A. Faivre-Bauman

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.

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.

MOLECULAR FORMS OF ACETYLCHOLINESTERASE: THEIR DE NOVO SYNTHESIS IN MOUSE NEUROBLASTOMA CELLS

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre‐existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form.

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.

Developmental and functional studies of parvalbumin and calbindin D28K in hypothalamic neurons grown in serum-free medium

The Ca2+‐binding proteins parvalbumin (Mr= 12K) and calbindin D28K [previously designated vitamin D‐dependent Ca2+‐binding protein (Mr= 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum‐free cultures of hypothalamic cells from 16‐day‐old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin‐immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti‐parvalbumin serum seems to recognize only the metal‐bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+‐dependent processes associated with neurotransmitter release.

Expression of microtubule-associated proteins during the early stages of neurite extension by brain neurons cultured in a defined medium

Abstract Immunoblotting analysis was used to identify the microtubule‐associated proteins (MAPs) present in cultures of mouse brain neurons. Polyclonal antibodies were raised against the two main adult brain MAPs, i.e., MAP2 (300 kDa) and τ (60–70 kDa). Whatever the stage of the culture, which was performed in a defined medium (3 or 6 days), the anti‐MAP2 serum detected several high‐molecular‐weight components (including MAP2) and an entity with 62–65 kDa. Anti‐τ revealed essentially a major peak of 48 kDa (young τ) but also slightly cross‐reacted with the 62–65 kDa entity. During the culture period (0–6 days) the cells developed progressively a dense neuritic network; the concentration of the different MAPs increased in parallel but at different rates depending on the different species. The in crease in concentration of the high‐molecular‐weight components occurred before that of 48‐kDa τ. This suggests that high‐molecular‐weight MAPs and 48‐kDa τ might be involved respectively in the initiation and elongation of neu‐rites. In contrast, and since the main developmental changes in τ composition seen in vivo did not occur during the time course of the culture, this transition might be related to later events of neuronal differentiation.

Immunological evidence for thyroliberin (TRH) neurons in primary cultures of fetal mouse brain cells. Ontogenic aspects

Primary cultures of dissociated cells were initiated from fetal mouse hypothalami and brain hemispheres, on the 13th and the 16th day of gestation (respectively 12-day and 15-day-old fetuses). After 10 days in vitro, the cultured cells were collected, pooled in an appropriate medium and thyroliberin (TRH) was assayed in the cell extracts using a specific radioimmunoassay. TRH was found in every type of culture. For hypothalami, higher levels of TRH were found when when starting from older embryos, while in brain hemisphere cultures the TRH content increased in culture of 12-day-old fetal cells only, whereas it decreased in cultures of 16-day-old fetal cells. Immunocytochemical staining allows visualization of TRH positive cells in all cultures except in hypothalamic cultures from 12-day-old embryos. This is consistent with the radioimmunoassay data. TRH was localized exclusively in some of the overlying cells, whereas the basal cells were always negative. Specificity of the staining was assessed by immunochemistry and radioimmunoassay. At the electron microscope level, the positive cells display neuronal features. The immunoprecipitate was found in both perikaryon and axons as well as in axonal dilatations.

Evolution of triiodothyronine nuclear binding sites in hypothalamic serum-free cultures: evidence for their presence in neurons and astrocytes.

[125I]Triiodothyronine (T3) nuclear binding was studied in hypothalamic cultures from fetal mouse grown in serum-free medium. In enriched neuronal cultures, the apparent dissociation constant of the binding does not change with time in vitro (7 X 10(-11) M), but the maximum binding capacity (MBC) doubles between day 7 and day 14 in vitro. We show here for the first time that homologous astrocyte cell cultures, devoid of neurons as checked by tetanus toxin binding, also display T3 nuclear binding, with the same affinity as neuronal cultures. However, their MBC is 3 times lower than that of neurons after a week in vitro, and increases more quickly thereafter.