Pierrette Dubourg

Distribution and characterization of neuropeptide Y-like immunoreactivity in the brain and pituitary of the goldfish

SummaryThe distribution of neuropeptide Y (NPY) immunoreactivity has been studied by means of immunocytochemistry and radioimmunoassay in the brain of the goldfish. It was found that NPY had a widespread distribution in the entire brain in particular in the telencephalon, diencephalon, optic tectum and rhombencephalon. In the pituitary gland, positive type-B fibers were observed in the various lobes frequently in direct contact with secretory cells, in particular the gonadotrophs, somatotrophs and MSH (melanocyte-stimulating hormone) secreting cells. When measured by radioimmunoassay, the highest NPY concentrations were found in the pituitary and telencephalon, confirming the results of immunocytochemistry. The displacement curves obtained with serial dilutions of brain extracts were parallel to that of synthetic porcine NPY. Following high performance liquid chromatography, the NPY-like material extracted from goldfish brain co-eluted as a single peak with synthetic porcine NPY. These data demonstrate the presence of an NPY-like substance widely distributed in the goldfish brain. The observation of NPY-immunoreactive fibers in the pituitary gland suggests that, among its other functions, NPY may play a role in the neuroendocrine regulation of pituitary function.

Simultaneous immunogold labeling of GABAergic terminals and vasopressin-containing neurons in the rat paraventricular nucleus.

SummaryThe GABAergic innervation of vasopressin-containing cells in the magnocellular part of the paraventricular nucleus was studied at the electron-microscope level using antibodies against GABA and vasopressin. The detection of both GABA and vasopressin on the same ultrathin section, performed with a double-labeling immunogold method, revealed GABAergic terminals in symmetrical synaptic contact with vasopressin-containing neurons. These GABAergic terminals displayed mitochondria, clear synaptic vesicles and varying numbers of electron-dense vesicles. Vasopressin-immunoreactivity was associated with neurosecretory granules, whereas GABA-immunoreactivity was found above mitochondria, clear synaptic vesicles and some electron-dense vesicles. This study, demonstrating the extensive participation of GABA in the innervation of magnocellular vasopressin-secreting neurons, suggests that this inhibitory neurotransmitter regulates vasopressin secretion at the level of the paraventricular nucleus.

The dopaminergic innervation of the goldfish pituitary

SummaryThe dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocyto-chemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.

Effect of osmotic pressure on prolactin release in rainbow trout: in vitro studies.

To investigate a possible effect of osmotic pressure on prolactin (PRL) release in rainbow trout, we developed a technique for in vitro perifusion of trout pituitaries. Changes in osmotic pressure similar to those observed in fish plasma during transfer experiments did not induce significant modifications of PRL release. In contrast, high-amplitude variation of osmotic pressure resulted in clear modifications of PRL secretion: hyperosmotic medium caused a reduction in PRL release, while infusion of hyposmotic medium induced a transitory increase in PRL release. By using different concentrations of mannitol, we found that the modifications of prolactin secretion could not be ascribed to alterations of the ionic composition of the medium but actually resulted from variations in the osmotic pressure of the incubation medium. In further experiments osmotic pressure was decreased from 300 to 220 mOsm/kg or from 400 to 300 mOsm/kg; a similar transitory increase in PRL release was observed. Measurement of gonadotropin (GtH) in the perifusion effluent medium showed that PRL and GtH secretion followed similar patterns. Thus, our results suggest a possible mechanical effect of wide changes in osmotic pressure on pituitary cell membranes. These data indicate that the rainbow trout differs notably from nonsalmonid teleost species thus far studied in the lack of sensitivity of its PRL cells to osmotic pressure.

Ultrastructural changes in the calcium-sensitive (PAS-positive) cells of the pars intermedia of eels kept in deionized water and in normal and concentrated sea water.

SummaryThe ultrastructure of the calcium-sensitive (Ca-s) (PAS-positive) cells of the pars intermedia was investigated in eels kept in hypo and hyperosmotic environments. Although the cells were moderately active in fresh water (FW), they were highly stimulated in deionized water (DW) and displayed an enlarged Golgi apparatus, a distinct rough endoplasmic reticulum, few secretory granules, some microtubules and an extended area of contact with the basal lamina that separates nervous and glandular tissues. Some mitosing cells were seen. A similar picture was observed in eels kept in sea water (SW) for 45 days, returned to FW and subsequently to DW for 21 days. In SW (30 and 33‰), and particularly in concentrated SW (50, 60 and 63‰), the Ca-s cells were inactive. Their granules were significantly smaller than in eels kept in FW, and the area of contact with the basal lamina was greatly reduced. However, signs of granule-release were seen in eels adapted to 50 and 60‰ SW. Nerve fibers rarely contacted the Ca-s cells and did not synapse with them. The ultrastructural data support the hypothesis that the Ca-s cells of Anguilla, like those of Carassius, are involved in ionic regulation. MSH cells were not greatly affected by the present experiments.

Light and electron microscopic identification of gonadotrophic cells in the pituitary gland of the goldfish by means of immunocytochemistry.

Immunocytochemical techniques were used at the light and electron microscopical levels in order to localize and to characterize the gonadotrophs in the goldfish pituitary gland by means of antibodies to carp gonadotrophin (c-GTH) or its subunit (c-GTH beta). At the light microscopical level antibodies to c-GTH reacted weakly with cells located in the rostral pars distalis (RPD) and strongly with cells of the proximal pars distalis (PPD). The labeling was restricted to the proximal pars distalis when antibodies to c-GTH beta were employed. The PAP and colloidal-gold postembedding procedures demonstrated that two cell types of the PPD react with both immune sera. These cells correspond to the so-called globular and nonglobular basophils of the goldfish pituitary. The labeling was located over the small secretory granules and the large globules. A relationship was noted between the intensity of the labeling and the electron density of the globules.

Effects of estradiol and mammalian LHRH on the ultrastructure of the pars distalis of the eel

SummaryMale silver eels were injected with estradiol-17β (E2) to induce the development of gonadotropic (GTH) cells. They were subsequently injected with luteinizing hormone-releasing hormone (LHRH). Exocytotic figures and the lysis of some large globules and granules were observed. Morphometric studies showed a significant increase in the percentage of vacuoles after 4 and 6 injections of LHRH and a slight but significant decrease of granules. This response did not, however, occur in all GTH cells which never appeared completely degranulated and did not reach a vesicular stage.Hemi-pituitaries of E2-pretreated eels were incubated with or without LHRH (20 min to 2 h). Although typical exocytoses were not detected, an increased number of small granules near the basal lamina and lytic processes (globules with a raspberry-shaped structure, granules with variable electron density) were observed in the LHRH-incubated hemi-pituitaries compared with those kept in a control medium. The structure of GTH cells and their response to LHRH has been studied only under conditions of artificial stimulation, and their functional similarity to GTH cells of spontaneously maturing eels is discussed.Large female eels had unstimulated GTH cells. Growth hormone (STH), thyrotropic (TSH) and prolactin (PRL) cells were stimulated after E2 and LHRH. As with GTH cells, they regressed slowly after treatment was discontinued.

Double immunocytochemistry in pre-embedding electron microscopy for the detection of neurotensin and tyrosine hydroxylase in the guinea pig, using two primary antisera raised in the same species.

In this study we identified for electron microscopy two different antigens (neurotensin and tyrosine hydroxylase) in the same pre-embedding section of nervous tissue, using two antibodies obtained in the same species. Optimal ultrastructural results were obtained without adding to the fixative either glutaraldehyde or acrolein (normally used for electron microscopy techniques). The different developing methods used in this study (DAB in combination with either 1 nm silver-enhanced colloidal gold or benzidine dihydrochloride) are perfectly distinguishable at the ultrastructural level, and show some advantages over other previously described developing procedures. For instance, the use of small gold particles (1 nm) reduces the severity of membrane damage caused by tissue penetration of the bigger gold particles (5 nm). In addition, the reaction products are stable, so there is no need to stabilize them before osmication, as is necessary in other developing methods such as the TMB procedure. The immunolabeling results obtained in this study were similar in both developing methods, although synaptic profiles were more readily visible when the DAB/colloidal gold procedure was used. Using electron microscopy, we have detected TH immunoreactivity in dendrites and perikarya receiving synaptic contacts from NT-positive terminals, as well as TH-immunoreactive inputs on NT-positive neurons, at both the somatic and dendritic levels.

The daily pattern of Fos synthesis by hypothalamic pro-opiomelanocortin neurons is unaffected by adrenalectomy in the rat.

At the onset of dark, a large population of rat mediobasal hypothalamic (MBH) pro-opiomelanocortin (POMC) neurons starts spontaneously expressing Fos-immunoreactivity (Fos-IR). Here we studied the effect of adrenalectomy upon this expression since circulating corticosteroids, which increase in the rat with the onset of behavioural wakening, are thought to modulate the basal expression of MBH POMC mRNA. Hence, groups of intact, adrenalectomised and sham-operated rats were sacrificed at times when Fos synthesis by POMC neurons is known to show either nadir (at light-offset) or peak (6 h after light-offset) values. Brains were processed for Fos- and/or POMC immunohistochemistry. This allowed us to show that, in all experimental groups, Fos-IR is hardly expressed in MBH POMC neurons at the onset of dark, whereas it is strongly induced 6 h later. We concluded that such an induction is not triggered through the known evening rise of plasma corticosteroid levels.

Response of prolactin cells in the goldfish adapted to diluted seawater with or without calcium

The activity of prolactin (PRL) cells was investigated in goldfish gradually adapted to diluted (30%) artificial seawater (ASW) or directly immersed in 23% ASW and in similar saline solutions free of calcium. When Ca2+ is omitted, PRL cells appear slightly more active than those in saline solutions with calcium, nuclear areas having intermediate values between those in freshwater (FW) and those in diluted SW. Nuclear areas are also slightly larger in Mg-free SW. Other signs of stimulation are not apparent. These data are compared to those obtained in other teleost species. The lack of stimulation in PRL cells of goldfish adapted to deionized water (DW) suggests that calcium plays a minor role in controlling prolactin secretion in this teleost.