Biomedical subjects
Publications and source records attributed to M Mallat.
Amoeboid microglial cells purified from primary cultures of embryonic rat brains and stimulated with bacterial lipopolysaccharides released high amounts of nerve growth factor. This finding suggests that brain macrophages play a major neurotrophic role during development, in adulthood, and in the regenerative events that follow localized lesions.
We have investigated the production of C3, C4, and factor B complement components in primary cultures of murine astrocytes and in clonal cell lines belonging to the astrocytic lineage by immunoprecipitation of secreted labeled polypeptides. Although C4 has not been detected, C3 appeared to be constitutively synthesized both by two transformed astroblastic cell lines and by astrocytes in primary cultures. In contrast, factor B was only secreted upon lipopolysaccharide stimulation both in astroglial primary cultures and in an immortalized astrocytic cell line. The eventual physiologic relevance of an endogenous brain production of components of the alternative pathway of complement activation is discussed.
Dissociate cultures were initiated from embryonic rostral mesencephalic and striatal tissues dissected from the mouse brain and previously incubated with a simian virus 40 (SV40) suspension. After several weeks in culture foci of fastly dividing cells were resuspended and cloned by successive dilutions. Several clones expressing the SV40 nuclear T antigen were obtained by these procedures and two of them, one mesencephalic (F7-Mes) and one striatal (F12-Str) were screened for the expression of glial or neuronal characters. Both clones possess adenylate cyclase-linked beta 2-adrenergic receptors. They also take up and synthesize gamma-aminobutyric acid (GABA) in amounts compatible with a glial origin. As is the case for astrocytes, the uptake of GABA is inhibited by beta-alanine and rather insensitive to the presence of diaminobutyric acid (DABA), a specific inhibitor of the neuronal GABA carrier. The most convincing evidence that F7-Mes and F12-Str belong to the astrocytic lineage comes from the fact that the two cell lines synthesize glial fibrillary acidic protein (GFAP) as demonstrated by immunofluorescence and immunoblotting. In an accompanying paper we also show that these lines behave like astrocytes when considered from the point of view of neuroglial interactions.
In an accompanying paper we report the characterization on the basis of pharmacological and immunological criteria of two astrocytic cell lines originating from the rostral mesencephalon and the striatum of the embryonic mouse (F7-Mes and F12-Str). This report compares the interactions of primary mesencephalic neurons with the astrocytic clones to that displayed with either an SV40-transformed fibroblastic clone (BT2) or primary mesencephalic (G-Mes) and striatal (G-Str) astrocytes. We show that BT2 differs from all other cell types (F7-Mes, F12-Str, G-Mes and G-Str). Indeed, as opposed to these cells BT2 is a poor substratum for neuronal adhesion or neuritic growth. This was clearly demonstrated by morphological examination of cocultures of the tested cells with either mesencephalic explants or dissociated cells. In addition a statistical analysis is provided which only concerns the dopaminergic (DA) neurons visualized by autoradiography after specific uptake of [3H]DA. The number of DA cells attached, the total length of their neurites and the degree of branching behaviour were examined. With the help of these criteria we show that F7-Mes and F12-Str are very similar to primary astrocytes and differ highly significantly from BT2. However, although sharing the main astrocytic features, F7-Mes and F12-Str do not differ from one another in their ability to induce the branching of DA neurites as their non-transformed counterparts do.
In two preceding papers we described the cloning of two astrocytic cell lines by simian virus 40 (SV40) transformation of embryonic mouse mesencephalon (F7-Mes) and striatum (F12-Str). The characterization of these lines as belonging to the astrocytic lineage is based on pharmacological, immunocytochemical and physiological data. Here we present quantitative and qualitative data on the morphological aspects of these two astrocytic clones observed under light and electron microscopy. We show that the clones present ultrastructural characters reminiscent of the morphology of young astrocytes. On one hand, they are rather similar to primary astrocytes in culture; on the other, they differ both from a clonal fibroblastic cell line (BT2) and from embryonic mouse fibroblasts in primary culture. These astroblastic clones display 4 morphologically different cell populations which we called types I, II, III and IV. Types II and III are very similar and represent the most predominant cells; their morphologies strongly remind of that of astroblasts. Type I corresponds to glioblasts and does not account for more than 15-20% of the total population. Type IV, which is very similar to differentiated velamentous astrocytes, normally represent ca. 5% of the cells. However, when the transformed cells are treated with mitomycin or mitomycin + dibutyryl cyclic AMP (dbcAMP), the proportion of type IV cells increases very much (up to more than 50% of the cells) while types I, II and III become less numerous. Morphological analysis therefore confirms that the two cell lines derived from the SV40 transformation of 14-day-old embryonic mesencephalic and striatal cells belong to the astrocytic lineage. Moreover, it seems that they can differentiate in vitro in cell culture conditions either spontaneously or under the action of pharmacological treatments known to enhance normal astrocyte maturation.
Fetal rat dopamine (DA) neurons were cultured in vitro for a 6-day period and transported, after redissociation, for 2 days prior to being grafted to the neostriatum of adult rats with 6-hydroxydopamine lesions of the ascending nigrostriatal pathway. In 2 of the 5 graft recipients that were tested for amphetamine-induced motor asymmetry, the grafts eliminated the lesion-induced turning behaviour within 3-6 weeks after transplantation. Fluorescence histochemistry revealed surviving grafts in all 6 recipients at 7 weeks after transplantation, containing between 42 and 125 DA neurons. The number of surviving DA neurons in the 3 non-compensated rats was below the minimum number of cells previously found to be necessary for functional effects on turning behaviour to occur.
We have analysed the isotubulin pattern of three astrocytic cell clones, derived from spontaneously established permanent cell cultures originating from 8-day postnatal mice cerebellar explants, in comparison with that of primary astroglial cultures from embryonic brain and cerebellum. These astrocytic clones, which may represent the different astroglial cell types of mouse cerebellum, did not produce the alpha- and beta-acidic isoforms, these being found only in cells of neuronal lineage. However, the three astrocytic clones, but not the primary astroglial cultures, did synthesize the beta'-tubulin isoforms; in addition quantitative analysis of the beta' proteins showed a positive correlation between the ability of the cells to extend processes and their synthesis of the beta' isoforms. These data suggest that the presence of beta'-tubulin is not specific for neuronal cells but may be related to the ability of cells from the nervous system to extend processes.
The use of neural culture is illustrated by the study of the in vitro development of a well defined neuronal system in the brain: the mesencephalic dopaminergic neurons of the rodent species. After a brief survey of its ontogenetic development, the various experimental techniques for its study in vitro are described. Such an approach has allowed the discovery of influences brought by a target area, like the striatum, on the maturation of these neurons. A new kind of local morphogenic interactions between these neurons and their glial partners is also reported.
The microheterogeneity of the alpha and beta isoforms of tubulin in brain cells in culture was studied. The cells were prepared from two precise regions of the embryonic mouse brain (ED15), the striatum and the mesencephalon. It was possible to maintain virtually pure cultures of neuronal or glial cells up to 1 and 4 weeks in vitro, respectively. The tubulin heterogeneity of striatal and mesencephalic neurons was found to be very similar after a few days in culture. More precise examination of pure neurons from the striatum revealed that their tubulin content after 7 days in vitro exhibited the same degree of complexity as a control extract from a 4 day-old mouse brain. In fact, we could detect the presence of at least six alpha and nine beta tubulin isoforms. Among these isoforms a specific family of beta proteins (beta' tubulin) and the more acidic alpha proteins were present. Since these isoforms have, up to now, been found only in tubulin extracts prepared from the nervous system, our experiments suggest that they belong to the neuronal subpopulation of this tissue. This point is reinforced by their complete absence from the tubulin proteins extracted from pure glial cells even after several weeks in vitro. These results lead us to propose that brain tubulin microheterogeneity is associated with the presence of neurons and not of glia and may, therefore, play a specific role in maintaining neuronal shape and function.
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A specific binding site for [3H]metergoline characterized by a KD of 0.5-1.0 nM was detected in microsomal and synaptic plasma membranes from various areas of the adult rat brain. Experiments with 5,7-dihydroxytryptamine- and kainic acid-induced lesions indicated that this specific binding site was localized post-synaptically with respect to serotoninergic neurons. The pharmacological characteristics of [3H]metergoline binding to microsomal membranes from the whole forebrain strongly suggest that this ligand labels a class of serotonin receptors. This was particularly obvious in the hippocampus in which serotonin was about 400 times more potent than dopamine and noradrenaline for displacing bound [3H]metergoline. In the striatum, serotonin was only 10 times as potent as dopamine in inhibiting [3H]metergoline binding, suggesting that this ligand may also bind to dopamine receptors. Striking similarities between the binding sites for [3H]metergoline and [3H]serotonin were observed in the hippocampus. Thus, not only the total numbers of binding sites for these two ligands in control rats but also their respective increases following intracerebral 5,7-dihydroxytryptamine treatment were very similar. Therefore, at least in the hippocampus, [3H]metergoline might well be the appropriate ligand for studying the characteristics of the 'antagonist form' of serotonin receptors postulated by Bennett and Snyder.
Amoeboid microglial cells (brain macrophages) were purified from early post-natal mouse brain cultures. The percentage of cells stained with an anti-Mac-1 antibody was greater than 95%. Stimulation of these brain macrophages by lipopolysaccharides induced the synthesis of interleukin-1 (IL-1), which, in part, remained associated with the cell surface and, in part, was released into the culture medium. In contrast, pure primary astrocyte cultures and cell lines of transformed or immortalised astrocytes did not synthesise significant amounts of IL-1, demonstrating that amoeboid microglia and not astrocytes synthesise IL-1 in vitro. These physiological data were confirmed by RNA hybridisation studies showing that, on LPS treatment, brain macrophages synthesise significant amounts of IL-1 alpha and IL-1 beta mRNAs.
In the adult cat, axons running through the corpus callosum interconnect the border between the visual cortical areas 17 and 18 (A17 and A18) of both hemispheres. This specific pattern emerges during postnatal development, under normal viewing conditions (NR), from the elimination of initially exuberant callosal projections. In contrast, if the postnatal visual experience is monocular from birth (MD), juvenile callosal projections are stabilised throughout A17 and A18. The present study aimed at using such a model in vivo to find indications of a contribution of glial cells in the shaping of projections in the developing CNS through interactions with neurones, both in normal and pathological conditions. As a first stage, the distribution and the morphology of microglial cells and astrocytes were investigated from 2 weeks to adulthood. Microglial cells, stained with isolectin-B4, were clustered in the white matter below A17 and A18. Until one month, these clustered cells displayed an ameboid morphology in NR group, while they were more ramified in MD animals. Their phenotype thus depends on the postnatal visual experience, which indicates that microglial cells may interact with axons of visual neurones. It also suggests that they may differentially contribute to the elimination and the stabilisation of juvenile exuberant callosal fibres in NR and MD animals respectively. Beyond one month, microglial cells were very ramified in both experimental groups. Astrocytes were labelled with a GFAP-antibody. The distributions of connexins 43 (Cx43) and 30 (Cx30), the main proteic components of gap junction channels in astrocytes, were also investigated using specific antibodies. Both in NR and MD groups, until 1 month, GFAP-positive astrocytes and Cx43 were mainly localised within the subcortical white matter. Then GFAP, Cx43 and Cx30 stainings progressively appeared within the cortex, throughout A17 and A18 but with a differential laminar expression according to the age. Thus, the distributions of both astrocytes and connexins changed with age; however, the monocular occlusion had no visible effect. This suggests that astrocytes may contribute to the postnatal development of neuronal projections to the primary visual cortex, including visual callosal projections.
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