A. Tixier-Vidal

Vasoactive intestinal peptide (VIP) stimulates prolactin (PRL) release and cAMP production in a rat pituitary cell line (GH3/B6). Additive effects of VIP and TRH on PRL release

The recent localization of VIP [l] (a peptide initially discovered in the gastrointestinal tract) within the hypothalamus, the hypothalamo-hypophyseal portal blood and the adenohypophysis [2-61, strongly suggested its possible regulatory role on the pituitary gland. Kato et al. [7] and Vijayan et al. [8] have shown that VIP was actually able to stimulate the release of several adeno-hypophyseal hormones including prolactin (PRL) in vivo, but not in vitro. They concluded, therefore, that VIP was acting on the secretion of pituitary hormones by means of indirect mechanisms [7,8]. Ruberg et al. nevertheless, demonstrated a direct stimulating effect of VIP on PRL release by incubated rat hem&pituitaries [9]. We have investigated the responsiveness to VIP of a rat prolactin-secreting celI line, the GH3/B6. The hormonal secretion of these cells, or of related strains, has already been shown to be sensitive to various physiological modulators of PRL secretion in vivo, including thyroliberin (TRH) [lo] (for recent reviews see refs. [ 11,121). Such an homogeneous population of target cells provides, therefore, a suitable model for investigating a hypothetical effect of VIP on prolactin cells at the cellular and subcellular levels. Here, we show that the GH3/B6 cells are highly responsive to VIP which stimulates in a concomitant manner both their PRL release and 3’,5’cyclic adenosine monophosphate (CAMP) production. These effects of VIP were compared to those of TRH, a well-established hypothalamic PRLstim-

Glial Fibrillary Acidic Protein

Since it was discovered, twenty years ago, the glial fibrillary acidic protein has been the subject of more than 500 publications. The huge interest it has raised up is probably due partly to its abundance in the central nervous system and also, above all, to its cellular specificity which makes it the universally recognized marker of astrocytes. It has been used by biologists as a tool to follow the normal or pathological glia cell differentiation in animal models and human pathology, particularly in the fields of neuropathology and neuro-oncology.

Differentiation of Fetal Mouse Hypothalamic Cells in Serum-Free Medium

We show here that cells dissociated from fetal mouse hypothalamus and cerebral hemispheres can be grown in primary culture in a serum-free medium (SFM). We describe several properties of these cultures and compare them to those in serum-supplemented medium (SSM). The SFM used is a modification of that described for neuroblastoma cells: neuronal survival is improved when 17 beta-estradiol is added. Initial events in culture development were similar to those observed in SSM. However, after 1 week, several differences could be noted: in SFM, the proportion of neuron-like cells was increased while the basal glial layer was noticeably reduced, and the neurite network remained less developed than in SSM. These findings demonstrate that the use of SFM permits manipulation of the types and proportions of cells in these primary cultures. This point has been already made. Several neuronal activities were studied. In cultures from both hypothalamus and cerebral hemispheres, thyroliberin (TRH)-immunoreactive cells were visualized by immunohistochemistry, and TRH was radioimmunoassayed in cell extracts and in the medium. In hypothalamic cultures, tyrosine hydroxylase was shown to remain stable for 1 week, and then declined. Glutamic acid decarboxylase disappeared very quickly in vitro, whereas choline acetyltransferase activity increased more rapidly in SFM than in SSM. It is concluded that the use of an SFM for growing normal fetal hypothalamic cells offers a promising model for studying neuroendocrine regulatory mechanisms in culture.

Effect of Polyunsaturated Fatty Acids on Fetal Mouse Brain Cells in Culture in a Chemically Defined Medium

Abstract: The biochemical and morphological effects of polyunsaturated fatty acids on fetal brain cells grown in a chemically defined medium were studied. Fetal brain cells were dissociated from mouse cerebral hemispheres taken on the 16th day of gestation. After cells had grown in chemically defined medium for 8 days, the proportion of polyunsaturated fatty acids of cultured cells was only one‐half of that observed at day 0 and about 1.5 times less than that of cells grown in serum‐supplemented medium. Fatty acid 20:3(n‐9) was present in cultured cells grown in either chemically defined or serum‐supplemented medium, demonstrating the deficiency of essential fatty acids. The reduced amount of polyunsaturated fatty acids in cells grown in the chemically defined medium was balanced by an increase in monounsaturated fatty acids. The saturated fatty acids were not affected. When added at the seeding time, linoleic, linolenic, arachidonic, or docosahexaenoic acid stimulated the proliferation of small dense cells. Besides, we demonstrate that each of the four fatty acids studied was incorporated into phospholipids. Adding fatty acids of the n‐6 series increased the content of n‐6 fatty acids in the cells, but also provoked an increase in the n‐3 fatty acids. Among several combinations of fatty acids, only 20:4 and 22:6, when added to the culture in a ratio of 2:1, restored a fatty acid profile similar to controls (i.e. in vivo tissue taken at postnatal day 5).

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.

Immunoelectron microscopic localization of synaptophysin in a golgi subcompartment of developing hypothalamic neurons

Synaptophysin, previously identified as an integral membrane glycoprotein (mol. wt 38,000) characteristic of presynaptic vesicles of mature neurons, provides a molecular marker to study the origin, formation and traffic of synaptic vesicles. Using the monoclonal antibody SY38 against this polypeptide we have localized synaptophysin by immunofluorescence and electron microscope immunoperoxidase methods in cultured mouse hypothalamic neurons taken from 16-day-old fetuses which achieve synaptogenesis after 10-12 days in vitro. We have compared the localization of synaptophysin in perikarya and nerve endings as a function of age (2-19 days in vitro) and of treatment of mature neurons with nocodazole. Using immunofluorescence microscopy, synaptophysin was already detected in neuronal soma at 2 days in vitro, where the initiation of neurite development is observed. At the electron microscope level, virtually all mature synaptic boutons and varicosities showed an extensive synaptophysin labeling of synaptic vesicles at 12-13 days in culture whereas neurites showed only very few labeled vesicles. In neuronal soma taken before synapse formation (6 days in vitro), synaptophysin was selectively localized in membranes of the innermost cisternae of the Golgi zone and in vesicles of variable size and shape in the core of the Golgi zone. In contrast, after synapse formation, synaptophysin labeling was barely detected in the Golgi zone of neurons but a very strong labeling of synaptic vesicles in synaptic boutons was observed. Treatment of mature neurons (12 days in vitro) with nocodazole (10(-5) M) resulted in a conspicuous synaptophysin staining of the innermost trans-Golgi cisternae and numerous vesicles in the cytoplasm. Furthermore, an accumulation of labeled synaptic vesicles on the presynaptic membrane of nerve terminals was found. The data suggest that synaptophysin is released from the Golgi apparatus in a vesicular form, after glycosylation, and is then transported to nerve endings by a mechanism which requires integrity of microtubules.

Dissociated cell cultures from fetal mouse hypothalamus patterns of organization and ultrastructural features

SummaryDissociated fetal hypothalamic cells mainly taken from 14 day-old mouse fetuses were grown in vitro for increasing time (9 to 60 days). Soon after inoculation the cells partly reaggregated and attached. The small reaggregates were then interconnected by fibers bundles. After the first week the cultures were composed of a continuous basal monolayer of flat and transparent cells, over which various types of refractile cells were lying in discontinuous areas. The ultra-structural study enabled us to identify these cell types, to describe their spatial relationships, and to follow their evolution with time in culture.The basal cell formed several superimposed layers. With increasing age, they displayed typical features of astrocytes and of ependymal cells. The latter exhibited rhythmic ciliary movements in culture.The overlying cells corresponded to three types which were associated in small clumps: primitive neuro-epithelial cells, maturing as well as mature neurons and typical neurosecretory cells. The latter cells were found as early as 9 days of culture of 14 day-old fetal hypothalamic cells and retained their typical features up to two months.Neuronal processes formed very dense networks at the surface of the cultures and terminated within the basal layers. Axon and dendrites were precociously found and were still present after two months. Within axon terminals dense-core vesicles appeared at the same time as neurosecretory cells. Synaptic vesicles and synaptic junctions were found later on.

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.

Binding of a tritiated thyrotropin-releasing factor to a prolactin secreting clonal cell line (GH3)

Abstract The GH3 pituitary clonal cell line, which secretes prolactin and is responsive to thyrotropin-releasing factor, TRF, has been used to study the binding of a strongly labelled 3H-TRF (60 Ci/mM). This binding is time-dependent. It increases linearly in the first 15 min of incubation, thereafter reaching a plateau up to 60 min, independent of the dose (from 0.27 to 27 nM). This kinetic is similar to that of the increase of prolactin release induced by TRF. The specificity has been tested by comparing the affinity of GH3 pituitary cells with those of 3T3 fibroblasts and C6 glial cells and by competition experiment with unlabelled TRF and other peptides. After a constant period of incubation (30 min), the amount of bound molecules is dose-dependent. The two different rates of augmentation with increasing doses for low (1.35 to 155 nM) and high (155 to 1 080 nM) concentrations of 3H-TRF, suggest the involvement of at least two components in the3H-TRF binding to GH3 cells. Moreover, the presence of radioactive material within the cells is revealed by an autoradiographic study performed after a 30 min incubation with 3H-TRF.

Immunocytochemical localization of glycoprotein hormones in the rat anterior pituitary. A light and electron microscope study using antisera against rat bet subunits: a comparison between preembedding and postembeeding methods.

The binding sites of antisera (anti) to the beta (beta) subunits of rat follicle-stimulating hormone (rFSH), rat luteinizing hormone (rLH), and rat thyroid-stimulating hormone (rTSH) have been localized in rat anterior pituitaries by immunocytochemistry using light and electron microscopy. With the light microscope, LHbeta and FSHbeta were found in the same cells, which were violet after the alcian blue-periodic acid Schiff (AB-PAS) staining. TSHbeta was found in polygonal or stellate cells that were blue after AB-PAS. With the electron microscope, the thyrotropic cells contained very small secretory granules. LHbeta and FSHbeta were found in various types of cells (types A and B and their intermediate forms), which had previously been identified as gonadotropic cells. On serial ultrathin sections using the postembedding method the same cells and even some granules inside these cells were stained by both anti-rLHbeta and anti-rFSHbeta. A comparison of binding sites of anti-rLHbeta was performed using the preembeeding and the postembeeding methods. Antigenicity was observed on secretory granules whatever the method used. However, binding sites of anti-rLHbeta were detected inside the cisternae of the rough endoplasmic reticulum only with the preembedding method.