Evidence for corticotropin releasing factor (CRF) synthesis in the preoptic nucleus of Xenopus laevis tadpoles

Summary For the study of the hypothalamo-hypophysial system of Xenopus laevis tadpoles, hypothalamic lesions were made by means of the electrocoagulation technique. Lesioning of the ventral region of the preoptic nucleus resulted in a decrease of the number of ACTH cells in the pars distalis of the pituitary gland and in a diminution of the PAS-positive reaction of these cells. In addition, regeneration of the neurosecretory cells of the ventral region of the preoptic nucleus observed 6 weeks after lesioning was accompanied by the reappearance of normal PAS-positive ACTH cells in the pars distalis. It is suggested that the neurosecretory cells of the ventral region of the preoptic nucleus of Xenopus laevis tadpoles are related to the ACTH synthesizing cells, probably by producing CRF.Dedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthdayThe authors thank Prof. Dr. J.C. van de Kamer for his interest, Prof. Dr. P.G.W.J. van Oordt for his many helpful comments, and Messrs. H. van Kooten, E. van der Vlist, J.J. van der Vlis and M.C.A. van Pinxteren for preparing the illustrations     

The distribution of monoamine oxidase and acetylcholinesterase in the brain of Xenopus laevis tadpoles

Summary The distribution of monoamine oxidase (MAO) in the brain of Xenopus laevis tadpoles (stage 52–56) was studied histochemically with a modified Glenner's tryptamine-tetrazolium method. A moderate activity was observed in fibre regions of the striatum and septum (including the medial and lateral forebrain bundles), in the neuropil of the nucleus amygdalae, in the commissura anterior and commissura hippocampi, in the fibre regions of the diencephalon (including the optic chiasma), in the fibre regions of the tectum opticum and the tegmentum of the mesencephalon and in the white substance of the ventral half of the medulla oblongata. A greater MAO activity was found in the neuropil of the entire nucleus praeopticus. In the partes anterior and magnocellularis of this nucleus, MAO positive fibres are present in close contact with the perikarya, indicating a monoaminergic innervation of these neurons. The perikarya themselves did not show MAO activity. In the neurons of the nucleus praeopticus epichiasmaticus, the paraventricular organ (PVO) and nucleus infundibularis dorsalis (NID), only a slight MAO activity has been demonstrated in the perikarya, whereas a strong MAO positivity was found in the intraventricular protrusions and the neuropil. These data indicate the aminergic character of the neurons of these nuclei. From the postoptic fibre region a MAO positive tract was observed towards the developing median eminence and pars intermedia of the hypophysis. The pars nervosa and some cells of the pars distalis also contained MAO. Along the border of the aquaeduct of Silvius and the fourth ventricle, MAO positive liquor-containing neurons are also present.The distribution of acetylcholinesterase (AChE) was investigated in the hypothalamohypophysial region. AChE activity was found in the neuropil of the nucleus praeopticus magnocellularis, in the fibres of the optic chiasma and in the postoptic fibre region. The neurons of the PVO and NID were AChE negative. An AChE positive tract could be traced from the postoptic fibre region to the developing median eminence and pars nervosa. The pars distalis did not show AChE activity. However, in tadpoles reaching the metamorphic climax, ChE activity appeared in certain cells of the pars distalis; this might be related to degenerative phenomena in the acidophilic cells. The absence of AChE activity in the pars intermedia indicates a regulation of MSH release by peptidergic nerves to be unlikely.The stimulating interest and helpful advice of Prof. Dr. P. G. W. J. van Oordt is gratefully acknowledged. Thanks are also due to Mr. H. van Kooten and his co-workers for making the photographs.     

An immunohistochemical study of adenohypophyseal cells in the viviparous reptile Chalcides chalcides

The morphology of the hypophysis and the immunocharacteristics of the adenohypophyseal cells in the viviparous reptile Chalcides chalcides were studied by light microscopy, using conventional staining methods and an indirect antibody technique (ABC method), respectively. The general morphology of the C. chalcides hypophysis was comparable to that of other reptiles, showing three main regions: the pars distalis, the pars intermedia and the pars nervosa. The gland appeared as an elongated body in a cephalic-caudal direction and was almost completely enclosed in the sella turcica. For this reason, the hypophysis was studied in toto with the brain in decalcified specimens. The pars distalis accounted for most of the whole organ. The pars intermedia surrounded the pars nervosa as a goblet. The pars tuberalis was lacking.The immunohistochemical identification of the adenohypophyseal cells was performed using rabbit antisera against mammalian/synthetic hypophyseal hormones. Prolactin cells were clustered in small cellular cordons in the rostral pars distalis and in the medial pars distalis in both male and female specimens. Somatotropic cells were found in the caudal pars distalis. Corticotropic cells were observed in the medio-rostral pars distalis, as well as in the pars intermedia, where melanotropic cells were also present. Melanotropic cells were confined to the pars intermedia. Gonadotropic cells were mostly distributed in the ventral and lateral portions of the pars distalis, where they were found isolated or in small clusters. Thyrotropic cells were detected in the pars distalis with a distribution similar to that of the gonadotropic cells; however, atypically, they were also found in the pars intermedia. Therefore, the cytological characteristics of the adenohypophyseal cells appeared mostly conserved.     

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study

Neuroanatomic comparison of the binding capability of 2-[¹²⁵I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[¹²⁵I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.     

Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development

Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat α-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II–III tadpoles and in successive larval stages. Soon after hatching, stages II–III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI–XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone. Received: 28 July 1997 / Accepted: 8 December 1997     

Some morphogenetic features of the adenohypophysial primordium of early Xenopus laevis tadpoles

Summary The adenohypophyses of Xenopus laevis tadpoles at developmental stages 20 to 46 (Nieuwkoop and Faber, 1956) were studied. From its first appearance at about stage 20 to 21, the adenohypophysial primordium passes through four morphogenetic phases, each characterized by internal events. The first phase (stages 20 to about 33/34) is characterized by extensive proliferation of the primordium. During the second phase (stages 33/34 to about 37/38), the growth of the primordium is arrested. This arrest coincides with the attainment of secretory function. The primordium is claviform in shape at these stages. The third phase, roughly stage 39, is characterized by a thorough reorganization of the adenohypophysial cells, leading to the formation of the pars distalis and pars intermedia. The shape of the primordium changes, and its volume temporarily increases. The last phase is characterized by the organization of the pars distalis cells into cell cords which possibly demonstrate a functional relation to a specialized region (the hilus) of the adenohypo-physis-brain interspace. Acknowledgements. Grants from the Faculty of Mathematics and Science, University of Lund, the Royal Physiographic Society, Lund, and the Swedish Natural Sience Research Council are gratefully acknowledged     

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, α-melanophore-stimulating hormone (α-MSH), γ-MSH, acetylated and non-acetylated endorphins and adrenocorticotropic hormone (ACTH). With the exception of γ-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, α-MSH, γ-MSH, non-acetylated endorphins and ACTH, neurones in the anterior preoptic area reveal POMC, α-MSH, γ-MSH and non-acetylated endorphin, neurones in the suprachiasmatic nucleus contain α-MSH, non-acetylated endorphin and ACTH and neurones in the posterior tubercle show α-MSH, non-acetylated endorphin and ACTH immunoreactivities. In the locus coeruleus POMC and ACTH coexist, whereas α-MSH and non-acetylated endorphin occur together in the nucleus accumbens, the striatum and the nucleus of the paraventricular organ. Finally, α-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.     

Comparison of binding and cyclic GMP accumulation by atrial natriuretic peptides in endothelial cells

Rat 125I-labeled atrial natriuretic factor (ANF (8-33)) was used to identify ANF receptors on cultured bovine aortic endothelial cells. Specific binding of 125I-ANF at 37 degrees C to confluent endothelial cells was saturable and of high affinity. Scatchard analysis of the equilibrium binding data indicated that endothelial cells contain a single class of binding sites with a Kd of 0.1 +/- 0.01 nM. This particular clone of endothelial cells had 16000 +/- 1300 receptors per cell. The order of potency for competing with 125I-ANF binding was human atrial natriuretic peptide (hANP) = atrial natriuretic factor (ANF (8-33)) greater than atriopeptin II greater than atriopeptin III greater than atriopeptin. The weakest competitor, atriopeptin I, had a K1 of 0.45 nM, which was only 6-fold higher than the K1 for hANP and ANF (8-33). ANF (8-33) and hANP in the presence of 0.5 mM isobutylmethyl-xanthine produced a 15-20-fold increase in cyclic GMP content at 10 pM and a maximal 500-fold elevation of cyclic GMP at 10 nM. The concentrations required to elicit a half-maximal increase in cyclic GMP for hANP, ANF (8-33), atriopeptin I, atriopeptin II and atriopeptin III were 0.30, 0.35, greater than 500, 4.0 and 5.0 nM, respectively. Although atriopeptin I acted as a partial agonist, it was unable to antagonize the effect of ANF (8-33) on cyclic GMP formation. These findings suggest that endothelial cells have multiple and functionally distinct ANF-binding sites.     

Brain and Spinal Cord Distribution of Biphalin: Correlation with Opioid Receptor Density and Mechanism of CNS Entry

Abstract: Biphalin [(Tyr-d -Ala-Gly-Phe-NH)₂] is a bivalent, opioid peptide containing two pharmacophores linked by a hydrazine bridge. When administered intracerebroventricularly, it has been shown to be more potent than morphine and etorphine at eliciting antinociception. Biphalin has also been shown to cross both the blood-brain and blood-cerebrospinal fluid barriers. To understand the basis of biphalin's potency, regional brain and spinal cord distribution studies with [¹²⁵I-Tyr¹]biphalin were performed 5, 20, and 40 min after intravenous bolus injections. A statistically greater amount of [¹²⁵I-Tyr¹]-biphalin was detected in the nucleus accumbens compared with other brain regions (p < 0.05). This correlates with the high density of δ- and μ-opioid receptor mRNA and binding sites shown to be expressed in the nucleus accumbens. Also, a statistically greater amount of [¹²⁵I-Tyr¹]biphalin was detected in two other circumventricular organs, the choroid plexus and pituitary, when compared with other brain regions. These studies provide evidence that biphalin can reach not only brain sites, but also spinal sites to elicit antinociception. The overall CNS distribution of [¹²⁵I-Tyr¹]biphalin was decreased with naloxone, d -Phe-Cys-Tyr-d -Trp-Arg-Thr-Pen-Thr-NH₂, or naltrindole pretreatment, showing that biphalin detected in the brain and spinal cord is binding to δ- and μ-opioid receptors. Additional in situ brain perfusion experiments identified a saturable component contributing to CNS entry of [¹²⁵I-Tyr¹]biphalin, which could be described by Michaelis-Menten kinetics with a K_m of 2.6 ± 4.8 µM, V_max of 14.6 ± 2.89 pmol^?1·min^?1·g^?1, and K_d of 0.568 ± 0.157 µl·min^?1·g^?1. Brain entry of [¹²⁵I-Tyr¹]biphalin was sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and l -phenylalanine, suggesting use of the large neutral amino acid carrier. This work provides evidence that biphalin is a promising, potent analgesic that has a unique mechanism for reaching both spinal and supraspinal opioid receptor sites.     

Visualizing Dopamine and Serotonin Transporters in the Human Brain with the Potent Cocaine Analogue [125I]RTI-55: In Vitro Binding and Autoradiographic Characterization

Abstract: The cocaine analogue RTI-55 was evaluated as a probe for in vitro labeling and localization of dopamine and serotonin transporters after death in the human brain. Kinetic, saturation, and competition binding experiments indicated complex interactions of the radioligand with the identification of multiple recognition sites. In membrane binding assays, the association of [¹²⁵I]RTI-55 at 25°C to putamen membranes was monophasic. In contrast, dissociation of [¹²⁵I]RTI-55 occurred in two phases with t_1/2 values of 9.4 and 36.5 min, respectively. Saturation analysis of [¹²⁵I]RTI-55 binding demonstrated two binding sites in the human putamen with K_D values of 0.10 ± 0.02 and 1.81 ± 0.46 nM. The binding of [¹²⁵I]RTI-55 was displaced by a wide range of cocaine analogues and monoamine uptake inhibitors. The rank order of potency demonstrated in competition assays with human putamen membranes indicates that the radioligand labels cocaine recognition sites on the dopamine transporter (mazindol > GBR 12909 > GBR 12935 > paroxetine > nisoxetine > desipramine ≥ fluoxetine > citalopram). In the human occipital cortex, [¹²⁵I]RTI-55 recognized multiple binding sites with K_D values of 0.02 ± 0.01 and 4.18 ± 0.46 nM. The rank order of potency for inhibition of [¹²⁵I]RTI-55 binding to cerebral cortex membranes (paroxetine > citalopram > GBR 12909 ≥ mazindol ≥ nisoxetine > benztropine) suggests that [¹²⁵I]RTI-55 labels the serotonin transporter in the human occipital cortex. Autoradiographic mapping of [¹²⁵I]RTI-55 revealed very high densities of cocaine recognition sites over areas known to be rich in dopaminergic innervation, including the caudate, putamen, and nucleus accumbens. Moderately elevated densities of [¹²⁵I]RTI-55 binding sites were also seen throughout the thalamus, hypothalamus, and substantia nigra. [¹²⁵I]RTI-55 binding sites were prevalent throughout the cerebral cortex and amygdala. In autoradiographic studies, the addition of the selective serotonin transport blocker citalopram completely prevented [¹²⁵I]RTI-55 labeling in the thalamus, hypothalamus, and throughout most of the cerebral cortex. In the presence of citalopram, [¹²⁵I]RTI-55 binding site densities remained elevated over the striatum and substantia nigra, with selective residual labeling also seen in the external segment of the globus pallidus and the lateral nucleus of the amygdala. These results demonstrate that in the human brain, [¹²⁵I]RTI-55 labels multiple recognition sites on dopamine and serotonin transporters.     

Characterization and distribution of bombesin binding sites in the goldfish hypothalamic feeding center and pituitary

Bombesin (BBS)/gastrin-releasing peptide (GRP) binding sites were characterized and their distribution examined in the goldfish brain and pituitary by radioligand binding and autoradiography. Binding of ¹²⁵I-[Tyr⁴]-BBS-14 to tissue sections was found to be saturable, reversible, time-dependent and displaceable by BBS/GRP-like peptides. Analysis of saturable equilibrium binding revealed a one-site model fit with a K_d of 0.665 ± 0.267 nM. This binding site displayed high affinity for members of the BBS subfamily of peptides, including GRP10 (K_i; 0.292 ± 0.038 nM) and GRP27 (K_i; 2.034 ± 1.597 nM), but showed no affinity for the BBS_8–14 fragment. While an approximate 100-fold lower binding affinity was displayed by the binding site for neuromedin B (K_i; 61.5 ± 28.2 nM), litorin was highly effective in displacing radiolabeled BBS binding (K_i; 1.469 ± 0.427 nM). The localization of saturable and high affinity BBS/GRP binding sites in specific areas of the goldfish brain and pituitary generally revealed a similar anatomical distribution to BBS/GRP-like immunoreactive material reported previously by our laboratory. Quantitative densitometric analysis of radiolabeled BBS binding to brain nuclei and the pituitary revealed a moderate concentration of BBS/GRP binding sites in the hypothalamic feeding area, including the nucleus diffusus lobi inferioris, nucleus recessus lateralis, nucleus lateral tuberis, and nucleus anterior tuberis. Other brain nuclei known to influence the brain feeding center which contained a high density of BBS/GRP binding sites included nuclei of the dorsal and ventro-medial telencephalon, the preoptic hypothalamus, and the optic tectum. High densities of BBS/GRP binding sites were also localized in the dorsal cerebellum, and nucleus habenularis. In the pituitary, BBS/GRP binding sites were present in high concentration in the neurointermediate lobe, with a relatively lower density localized in the pars distalis. The present study further supports a role for BBS/GRP-like peptides in the regulation of feeding behavior and anterior pituitary hormone secretion in teleosts.     

Estrogen target cells in the pituitary of platyfish,Xiphophorus maculatus

Summary The distribution of estradiol-concentrating cells in the pituitary of the platyfish, Xiphophorus maculatus, is studied after the injection of ³H estradiol-17 by thaw-mount autoradiography. Autoradiograms prepared 2–8 h after the injection show nuclear concentration of radioactivity in certain cells of the proximal pars distalis, while no nuclear labeling is found in cells of the rostral pars distalis, pars intermedia and pars nervosa. Radioactively labeled cells are identified as gonadotropes by a combined technique of thaw-mount autoradiography and immunoperoxidase staining with antiserum to ovine LH. Approximately 80% of the immunoreactive LH cells show a concentration of radioactivity in their nuclei. These observations indicate that in teleosts, as in mammals, estradiol has a direct effect on pituitary gonadotropes.Supported by PHS grant NS09914. The authors are grateful to Dr. D.G. Humm for providing the platyfish     

The histochemical localization of steroid binding sites in the pituitary gland of a teleost (the platyfish)

Summary Specific binding sites for estrogen, testosterone, and progesterone have been demonstrated in the pituitary gland of mature male and female platyfish (Xiphophorus maculatus). With a histochemical procedure, fluorescent-steroid-hormone conjugates were localized in the cytoplasm and nucleus of the gonadotrops of the caudal pars distalis (CPD) and in cells of the pars intermedia (PI) previously demonstrated to contain immunoreactive gonadotropin. The specificity of the response was confirmed by means of competitive binding analyses and by using fluoresceinated BSA not linked to steroids. The physiological significance of steroid binding in the PI, as well as in the CPD, is discussed in the light of other recent studies on the pituitary gland of the platyfish.     

Immunohistochemical demonstration of oxytocin-like, neuropeptide Y-like and gonadotropin-releasing hormone-like substances in the hypothalamo-hypophysial system of ayu,plecoglossus altivelis altivelis, in osmotically different environments

To ascertain the role of neuropeptides on the hypothalamo-hypophysial system of a fish in osmotically different environments, an immunohistochemical study of oxytocin (OXT), neuropeptide Y (NPY) and gonadotropin-releasing hormone (GnRH) was carried out on the anadromous salmonoid fish,Plecoglossus altivelis altivelis, commonly known as Ayu. River fish caught were acclimatized in a freshwater aquarium, half of them being subsequently kept as a control group and the remainder being transferred to a sea water aquarium, through 1/3 diluted sea water, as an experimental group. OXT-like immunoreactivity as demonstrated in the neurosecretory pathway, having the same pattern was that shown by aldehyde fuchsin staining. Noticeably, a mass of nucleus preopticus (NPO) and a marginal portion of the pars nervosa in the control group became strongly immunoreactive, whereas a very weak reaction was obtained in the sea water-retained fish, suggesting the release of the labelled substance. In the latter, NPY-like substance was widely distributed in the brain without NPO, with the positive substance being dense in the terminal rami of the pars nervosa bordering the pars distalis. However, no remarkable difference in GnRH-like and NPY-like immunoreactivities in the hypothalamo-hypophysial system was apparent between the two groups. These results suggested that OXT (probably isotocin)-like substance may play a role in osmoregulation.     

Localization of acetylcholinesterase and choline acetyltransferase in the rat pituitary gland

Summary Experiments were conducted to determine the presence of two cholinergic biomarkers, acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the rat pituitary. A histochemical procedure for AChE was used to provide visualization of structures containing this enzyme. Radiochemical methods provided a sensitive assay for measuring ChAT activity. Nerve fibres staining for AChE activity were observed in the neurointermediate lobe, with the greatest concentrations appearing at the junction region with the pituitary stalk. Cells staining for AChE were found in the pars distalis and pars intermedia. ChAT activity correlated well with AChE distribution in pars nervosa and pars intermedia but not in pars distalis. The greatest levels of ChAT activity were in pars intermedia and the region where the stalk joins the pituitary. Significant values were also found for the pars nervosa. The presence of AChE and ChAT in pars intermedia and pars nervosa is evidence for a cholinergic innervation to these regions. In pars distalis, where other investigators have found muscarinic receptors, intense staining for AChE and absence of ChAT activity may indicate non-innervated, acetylcholine-sensitive sites.     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

Characterization and localization of gonadotropin-releasing hormone in the brain and pituitary of the three-spined stickleback,Gasterosteus aculeatus

Radioimmunoassay (RIA) studies on highperformance liquid chromatography (HPLC) fractions of brain extracts of the three-spined stickleback, Gasterosteus aculeatus, provided evidence for at least two forms of gonadotropin-releasing hormone (GnRH). One form showed chromatographic and immunological properties similar to that of synthetic salmon GnRH (sGnRH). A second, unidentified form of GnRH eluted in the same position as chicken GnRH I (cGnRH-I); however, it did not cross-react in a cGnRH-I RIA. Furthermore, it cannot be excluded that chicken GnRH II (cGnRH-II) and maybe one other unidentified form are present in the stickleback. The distribution of GnRH in the brain of breeding adult male sticklebacks was studied by use of immunohistochemistry. Two antisera against sGnRH and antisera against mGnRH and cGnRH-II were applied on cryosections and visualized using the peroxidase-antiperoxidase method. Staining patterns were similar after incubations with all four antisera. Immunoreactive fibers were found in most parts of the brain. Three distinct groups of GnRH-immunoreactive perikarya were found in the nucleus olfactoretinalis, in the nucleus anterior periventricularis, and in the nucleus lateralis tuberis. Moreover, weakly stained cells occurred in a periventricular position in the midbrain. The proximal pars distalis of the pituitary, housing the gonadotropic cells, was richly innervated by GnRH-positive fibers. In the pars intermedia and in the rostral pars distalis, immunoreactive fibers were absent.     

Galanin: presence and distribution in the brain and pituitary of Rhinella arenarum (Amphibia: Anura) during development

The immunohistochemical distribution of galanin (Gal) in the brain and pituitary of Rhinella arenarum was studied during development. Gal-immunoreactivity was first observed in the brain just after hatching in anterior preoptic area, infundibular area, median eminence and pars distalis of the pituitary as well as in the olfactory epithelium. At the beginning of prometamorphosis new Gal-immunoreactive (ir) cells were observed in the olfactory nerve and bulb. Later in prometamorphosis new Gal-ir cells were observed in the telencephalon, suprachiasmatic nucleus, rostral rhombencephalon and in the pars nervosa of the pituitary. The most numerous accumulations of Gal-ir neurons throughout the larval development were observed in the ventral hyphothalamus where numerous Gal-ir cells of cerebrospinal fluid-contacting type were found. During metamorphic climax and soon after we did not detect Gal-ir neurons in the pallium, medial or pretectal dorsal thalamus. In the median eminence and pars distalis of the pituitary many Gal-ir fibers were found during development indicating that Gal may play a role in the modulation of hypophyseal secretion. Furthermore, the distribution of Gal-ir elements observed throughout larvae development indicates that galaninergic system maturation continues until sexual maturity.     

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species

Summary The distribution of somatostatin (SRIF) — and corticotropin-releasing factor (CRF)-like — immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.This work was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek and the CNRS     

Hypophysiotropic neurons in the goldfish hypothalamus demonstrated by retrograde transport of horseradish peroxidase

Summary The horseradish-peroxidase (HRP) technique was used to visualize the cell bodies of axons projecting to the goldfish pituitary. Following intravenous injections of HRP, HRP reaction products were observed in axons of the rostral pars distalis, proximal pars distalis, neurointermediate lobe, pituitary stalk and in axons coursing from the pituitary into the hypothalamus. HRP-labelled cells in the brain were localized in two regions only — the nucleus preopticus (NPO) pars magnocellularis and pars parvocellularis, and the nucleus lateralis tuberis (NLT) of the hypothalamus. These observations suggest that the NPO and NLT are the source of the neurosecretory innervation of the goldfish pituitary.