei. A positive hybridization signal with a Xenopus-specific probe for preproneuropeptide Y-RNA was found in the diencephalic ventromedial thalamic nucleus and in the suprachiasmatic nucleus. With both immunocytochemistry and in situ hybridization, suprachiasmatic neurons appeared to be stained only in animals adapted to a white background; animals adapted to a black background showed no staining. Quantitative image analysis revealed that this effect of background adaptation is specific for suprachiasmatic neurons because no effect could be demonstrated of the background light condition on the ventral infundibular nucleus (immunocytochemistry) or the ventromedial thalamic nucleus (in situ hybridization). These results indicate that neurons in the suprachiasmatic nucleus enable the adaptation of X. laevis to a white background, by producing and releasing neuropeptide Y that inhibits the release of melanophore-stimulating hormone from the melanotrope cells in the pars intermedia of the pituitary gland.

Distribution of tyrosine hydroxylase and dopamine immunoreactivities in the brain of the South African clawed frog Xenopus laevis.

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.

Cloning and sequence analysis of hypothalamic cDNA encoding Xenopus preproneuropeptide Y.

Neuropeptide Y (NPY) consists of 36 amino acids and it constitutes one of the most conserved neuropeptides. Here we report the complete sequence of the first amphibian NPY precursor by cloning of a hypothalamic cDNA encoding Xenopus laevis preproNPY. The overall amino acid sequence identity between Xenopus and other known NPY precursor proteins ranges from 59% (fish) to 82% (chicken); a low degree of identity was found for the signal peptide sequence (32-75%) and for the carboxy-terminal peptide of NPY (CPON; 43-73%), while the NPY peptide sequence itself constitutes the most highly-conserved region (89-100%) within the preproNPY structure.

Subicular efferents to histaminergic neurons in the posterior hypothalamic region of the rat studied with PHA-L tracing combined with histidine decarboxylase immunocytochemistry.

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.