C. Loudes

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.

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.

Effects of polyunsaturated fatty acids and hormones on synaptogenesis in serum-free medium cultures of mouse fetal hypothalamic cells

The effects of soluble factors on synaptogenesis by mouse fetal hypothalamic cells cultured in chemically defined conditions have been examined using transmission electron microscopy. Hypothalami taken on the 16th day of gestation were mechanically dissociated and cells were seeded in a minimum serum-free medium supplemented or not with the following components: triiodothyronine, corticosterone and a mixture of polyunsaturated fatty acids (arachidonic acid plus docosahexaenoic acid bound to defatted bovine serum albumin). In the minimum serum free medium synapses were found after 10 days in culture. However, the development of synaptic vesicles was very limited, whereas that of the presynaptic and postsynaptic densities was apparently normal. Supplementation of the minimum serum-free medium with triiodothyronine, corticosterone and polyunsaturated fatty acids added simultaneously, permitted a full development of synapses as attested to by the increase in number and the regular shape and diameter of synaptic vesicles as well as by the complexity and diversity of synapse configurations. Among those three factors, polyunsaturated fatty acids clearly played a key role. The ability of synapses formed in culture to respond to potassium evoked depolarization was examined on cultures grown for 12 days in the simultaneous presence of the three above mentioned supplements. Exposure for 3 min to 60 mM potassium chloride induced in synaptic boutons vesicular depletion, apposition of vesicle clusters onto the presynaptic grid, appearance of a rich filamentous network and of some coated vesicles. Return to 3mM potassium chloride induced in 3 min a massive restoration of the population of vesicles which slightly differed from synaptic vesicles in control cultures. These results show that: (1) the formation of synaptic vesicles in this system is regulated by soluble factors among which polyunsaturated fatty acids play a major role, and (2) synapses formed de novo in chemically defined conditions of culture display the same ability to respond to and to recover from potassium evoked depolarization as adult axon terminals. Thus, they offer a suitable model for analysis of the mechanisms involved in membrane traffic in central neurons.

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.

Involvement of DHP voltage-sensitive calcium channels and protein kinase C in thyroliberin (TRH) release by developing hypothalamic neurons in culture.

The intracellular mechanisms regulating the process of thyroliberin (TRH) release were studied using fetal hypothalamic neurons grown in serum-free medium. In particular, we compared the effects of dihydropyridine (DHP) derivatives, omega-conotoxin and phorbol esters on basal and K+-evoked TRH release from 12 days in vitro (DIV) neurons. BAY K 8644, a DHP calcium channel agonist increased in a dose-related manner basal and K+-evoked TRH release. PN 200-110, an antagonist of DHP-sensitive calcium channels, completely suppressed the effect of BAY K 8644, whatever the extracellular K+ concentration, but did not modify basal or K+-evoked TRH release. In contrast, omega-conotoxin partially inhibited the two latter processes. The active phorbol ester 12-O-tetradecanoyl-phorbol-beta-acetate (TPA), and to a lesser extent Sn-1,2-dioctanoylglycerol (DAG), triggered TRH release. This effect was specific, time and dose dependent and only partly dependent on extracellular calcium. Simultaneous addition of BAY K 8644 and TPA to the cells displayed a synergistic effect. The same compounds were studied on younger neurons (6-DIV cultures): BAY K 8644 stimulated TRH release whereas neither 60 mM K+ nor TPA did. These results suggest that TRH release can be mediated at least by two intracellular routes: (i) increase of intracellular calcium mediated by the opening of different types of voltage sensitive calcium channels, and (ii) activation of protein kinase C (PKC). The asynchrony in the maturation of the intracellular mechanisms underlying TRH release may be explained by different subcellular localizations of these mechanisms in neurons and is discussed in relation to synapse differentiation.

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.

Does triiodothyronine influence the morphogenesis of fetal mouse mesencephalic dopaminergic neurons cultured in chemically defined medium

Serum-free cultures of mouse mesencephalic cells were used for studying the influence of triiodothyronine (T3) on the morphogenesis of dopaminergic neurons. Supplementation of culture medium with T3 (10(-9) M) did not modify the number, but increased the size of the perikarya without affecting the neurite density of mesencephalic dopamine (DA) neurons. Furthermore, T3 had no effect on the uptake of [3H]DA in these cultures. These findings, taken together with previous results obtained with hypothalamic cell cultures, show that T3 affects the morphogenesis of mesencephalic DA neurons in a different manner than that of hypothalamus and that the perikarya size on the one hand, and the neurite elongation on the other hand, seem to be independently regulated by T3.

Thyroliberin (TRH) biosynthesis by hypothalamic cells in serum-free medium cultures

The terminal step of thyroliberin (TRH) biosynthesis was studied in fetal mouse hypothalamic cultures by incorporation of labelled amino-acids. Neosynthetized labelled TRH was identified by both biochemical and immunological techniques in cell and media extracts. Three possible stimuli of TRH biosynthesis were investigated: maturation of TRH neurons in culture, acceleration of the last processing step by ascorbate supplementation of the culture medium, and K(+)-evoked release of TRH. In all three cases, rate of labelled TRH synthesis was not changed while the amount of unlabelled TRH was increased. Moreover, in the case of K(+)-evoked release of TRH, the preferential release of a preformed unlabelled pool of TRH was observed. These results suggest that regulatory mechanisms are more likely to occur at the level of the processing of stored precursor(s) rather than on their neosynthesis.