Immunocytochemical localization of a galanin-like peptidergic system in the brain of two urodele and two anuran species (Amphibia).

Galanin-like immunoreactivity was localized in the brain of Urodela (Ambystoma, Pleurodeles) and Anura (Bufo, Xenopus) by immunocytochemistry with anti-porcine galanin antiserum. In the four species, immunoreactive perikarya were observed in the telencephalon (striatum, amygdala), diencephalon preoptic area mainly along the anterodorsal wall of the preoptic recessus, suprachiasmatic nucleus, lateral hypothalamus, ventral and dorsal infundibular nuclei, paraventricular organ, and rhombencephalon (nucleus of the solitary tract). Galaninergic fibres extended in similar regions and in the medial septum, ventral telencephalon, ventral hypothalamus, median eminence, and various mesencephalic and rhombencephalic regions. Contacts with the cerebrospinal fluid cavity occurred along the preoptic recessus (Ambystoma) and the ventral infundibular wall (all species). Fibres were scarce in the neurohypophysis. The distal and intermediate lobes of the pituitary were virtually devoid of immunoreactivity. The galaninergic system appeared more developed in adult amphibia than in young animals, suggesting the stimulating influence of sex steroids on the expression of galanin as previously described in Anguilla. The extensive distribution of the galanin-like immunoreactive neurons in amphibian brains suggests that this peptide may act as a neuromodulatur and/or neurotransmitter.

Corticotropin-like immunoreactivity in the brain of intact, hypophysectomized, cortisol- and metopirone-treated eels. Comparison with changes in pituitary corticotropes and brain corticotropin-releasing factor.

An ACTH-like peptidergic system was demonstrated in the brain of three teleost species by immunocytochemistry. In order to investigate the origin of brain ACTH and factors modulating its synthesis, similar techniques were applied to the brain of eels (1) submitted to hypophysectomy in order to suppress pituitary ACTH and plasma cortisol, (2) injected with cortisol to inhibit pituitary ACTH synthesis and release, and (3) injected with metopirone to block cortisol synthesis and stimulate ACTH synthesis and release. Hypophysectomized eels showed a normal distribution of immunoreactive perikarya in the ventral hypothalamus and fibers in the brain, suggesting that brain ACTH does not arise from the pituitary. In cortisol-treated eels immunostaining was markedly reduced in brain perikarya and pituitary corticotropes, suggesting a reduced synthesis. In metopirone-injected eels, one third of the animals showed an increased immunostaining in perikarya and a dense network of immunoreactive fibers, suggesting that ACTH synthesis was increased. Brain ACTH was not affected in other animals. Pituitary corticotropes were rapidly degranulated. Responses of ACTH in the brain and pituitary occur independently when cortisol synthesis is inhibited. These responses are compared to those of the corticotropin-releasing factor system in the same eels.

Immunocytochemical localization of a galanin-like peptidergic system in the brain and pituitary of some teleost fish.

Immunostaining of brain and pituitary sections of teleost fishes (eels, salmonidae, cyprinidae, gourami, sculpin, mullet) with anti porcine galanin (GAL) revealed the presence of immunoreactive (ir) perikarya and a rich network of fibers. Ir-perikarya were located rostrodorsally to the recessus preopticus, and in the posterior tuberal hypothalamus. Ir-fibers were abundant in basal telencephalon and around diencephalic ventricular recesses but never contacted their lumen. Furthermore, they were observed in basal hypothalamus, brainstem and ventral medulla. Ir-fibers passed along corticotropic (ACTH), gonadotropic cells and somatotropes (GH cells) in eel and trout pars distalis, but rarely ended in caudal neurohypophysis. In goldfish pituitary ir-fibers occurred in neural digitations and among different cell types which however did not contain a GAL-like peptide. The relation GAL fibers/GH cells appeared more evident in species with a high growth rate. The other species showed a similar distribution of brain GAL. In eels and trout, ir-perikarya were not observed in areas containing somatostatin, GH- and ACTH-releasing factor, and ACTH-like perikarya, suggesting that GAL did not coexist with these peptides. The widespread distribution of a GAL-like peptide in teleost brain suggests that it could play a role of neurotransmitter and/or neuromodulator and regulate the secretion of adenohypophysial hormone(s).

Effect of hypophysectomy on the hypothalamic corticotropin-releasing factor (CRF) neuronal system in the eel: immunocytochemical study.

Male and female eels were hypophysectomized by a buccal approach and examined either 2 and 7 weeks (males) or 4 and 5 months (females) later. Intact and sham-operated eels served as controls. Fish were killed by decapitation, and the brains fixed in sublimated Bouin-Hollande solution and embedded in paraffin wax. Frontal and sagittal sections were immunostained by the peroxidase-antiperoxidase method, using antisera against cortiotropin-releasing factor (CRF) or arginine vasotocin (AVT). Immunoreactive (ir)-CRF was detected in parvo- and magnocellular perikarya of the preoptic nucleus. Sham hypophysectomy did not affect ir-CRF in cell bodies and fibres. After hypophysectomy, mainly after 4 or 5 months, there was an apparent increase in the number of ir-CRF cells. The intensity of immunostaining in the cell bodies was variable but not significantly different from the controls, but the two ventral axonal tracts were much thicker and contained more immunoreactive material than in sham-operated eels, while ir-CRF accumulated close to the sectioned pituitary stalk. The existence of negative feedback by cortisol on the CRF neurons is discussed.

[Ultrastructure of prolactin cells and survival of Gambusia sp. (teleost fish) in deionized water enriched in calcium or sodium]. / Ultrastructure des cellules à prolactine et survie de Gambusia sp. (poisson téléostéen) en eau désionisée enrichie en calcium ou en sodium.

The ultrastructure of the prolactin (PRL) cells of Gambusia was studied in animals kept in deionized water (EDes) and in EDes supplemented either with Ca2+ (10,2 and 15,3 mM) or with Na+ (10,2 and 15,3 mM). In environments supplemented with CaCl2 the maximal survival was 40 days. The stimulation of PRL cells was similar to that described in EDes. On the contrary, in environments supplemented with NaCl, 50% of the animals are still alive after 45 days. PRL cells are slightly stimulated and their ultrastructural aspect is similar to that observed in controls kept in freshwater. The significance of these results is discussed in relation with our present knowledge of the pituitary control of osmoregulation in teleost fish.

Response of prolactin cells to environmental calcium in the eel (Anguilla anguilla L.).

Prolactin (PRL) cell activity was investigated in eels kept in fresh water (FW), deionized water (DW) supplemented or not with Ca (2 mM), in Ca-enriched FW (10 mM), in normal (Ca 3.4 mM) or Ca-free 1/3 sea water (SW), and in SW (Ca 10.2 mM) or Ca-free SW (Ca 0.15 mM). Light-microscopic studies, including measurement of the nuclear area and cell height, showed that PRL cell activity, reduced in DW, is not affected by Ca supplementation. Activity is reduced in Ca-enriched FW, in 1/3 SW and in SW, conditions inducing an increase in the plasma sodium level. The lack of calcium in saline environments partly suppresses the nuclear atrophy occurring in SW. There is no significant correlation between external or total plasma calcium concentration and PRL cell activity. In artificial Ca-free SW, eels show a rapid increase in plasma osmolarity and sodium levels; there is a significant negative correlation between these two plasma values and the nuclear area or cell height of PRL cells. As in some other teleosts, plasma osmolarity and plasma sodium seem to play a more important role than external or internal calcium in controlling PRL secretion. This correlation is not apparent in eels kept in SW, having unstimulated PRL cells but active calcium-sensitive (Ca-s) cells in the pars intermedia.

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel. I. Responses to changes in the environmental calcium and magnesium.

The structure of the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia was investigated in eels kept in deionized water (DW) or fresh water (FW) supplemented with Ca2+ of Mg2+ x Ca2+ (2mM) reduces considerably the response to DW; plasma osmolarity, Na+ and Ca2+ levels are not significantly affected. In eels adapted to DW for 21 or 28 days, showing highly stimulated Ca-s cells, an addition of CaCl2 for 2 days inhibits the release of granules, but does not immediately block their synthesis and the mitotic activity. The nuclear area is reduced, osmolarity and plasma sodium increase, but the rise in calcium is not always significant. Magnesium, at a 10-fold greater concentration than in FW (2mM), slightly inhibits the release of secretory granules without reducing other indicators of stimulation. In Ca-enriched FW, the Ca-s cells appear inactive. These data show that the PAS-positive cells in the pars intermedia of the eel are calcium-sensitive, similar to those of the goldfish; their role in calcium regulation is briefly discussed.

[Response of the pars intermedia of the goldfish pituitary to deionized water, buffered or not, and to calcium and magnesium supplementation (author's transl)]. / Réponse de la pars intermedia de l'hypophyse du Cyprin à l'eau déau désionisée, tamponnée ou non, et à l'apport de calcium ou de magnésium.

In the goldfish, one cell type of the pars intermedia, slightly PAS positive, is highly stimulated in deionized water (E Dés). This response is not influenced by the pH of the water (E Dés pH 5.3, E Dés buffered with imidazole pH 6.7) for 20 days. Stimulation is prevented when the E Dés is supplemented with CaCl2 (2mM), but it is not affected by the addition of MgCl2 (2 mM). When goldfish are kept in E Dés for 20 days, and subsequently in E Dés supplemented with calcium gluconate (1 mM), a granule storage occurs in the cytoplasm of the enlarged cells within 48 hours. These cells are therefore sensitive to the lack or presence of calcium in the environment. The term of calcium-sensitive (Ca-s) cells appears justified in the goldfish, as in the eel; their putative role in the control of the pigmentation is not evident.

Specific effect of calcium ions on the calcium-sensitive cells of the pars intermedia in the goldfish.

Cytological changes in the calcium-sensitive (Ca-s) cells (formerly termed PAS-positive cells) of the pars intermedia were investigated in the goldfish after adaptation to deionized water (DW), with or without addition of sodium, potassium and magnesium. These ions were added as chloride salts at concentrations similar to those present in fresh water (FW). The marked stimulation of the Ca-s cells is not inhibited in DW supplemented with Na+ (0.35 mM/l), K+ (0.05 mM/l), and Mg2+ (0.2 mM/l) for a period of 24 days. The inhibition of the response to DW with calcium chloride (2 mM/l). These data show that chloride ions are not responsible for the regression of the Ca-s cells observed in goldfish kept in DW supplemented with calcium chloride. The effect of calcium ions on the Ca-s cells appears to be specific. These results support the hypothesis that the Ca-s cells synthesize a factor (hypercalcin?) involved in calcium regulation, and that its release is influenced by the calcium content of the environment. The role of the pars intermedia in calcium metabolism is strengthened by the present results. Biochemical data suggest the presence of a hypercalcemic factor in the pituitary of fish (Parsons et al. 1978) and are in agreement with the present cytological findings.

PAS-positive cells of the pars intermedia are calcium-sensitive in the goldfish maintained in a hyposmotic milieu.

Cytological changes in the pars intermedia of the goldfish were investigated after adding calcium to deionized water (DW). In fish maintained in DW, the PAS-positive cells are highly stimulated in comparison to cells of fish kept in fresh water (FW). In DW supplemented with calcium at the same concentration as in FW (2 mM/l), the hyperactivity of the PAS-positive cells is prevented. When calcium ions are added 60 h before the animals are sacrificed, the PAS positive cells start to show signs of regression and their granules are stored: the release of the granular material appears to be suppressed by calcium. In the goldfish, the PAS-positive cells, homologous to a similar cell type in the eel, react only very weakly with the PAS technique. The name "calcium-sensitive cells" appears to be more appropriate in the goldfish for this particular cell type, secreting an unknown factor. This factor, different from the prolactin produced in the rostral pars distalis of the hypophysis, might be an equivalent of a "hypercalcin".

Effect of pimozide on the cytology of the eel pituitary. I. Prolactin-secreting cells.

Pimozide, a specific blocker of dopaminergic receptors, was injected for 4 to 9 days in freshwater (FW) eels or eels acclimated to sea water (SW), for 10 to 30 days. The daily dose was 100 or 200 microgram/100 g. In FW, pimozide induces a nuclear hypertrophy in the prolactin (PRL) cells of eels; these elongated cells increase in height. The amount of erythrosinophilic granules in the cytoplasm, initially reduced, increases. Plasma electrolyte values are not modified: only the plasma sodium level slightly rises with the higher dose. In SW, PRL cells appear less active. After 10 days, this hypoactivity is not yet fully evident; pimozide stimulates PRL cells without affecting electrolyte values. After 1 month in SW, PRL cells are stimulated with pimozide and a slight regranulation may occasionally occur. The response in SW is never as marked as it is in FW; a high dose is not more effective than a low one. The higher dose significantly raises Na+, Ca2+ and Cl- plasma levels. These data suggest that prolactin synthesis and release increase with pimozide. They corroborate the hypothesis of a hypothalamic inhibitory control on PRL secretion mediated through dopaminergic fibers in the eel, but other factors may also be involved in this regulation in addition to the effect of salinity.