Alain Barret

Laminin promotes attachment and neurite elongation of fetal hypothalamic neurons grown in serum-free medium

Laminin, as a coating or in solution, allows a rapid attachment of fetal hypothalamic cells in serum-free medium, and strikingly enhances the neurite network development. As compared to cultures grown on a fetal calf serum coating, cells remain in clusters and astrocytes become fibrous. Laminin was visualized by immunocytochemistry in non-neuronal cells. The number of laminin-positive cells was lower in cultures grown in serum-free medium than in those grown in serum-supplemented medium. In both culture conditions, their number decreases with time in vitro.

Triiodothyronine enhances the morphological maturation of dopaminergic neurons from fetal mouse hypothalamus cultured in serum-free medium

In dissociated hypothalamic cell cultures of 16-day mouse embryos, growing in chemically defined medium, the catecholaminergic neurons were identified by autoradiography after labelling with [3H]dopamine and by immunocytochemistry with an anti-tyrosine hydroxylase antibody. Using selective inhibitors of amine transport and radioenzymatic determination of amine levels in these cultures, we show that these neurons were mostly dopaminergic. The number of dopaminergic neurons identified by the two techniques increased between days 5 and 8 and decreased after 15 days in vitro. The same number of neurons were identified by autoradiography and by immunocytochemistry and consisted of fusiform and multipolar neurons. The proportion of both types remained steady until 15 days in vitro. Under these conditions, the addition of triiodothyronine (10(-9) M) at the initiation of the culture increased the size but not the number of dopaminergic neurons after 8 days in vitro. Furthermore, triiodothyronine significantly increased the dopaminergic neurite length and arborization. This morphological effect of triiodothyronine was associated with an increase of 35% in [3H]dopamine uptake. Our study shows that hypothalamic dopaminergic neurons are responsive to triiodothyronine which acts as a maintenance or trophic factor having an effect on neurite extension and arborization.

Triiodothyronine stimulates the production of insulin-like growth factor (IGF) by fetal hypothalamus cells cultured in serum-free medium

Grown in serum-free medium, dissociated cells from fetal mouse hypothalami release insulin-like growth factors (IGFs) and their binding proteins (IGF BPs) into the culture medium. Addition of triiodothyronine (10-12-10-8 M), which enhances neuron maturation, resulted in a significant increase in IGF concentration. By contrast, there was no significant effect on IGF BP. These results suggest a role for thyroid hormone in the control of IGF biosynthesis in nerve cells.

Release of immunoreactive TRH in serum-free cultures of mouse hypothalamic cells

Serum-free cultures of mouse hypothalamic cells were used as a model for studying TRH (thyroliberin) secretion in vitro. Supplementation of the culture medium with triiodothyronine, corticosterone and polyunsaturated fatty acids is necessary to obtain a substantial release capacity of TRH neurons. Under these conditions depolarization of the cells with 60 mM K+ results in a calcium-dependent release of immunoreactive TRH.

Possible role of neuropeptide degrading enzymes on thyroliberin secretion in fetal hypothalamic cultures grown in serum free medium

In the present work, we have looked for the presence of two tissular neuropeptide degrading activities, the pyroglutamate aminopeptidase (PAP) and the post-proline cleaving enzyme (PPCE), in dissociated brain cell cultures. These two activities are present in extracts of cells grown in serum-free medium and are detected at a very low level in incubation media. Depolarization of hypothalamic neurons by 60 mM K+ does not specifically increase the level of PAP and PPCE in the medium. We have also used an inhibitor of PPCE: Z-Gly-ProCHN2. This compound can be left in contact with living cells without any toxicity, and in certain conditions of incubation blocks totally and irreversibly both PAP and PPCE. This blockade results in increased levels of TRH, intracellular as well as released into the medium, spontaneously and upon K+ depolarization. These results evidence the role of degradation processes in the mechanisms regulating peptide turn-over.

Differential expression and subcellular localization of secretogranin II and synaptophysin during early development of mouse hypothalamic neurons in culture

Mature neurons contain two distinct regulated secretory pathways, characterized electron microscopically by so-called large dense core vesicles and small synaptic vesicles, respectively. Each vesicle type is characterized by vesicle-specific proteins, such as the granins (chromogranins/secretogranins) for the matrix of large dense core vesicles and synaptophysin for the membrane of small synaptic vesicles. So far, no data exist on the biogenesis of these two distinct vesicle types during neuronal development. We have used secretogranin II and synaptophysin as markers for the biogenesis of these two vesicle types during the development of mouse hypothalamic neurons in culture, using immunocytochemistry and biochemical analyses. By immunofluorescence, we found that secretogranin II appears as early as synaptophysin, but in a subset of neurons only, and with different subcellular localizations. It was observed in cytoplasmic areas where little or no synaptophysin immunofluorescence was detected, such as lamellipodia, emerging neurites and growth cones. At later stages, the proportion of secretogranin II-containing varicosities remained steady whereas that of synaptophysin-containing varicosities increased dramatically. By quantitative analysis we found that the level of expression of synaptophysin increased several-fold during synaptogenesis whereas that of secretogranin II decreased. These data suggest that large dense core vesicles and small synaptic vesicles can be formed separately and expressed at different levels. They provide evidence for a differential biogenesis of these two distinct vesicle types.

Biochemical characterization of the uptake and release of [3H]dopamine by dopaminergic hypothalamic neurons: a developmental study using serum-free medium cultures.

The development of the biochemical properties of mouse hypothalamic dopaminergic neurons has been analyzed in vivo and in cultures of cell taken on the 16th day of gestation and grown in serum-free medium for up to 3 weeks. In the course of in vivo development, the dopamine (DA) content remains low during fetal life (10% of the adult value), beginning to increase on the 19th fetal day. In contrast, the specific accumulation of [3H]DA increased markedly during the last days of gestation from 20% of the adult value on the 16th fetal day to 70-80% of the adult value on Postnatal Day 3. Hypothalamic DA neurons in culture accumulate endogenous DA although at a lower level than in vivo. They take up [3H]DA by an active transport system which is specific for DA, and which shows time, temperature, and sodium dependency (Km = 1 microM). HPLC analysis showed that the newly taken up [3H]DA was not metabolized in the short run under the conditions used. It was stored in a form that could be released when neurons were depolarized in a high K+ (60 mM) medium. The K+-evoked [3H]DA release was found to be strictly dependent on extracellular Ca2+. Moreover the release of [3H]DA was also stimulated by veratridine in a Ca2+-dependent manner. Similar data have been obtained with the release of endogenous dopamine. No specific uptake and no K+-evoked dopamine release occurred in 2-day-old cultures. The specific [3H]DA uptake and the K+-evoked release appeared in 5-day-old cultures and increased with time in culture at least until Day 15. We examined the effects on [3H]DA release of polyunsaturated fatty acid, triiodothyronine, and corticosterone, all of which have been shown to play an important role in synaptogenesis in culture. These components, either separately or together, did not modify the percentage of the basal or the stimulated [3H]DA release. These results showed that hypothalamic DA neurons grown in serum-free medium progressively acquired the functional properties of adult DA neurons as concerns DA synthesis, DA uptake, and release. From a development point of view, this study suggests that the capacity to specifically take up [3H]DA and to respond to high K+ concentration is not expressed at early stages of neuronal development.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteolytic Processing of Sulfated Secretogranin II in the trans-Golgi Network of GH3B6 Prolactin Cells

Secretogranin II (SgII) is a protein specific to the matrix of the secretory granules in neurons and neuroendocrine cells. We have already demonstrated the precursor-product relationship between sulfated SgII and four N-terminal derived peptides in GH3B6 prolactin cells. In this study, we have investigated the subcellular compartment in which the cleavage of SgII is initiated by taking advantage of its tyrosine sulfation in the trans-Golgi network (TGN). In order to prevent export of radiosulfated SgII from the TGN, we used brefeldin A (BFA) as well as incubation at 20°C. BFA completely inhibited the cleavage of SgII when added immediately post-pulse. BFA added a few minutes post-pulse or after a 20°C incubation, however, permitted the cleavage of SgII in the presence of the drug. These SgII-derived peptides generated in the presence of BFA could not be released upon stimulation of the cells by either thyroliberin, a physiological secretagogue, or KCl. These results demonstrate that SgII can be cleaved in the TGN. They also evidence that the cleavage occurs in a distal compartment of the TGN different from the sulfation site. The transfer of SgII from the sulfation site to this distal compartment of the TGN involves BFA-sensitive membrane dynamics.

Ontogenesis of peptidylglycyl α-amidation activity in the mouse hypothalamus in vivo and in serum-free medium cultures. Relation with thyroliberin (TRH) accumulation and release in vitro

The influence of peptidylglycine alpha-amidating monooxygenase (PAM) and of its co-factor, ascorbate, were studied in relation to thyroliberin (TRH) activity during mouse hypothalamus development. In vivo, PAM activity developed slowly at fetal stages, and exhibited a sharp rise around the 5th-8th postnatal day, the adult level being reached around day 15. The same developmental pattern was observed when studied in serum-free cultures initiated from fetal mouse hypothalamus. Using this in vitro model, we investigated the effects of ascorbate, a necessary co-factor of PAM, on TRH. Upon ascorbate supplementation of the culture medium, the TRH accumulation normally occurring in our cultures was further enhanced. The half maximum effect was attained with 20 microM, and the amplitude of the response to ascorbate was maximum around 9-13 days in vitro. Moreover, ascorbate increased to an even larger extent the amounts of TRH released upon chemical depolarization. These results are consistent with a direct role of ascorbate on PAM activity, but other more general effects on the maturation of the neuronal response to physiological stimuli cannot be excluded.

STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN PROLACTIN CELLS FROM THE INNER AND OUTER ZONES OF THE MALE RAT ANTERIOR PITUITARY

Abstract.The aim of this study was to compare the ultrastructure of prolactin cells in the inner and outer zones of the male rat anterior pituitary, and to relate their morphological features to their secretory activity, by means of standard and ultrastructural reverse hemolytic plaque assays (RHPA). The immuno-ultrastructural study showed that in the inner pituitary small-granulated cells represented 52% of the prolactin cells, there being only 5% with large granules, whereas the prolactin cells with large granules accounted for 52% in the outer zone, with only 7% being small-granulated. Percentages of cells with intermediate-sized granules were 43% and 41%, respectively. Analysis of RHPA data revealed that, under basal conditions, prolactin cells secreted more actively in the inner zone than in the outer zone. Stimulation with thyrotropin-releasing hormone or KCl treatment increased the percentage of secretors and the sizes of hemolytic plaques in both zones. However, in response to thyrotropin-releasing hormone, the increase in number of secretors was always higher in the outer zone, whereas the enlargement of plaque sizes was greater for the ”inner” cells. These findings are in favor of the small-granulated cells, which predominate in the inner zone, being in a stage of active secretion and responsiveness.