Biomedical subjects

J Gabrion

Publications and source records attributed to J Gabrion.

At least 19 recordsLinked to original sources

PubMed1991

Gap junctional intercellular communication between cultured ependymal cells, revealed by lucifer yellow CH transfer and freeze-fracture.

In order to analyze intercellular communication between ependymal cells in mammalian brain, we have studied gap junctional communication of ependymal and glial cells in long term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus obtained in serum supplemented media with two complementary methods: 1) dye transfer of Lucifer Yellow CH after intracellular microinjection of the different cellular types, and 2) freeze-fracture of the same cultured ependymal cells. In our culture conditions, we have shown that the GJIC capacity to transfer dye was very different according to cellular types microinjected with Lucifer Yellow CH in the following respects: 1) in ependymal cells, GJIC was always important: ciliated ependymal cells, which are numerous in hypothalamic ependymal cultures (10-120 coupled cells), choroidal ependymocytes in plexus cultures (15-250 coupled cells), and non-choroidal ependymocytes in diencephalic roof cultures (10-30 coupled cells), and 2) in astroglial cells found in these primary cultures, no GJIC was observed in spite of the presence of well-differentiated gap junctions revealed by freeze-fracture replicas. All these results show a strong GJIC in ependymal cells and indicate the very good functional state of these cells in vitro.

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PubMed1991

Incorporation of D-[3H]-glucosamine and L-[3H]-fucose into the developing rat cochlea.

The uptake of two tritiated carbohydrates, D-[3H]-glucosamine and L-[3H]-fucose, to the developing rat cochlea was examined using light and electron microscopic radioautography. Both carbohydrates, administered to in vitro developing rat cochleas, shared a similar ultrastructural labeling pattern on the microvilli and apical cell region and on the tectorial membrane (TM) fibrils. On embryonic day 18, the radiolabeling appeared on the apical surface of the undifferentiated epithelium that will develop into both spiral limbus and Kölliker's organ (KO), while on postnatal day (PD) 1, it was only located on the apical surface of the KO. When D-[3H]-glucosamine was administered in vivo to newborn rats, the radiolabeling was observed in the TM covering the KO at PD 3. Lastly, D-[3H]-glucosamine administered in vivo to PD 7 rats, appeared at PD 9 in the TM region lying just above the organ of Corti. The present findings support the previously suggested leading role of the spiral limbus and KO in the secretion of the TM during cochlear development. The secretion of carbohydrates, and probably of other matrix components, starts on the spiral limbus and KO region and progressively extends to the organ of Corti.

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PubMed1991

Distribution of arrestin-like protein and beta-subunit of GTP-binding proteins in quail choroid plexuses.

Monoclonal antibodies (Mabs) directed against retinal arrestin (S-antigen) were used to detect and characterize this protein in choroid plexus (CP) of quails maintained during eight days, either under long-day photoperiods or in constant darkness. Immunocytochemistry and Western blotting confirmed the presence and the distribution of an arrestin-like protein in quail CP. Arrestin-like immunoreactivities in CP were compared with those obtained with Mabs to beta 36-subunit of G proteins (G beta), alpha-subunit of transducin and rhodopsin. Rhodopsin-like and transducin-like proteins could not be detected in choroidal cells, whereas intense positive reactions were observed with anti-G beta and anti-arrestin Mabs. The strongest immunoreactivities were found in choroidal ependymocytes of the lateral and IIIrd ventricles. In CP epithelial cells lining the IVth ventricle, very weak or no immunoreactivity could be detected with Mabs to arrestin, while Mab against G beta subunit always provided a positive reaction. In quails maintained in constant darkness, arrestin- and G beta-immunoreactivities of CP epithelial cells displayed changes in cellular distribution and intensity (decrease or disappearance of the immunoreactions). The strong arrestin-like immunoreaction located in the apical region of ependymocytes suggests the preferential association of the protein with choroidal microvilli and a possible role in cerebrospinal fluid production assumed by CP cells.

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PubMed1991

A common epitope is shared by ciliary rootlets and cell-cell adherens junctions in ciliated ependymal cells.

Using immunoblot, light and electron immunocytochemistry, we investigated the presence and the localization of polypeptides cross-reacting with the monoclonal antibody CC.310 (mAb CC.310), which is mainly directed against a 175K (K = 10(3) Mr) ciliary rootlet protein. In hypothalamic ependymal cultures, the unique antigen recognized by mAb CC.310 was associated with the Triton X-100-insoluble fraction in these cultures and electrophoretically migrated to these cultures and electrophoretically migrated to 94K. mAb CC.310, which appears to be a very suitable marker for ciliated ependymocytes, allowed us to observe ciliogenesis during the growth of the ependymal cultures, from a single spot in each undifferentiated ependymal cell to a massive labeling in ciliated ependymal cells. In fully differentiated ciliated ependymocytes, mAb CC.310 strongly reacted with fibrous structures corresponding to ciliary rootlets, as confirmed by ultrastructural observations. In addition, a weaker immunostaining was also found along the intercellular junctions, and showed that proteins sharing a common epitope are located in ependymal ciliary rootlets and near adherens-type junctional complexes. Immunofluorescence studies confirmed the presence of positive labeling at the level of junctional complexes between cells in two epithelial lines, HeLa and PtK2, in which mAb CC.310 mainly reacted with one polypeptide of 85K.

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PubMed1991

[Monoclonal antibodies obtained against G-protein epitopes: comparison of immunoreactions seen in the brain, retina and striated muscular tissue].

Monoclonal antibodies have been obtained against a purified fraction of brain G proteins containing the Gi alpha, G0 alpha, G beta, and G gamma subunits. After characterization, two monoclonal antibodies have been used to detect the cellular distribution of the two epitopes in neural, retinal and muscular tissues: ELISA, cross-dot and Western blot demonstrated that F.IV.5 is an anti-G beta antibody specific for the 36 kDa beta-subunit. ELISA, cross-dot and immunocytochemical distribution of the epitopes recognized by F.VII.9 suggested that this antibody recognizes epitopes which are also detected with polyclonal anti-G0 alpha antibodies. With both monoclonal antibodies, we confirmed that G proteins demonstrated a sub-membranous distribution as well as extensively cytoplasmic, axoplasmic or sarcoplasmic distributions in different cell types.

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PubMed1990

Establishment of a long-term primary culture of striatal neurons.

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.

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PubMed1990

Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage-dependent calcium channels.

GTP binding proteins have been proposed to play a role in excitation--contraction coupling. In a precedent study [Toutant et al., (1988), Biochem. J., 405-409], we determined that Bordetella pertussis toxin is able to catalyse ADP-ribosylation of two substrates in the detergent soluble fraction of total muscle extracts. Purified fractions of transverse tubule membranes (T-tubule membranes), a key element of the excitation--contraction coupling, were shown to exhibit a major ADP-ribosylated substrate at 40 kd and an immunoreactivity with antisera raised against purified bovine brain Go alpha or G beta. In the present study, we have investigated the cellular distribution of G protein subunits in comparison with that of the voltage-dependent Ca2+ channels by immunofluorescence on transverse and longitudinal sections of fast and slow muscles. With affinity-purified antibodies against G beta subunits, a fluorescent labelling underlined the myofibrils and sarcolemma, whereas a strong immunoreaction in a dotted pattern evoked the presence of the subunit in repetitive triadic structures. With anti-Go alpha antibodies, the immunofluorescence was more clearly focussed on a dotted pattern and the co-location with the voltage-dependent Ca2+ channel immunoreactivity indicates that both proteins were located in very close subcellular structures. Immunoblot analysis and PTX ADP-ribosylation of the purified light sarcoplasmic reticulum (LSR), heavy sarcoplasmic reticulum (HSR) and T-tubule subcellular fractions indicate the discrete presence of G proteins in LSR, an unambiguous labelling of the HSR fraction, while T-tubule membranes clearly appear very rich in a Go-like protein, confirming the observed preferential immunocytochemical distribution of G protein subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1990

Maturation of the endocrine pancreas in the sea bass, Dicentrarchus labrax L. (Teleostei): an immunocytochemical and ultrastructural study. I. Glucagon-producing cells.

The structure of the endocrine pancreas in the sea bass (Dicentrarchus labrax L.) was studied with special reference to glucagon-immunoreactive cells. As described in most of the teleosts, the sea bass was found to have a diffuse pancreas. In the adult, endocrine cells were clustered in a principal islet and numerous accessory islets where the glucagon A cells were localized peripherally. Under electron microscopy, the A cells displayed a clear hyaloplasm with granules having typical spherical or polyhedral cores, as in other vertebrates. The maturation of the endocrine pancreas was monitored under rearing conditions. The endocrine pancreas appeared during the prelarval stage, 3 days after hatching, and consisted of a single cluster of morphologically similar cells, containing very small cytoplasmic granules. During the larval stage, cytodifferentiation resulted in modifications of cell shape and increased granule size. Typical granules appeared in 8-mm-long larvae. Cells immunoreactive with mammalian glucagon antibodies appeared only at the beginning of the juvenile stage (3 months/20 mm). Electron microscope observations revealed that the storage of hormone in numerous cytoplasmic granules began at this stage.

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PubMed1990

Selective distribution of sugars on the tegumental surface of adult Bothriocephalus gregarius (Cestoda: Pseudophyllidea).

Whole specimens and histological and semi-thin sections of Bothriocephalus gregarius adults were exposed to lectins to identify carbohydrates present in the tegument and parenchyma. The sugars N-acetyl glucosamine, N-acetyl galactosamine, galactose, glucose (or mannose) and fucose were detected in the cestode using eight lectins: WGA (Wheat germ agglutinin), HPA (Helix pomatia agglutinin), SBA (Soy bean agglutinin), PHA (Phaseolus vulgaris agglutinin), RCA60 and RCA120 (Ricinus communis toxin and agglutinin), ConA (Concanavalin agglutinin) and UEA-I (Ulex europaeus agglutinin). Combined use of these methodological approaches (whole specimens, paraffin and semi-thin sections) revealed the presence of a gradient in the distribution of most of the sugars over the tegument, with the highest concentrations on the strobila (as shown by most of the lectins). Other sugars were specific for the scolex or strobila (as shown by UEA-I or HPA, respectively). The ultrastructural study showed that the distribution of glycoconjugates was associated with the presence of specific tegumental coats. The significance of this selective distribution and its relevance to cestode physiology and host-parasite relationships are discussed.

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PubMed1990

Choroidal ependymocytes in culture: expression of markers of polarity and function.

Long-term primary cultures derived from fetal mouse or rat choroid plexus were obtained in serum-supplemented media. Monoclonal and polyclonal antibodies to basolateral and apical membrane components were used to observe the expression of specific markers of polarity and function. Choroid plexus cultures and thin frozen sections of adult tissues were compared by immunocytochemistry. Two polyclonal antibodies directed against laminin and fibronectin were used on cultured choroid plexus and sectioned tissues, showing that fibronectin and laminin are located on the basolateral membrane domain in ependymocytes in vitro, as well as in vivo. Na(+)-K(+) ATPase was apically detected by light and electron microscopy with a monoclonal antibody (Mab H30) in both cultured cells and sectioned tissues. Double immunofluorescent staining with Mab H30 and affinity-purified polyclonal antibodies to the alpha subunit of G0 protein (G0 alpha) demonstrated the relatively similar distribution of the two antigens on the apical face of the choroidal tissue, both in vivo and in vitro. The distribution of these markers shows a typical differentiation with maintenance of polarized features in choroidal ependymocytes in culture, testifying that this cell culture system constitutes an interesting model for studying the functional characteristics of ependymal cells of the choroid plexus.

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PubMed1989

Isolation, characterization and immunocytochemical localization of caldesmon-like protein from molluscan striated muscle.

A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat-stable protein. It cross-reacts immunologically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2+-dependent manner, cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+-ATPase. The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.

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PubMed1989

A thyroid hormone-vasopressin interaction promotes survival and maturation of hippocampal neurons dissociated postnatally.

Hippocampal cells dissociated from 5-day-old rat pups were grown in a suitable chemically defined basal medium, supplemented or not with 3,3',5-triiodo-L-thyronine (T3), [Arg8]-vasopressin (AVP), or both, at different concentrations. Four days after plating, neuron-like cells began to degenerate drastically in the basal medium. Although AVP alone had no effect, T3, and to a greater extent T3 and AVP together, prevented their death. Moreover, T3 and AVP also acted synergically in promoting the maturation of surviving cells, especially AchE-positive neurons, either directly or through glial cells.

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PubMed1989

Characteristics and specific localization of receptors for atrial natriuretic peptides at non-neuronal cells in cultured mouse spinal cord cells.

Characteristics of atrial natriuretic peptide receptors were determined in cultured mouse spinal cord cells. Saturation and competition experiments demonstrated the presence of a single class of atrial natriuretic peptide binding sites with high affinity (KD = 0.054 nM) and a density of 1.92 fmoles/10(6) cells. A similar affinity (KD = 0.070 nM) was observed in rat spinal cord membrane preparations. These atrial natriuretic peptide binding sites were functional receptors since the treatment of cells with atrial natriuretic peptide increased cyclic guanosine monophosphate levels within these cells in a classical time-dependent manner. When atrial natriuretic peptide was applied onto the cell body of intracellularly recorded spinal cord neurons, this peptide did not evoke a change of the input resistance or of the resting membrane potential value. Light-microscopic autoradiography studies showed that no atrial natriuretic peptide binding could be detected on typical birefringent neurons but it could be located on astroglial and epithelial cells as identified by immunocytochemical markers. These results show that functional atrial natriuretic peptide receptors with high affinity exist in cultured mouse spinal cord cells and are not located on neurons. The presence of atrial natriuretic peptide receptors on astrocytes suggests that this neuropeptide might be a good candidate for neuron-glial communication. As the atrial natriuretic peptide binding sites previously shown in epithelia responsible for maintaining fluid and electrolyte gradients, the atrial natriuretic peptide receptors on epithelial cells in these spinal cord cultures may be involved in vivo in the control of water balance in the central nervous system.

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PubMed1989

Ultrastructural localization of the GTP-binding protein Go in neurons.

The ultrastructural localization of Go, a GTP-binding protein (G protein) highly expressed in nervous tissues, was performed in cultured fetal and adult murine neurons, using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha). These antibodies recognized denatured Go alpha and both the native Go alpha-subunit and the Go alpha beta gamma heterotrimer. At the ultrastructural level, the positive immunoreactivity detected in cultured cells as well as in thin frozen sections, showed that Go was largely distributed in cell bodies and neuritic cytoplasm. Labelling was principally noted on the cytoplasmic face of the plasma membrane lining the cell body and the neurites, especially in 'cell-cell' contacts, but also in the cytoplasmic matrix, between endoplasmic reticulum and Golgi cisternae. No immunoreactivity was observed on the inner face of the pre- or postsynaptic membranes in both adult brain and in cultured neurons. This last finding strongly suggests that the Go protein is not involved in transducing chemical signals at the level of synapses, but more probably modulates the synaptic functions by controlling the activity of effectors localized outside of the synaptic densities.

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PubMed1989

Endogenous amino acid release from cultured cerebellar neuronal cells: effect of tetanus toxin on glutamate release.

Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 mM K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, gamma-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 micrograms/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

Amino Acids↗

PubMed1989

Apical localization of the alpha subunit of GTP-binding protein Go in choroidal and ciliated ependymocytes.

The presence of GTP-binding proteins (G proteins) has been studied in murine adult choroid plexuses and cultured fetal choroidal or hypothalamic ependymal cells by ADP-ribosylation catalyzed by Bordetella pertussis toxin (PTX) and by immunodetection using affinity-purified polyclonal antibodies against the alpha subunit of the Go protein (Go alpha), the major brain G protein. ADP-ribosylation with 32P-NAD and PTX of choroid plexus revealed an intense labeling at the 40 kDa level in addition to the known PTX-substrates at 41 kDa (Gi alpha) and 39 kDa (Go alpha). This 40 kDa substrate was also predominant in cultured ependymal cells. However, a positive immunoreactivity with the anti-Go alpha antibodies was detected at the level of the 39 kDa faster component, indicating the presence of Go alpha in both choroid plexuses and cultured ependymal cells. In thin frozen sections as well as in cultured cells, Go alpha was mainly immunolocalized at the apical pole of choroidal ependymocytes and in the kinocilia of ciliated ependymal cells. At the ultrastructural level, using gold immunoprobes, the immunoreactivity of a Go alpha-like protein was detected on the cytoplasmic face of the apical plasma membrane, coated pits and vesicles, and in the apical cytoplasmic matrix. In ciliated ependymal cells, the positive immunostaining displayed a dotted pattern at the surface of demembranated axonema of apical kinocilia. These findings strongly suggest that G proteins, especially Go, are involved in transducing chemical signals that modulate traffic and exchanges between cerebrospinal fluid and ependyma through the apical membrane of ependymocytes.

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PubMed1988

The presence of non-neuronal cells influences somatostatin release from cultured cerebral cortical cells.

We examined the effect of non-neuronal cells on somatostatin release from cultured cerebral cortical cells. Three culture models were used: (1) neuron-enriched cultures obtained from cortex of 17-day-old rat embryos and exposed to 10 microM cytosine arabinoside (Ara C) for 48 h between days 3 and 5 after plating; (2) whole cell cultures obtained by using the same protocol but untreated with Ara C; (3) glial primary cultures obtained from newborn rats. We studied: (i) the cellular composition of the cultures by using two astroglial markers: vimentin and glial fibrillary acidic protein (GFAP); (ii) the spontaneous and forskolin-stimulated somatostatin release. In 8-day-old cultures morphological data revealed that Ara C treatment reduced glial cells to 6%. At 7 and 10 days of culture somatostatin spontaneously released from Ara C-treated cells was higher than that measured from untreated cells. On the 17th day of culture, neuron-enriched cultures contained a lower amount of somatostatin than whole cell cultures. Forskolin elicited a dose-dependent release of somatostatin from whole cell cultures, but had no effect on neuron-enriched cultures. Astroglial released media (ARM) from glial primary cultures exposed to forskolin for 20 min induced somatostatin release from neuron-enriched cultures. HPLC analysis of endogenous amino acids of ARM showed that glutamate, glutamine, glycine and alanine were significantly increased after forskolin stimulation. Our results suggest a functional interaction between glial cells and neurons secreting somatostatin.

Amino Acids↗

PubMed1988

Characterization of ependymal cells in hypothalamic and choroidal primary cultures.

Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in terms of marker expression and ultrastructural features.

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