Biomedical subjects

A Rabié

Publications and source records attributed to A Rabié.

At least 19 recordsLinked to original sources

PubMed2005

Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system.

Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments.

Animals↗

PubMed2000

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells.

To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [(3)H]-taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l(-1)) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl(-) channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume-sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC(3), phospholemman and VDAC(1), and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC(2). In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidentified proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Expression of the genes encoding the vasopressin-activated calcium-mobilizing receptor and the dual angiotensin II/vasopressin receptor in the rat central nervous system.

The distributions of two newly discovered receptors, the vasopressin-activated calcium-mobilizing receptor (VACM-1) and the dual angiotensin II/vasopressin receptor (AII/AVP), in the central nervous system (CNS) of the rat were determined using reverse transcriptase-polymerase chain reaction and in situ hybridization. The sequence of the rat VACM-1 cDNA was determined and found very homologous to the rabbit and human sequences. Both VACM-1 and AII/AVP receptor genes were widely expressed in the brain, but differed according to the cell type studied. Glial cells were very faintly labelled. The epithelial cells of the choroid plexuses, the ependymal cells and the pia mater were all labelled. Both genes were most active in neurones throughout the CNS. VACM-1 and AII/AVP receptors were detected in neurones previously shown to possess V1a and V1b vasopressin receptors, and/or the AT1 and AT2 angiotensin II receptors in many brain areas. This was the case for the magnocellular neurones of the supraoptic and paraventricular nuclei of the hypothalamus. We suggest that the VACM-1 and AII/AVP receptors may account for the V2-like responses to vasopressin by these neurones which lack a genuine V2 vasopressin receptor.

Animals↗

PubMed1998

A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons.

A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.

Animals↗

PubMed1998

Solid-phase synthesis of peptide nucleic acid (PNA) monomers and their oligomerization using disulphide anchoring linkers.

A new simple solid-phase method has been developed for synthesizing Boc-protected peptide nucleic acid (PNA) monomers. An immobilized backbone 3 was built on Expansin resin using an ester disulphide handle: 2-hydroxypropyl-dithio-2'-isobutyric acid (HPDI). The base acetic acids of thymine 5, Z-cytosine 9, Z-adenine 12, and 6-O-benzyl guanine 17 were prepared and coupled to the immobilized backbone. The HPDI handle was cleaved under mild conditions by cyanolysis or assisted hydrolysis with tris(2-carboxyethyl)phosphine (TCEP) to give undamaged PNA monomers. These monomers were coupled to form oligomers by solid-phase method with another disulphide linkage: aminoethyldithio-2-isobutyric acid (AEDI) grafted on an amino-functionalized TentaGel resin, using in situ neutralization and TBTU as activating reagent. Final cleavage of the AEDI linker gave PNA bearing a cysteamide residue that could be useful for optimizing PNA properties. Oligomers of up to 16 residues long were assembled.

DNA↗

PubMed1998

Synergy between chronic corticosterone treatment and cerebral ischemia in producing damage in noncalbindinergic neurons.

Administration of endogenous corticosterone to intact animals induces calbindin-D28k protein in the hippocampal CA1-CA2 subfields. The fact that this effect on calbindin-D28k was shown to be specific for the hippocampus argues for a receptor-mediated effect on gene expression. In addition, chronic pretreatment with corticosterone aggravates ischemia-induced neuronal damage in the CA3-CA4 subfields. This effect is similar to that of preischemic hyperglycemia, which also induces postischemic seizures and aggravates brain damage, since corticosterone raises blood glucose level and enhances tissue lactic acidosis during ischemia. The energetically compromising qualities of corticosterone indicates that it is a key factor in hippocampal vulnerability. We assume that the increase of calbindin-D28k expression in the CA1-CA2 subfields in corticosterone-treated animals is an adaptive response to the exogenous stress. The lack of adaptive response in CA3-CA4 neurons endangers them by impairing the ability of these neurons to counteract the deleterious effects of calcium. This finding, supports: (1) the hypothesis that corticosterone treatment, when paired with an ischemic insult, causes a prolonged elevation of neuronal [Ca2+]i, in an energy dependent manner, probably through the reduction of calcium efflux and (2) that neurons which do contain calbindin-D28k are particularly predisposed to ischemic insults. The CA1-CA2 neurons express high amounts of calbindin-D28k under stress conditions because their activity may involve a high rate of calcium buffering.

Animals↗

PubMed1998

New aspects of firing pattern autocontrol in oxytocin and vasopressin neurones.

In the rat, oxytocin (OT) and vasopressin (AVP) neurones exhibit specific electrical activities which are controlled by OT and AVP released from soma and dendrites within the magnocellular hypothalamic nuclei. OT enhances amplitude and frequency of suckling-induced bursts, and changes basal firing characteristics: spike patterning becomes very irregular (spike clusters separated by long silences), firing rate is highly variable, oscillating before facilitated bursts. This unstable behaviour which markedly decreases during hyperosmotic stimulation (interrupting bursting) could be a prerequisite for bursting. The effects of AVP depend on the initial phasic pattern of AVP neurones: AVP excites weakly active neurones (increasing burst duration, decreasing silences) and inhibits highly active neurones; neurones with intermediate phasic activity are unaffected. Thus, AVP ensures all AVP neurones discharge with moderate phasic activity (bursts and silences lasting 20-40 s), known to optimise systemic AVP release. V1a-type receptors are involved in AVP actions. In conclusion, OT and AVP control their respective neurones in a complex manner to favour the patterns of activity which are the best suited for an efficient systemic hormone release.

Animals↗

PubMed1998

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Animals↗

PubMed1995

The retro-inverso form of a homeobox-derived short peptide is rapidly internalised by cultured neurones: a new basis for an efficient intracellular delivery system.

The capacity of a retro-inverso form of a 16-residue peptide from the Antennapedia homeodomain to be taken up by cultured neurones was tested. Like its homologue made of L-amino acids, it was rapidly internalised and distributed throughout the cytoplasm and even the cell nucleus. The amount of retro-inverso peptide in cells after incubation in culture medium was 3.4 times that of the L-peptide form. With a cholesteryl moiety attached to the C-terminus to increase its lipophilicity, the retro-inverso peptide was internalised 8 times better than the L-form. The greater efficiency of the peptidomimetic is probably due to the resistance to proteolytic degradation conferred by the D-amino acids, as the sequence contains two sites sensitive to neuronal endoproteases. The peptide might be the basis for development of system for delivering a variety of molecules into cells.

Amino Acid Sequence↗

PubMed1994

Coordinated expression of five tropomyosin isoforms and beta-actin in astrocytes treated with dibutyryl cAMP and cytochalasin D.

Cytochalasin D and dBcAMP cause cultured astrocytes to change from flat cells to retracted process-bearing cells. F-actin was present throughout cells stimulated with dBcAMP for 16 h, whereas cytochalasin D caused F-actin to form massive aggregates at the tips of the cell processes. The two drugs differently regulated the expression of both beta-actin and tropomyosin genes in astrocytes cultured in the presence or absence of serum: dBcAMP caused down-regulation and cytochalasin D caused up-regulation. Northern blot analyses indicated that: (1) serum deprivation halved the concentration of all tropomyosin transcripts (TM-1, TM-2, TM-4, TMBr-1, TMBr-2). Serum induced TM-4 via transcriptional activation, independent of protein synthesis, (2) dBcAMP induced down-regulation of beta-actin (-50%) and tropomyosin transcripts (-35 to 52%) even in the presence of serum. The concentration of profilin mRNA decreased in dBcAMP-reactive astrocytes (-46%). The decrease in beta-actin mRNA concentration was not blocked by cycloheximide, whereas down-regulation of tropomyosin transcripts was completely reversed when protein synthesis was inhibited, and (3) cytochalasin D induced an increase in the concentration of tropomyosin transcripts (+69 to 185%) which was cumulative with serum stimulation. Cytochalasin D induction of both beta-actin and TM-4 operated through transcriptional activation, independent of protein synthesis. The production of all tropomyosin transcripts examined here were strictly coordinated with beta-actin expression in serum-, dBcAMP- and cytochalasin D-treated astrocytes. This indicates that the differential expression of tropomyosin isoforms occurring during astrocyte maturation is due to more complex regulation than that involved in serum- or cAMP-stimulated astrocytes.

Actin Cytoskeleton↗

PubMed1994

High gelsolin content of developing oligodendrocytes.

The actin-binding protein gelsolin that severs and caps the actin microfilaments under the control of the cytoplasmic free calcium and the membranous phosphatidylinositol 4,5-bisphosphate, is essentially restricted to the oligodendroglia in the central nervous system. Immunocytochemistry showed that gelsolin is an early marker of oligodendrocytes, both in vivo, in the rat cerebellum, and in vitro, in oligodendrocyte culture. We report the early appearance of gelsolin in A2B5-positive precursor oligodendrocyte cells and the specific expression of gelsolin in OL-1-, GC-, and MBP-positive oligodendrocytes in culture. The protein was distributed throughout the cell body and in the branched cell processes of cultured oligodendrocytes, but not in the MBP-positive membrane sheets. Gelsolin is thus cytosolic and not a myelin component. The quantitative study demonstrated that that the cerebellar gelsolin content changes significantly with age, with the maximal value at the age of 21 days, confirming that large amounts of gelsolin are transiently synthesized during development, especially from the first events of myelinogenesis. The results are consistent with gelsolin being involved, through its effects on the actin cytoskeleton, in the motile events occurring during the growth of the oligodendroglial processes towards the axons and the wrapping of the myelin sheaths around the axons.

Animals↗

PubMed1994

Tropomyosin isoforms in rat neurons: the different developmental profiles and distributions of TM-4 and TMBr-3 are consistent with different functions.

Antipeptide antisera specific for TM-4 and TMBr-3, the two tropomyosin isoforms in neurons, were used to investigate the concentrations and distributions of these F-actin-binding proteins in neurons in vitro and in vivo. TM-4 and TMBr-3 tropomyosins had different developmental profiles. TM-4 was found mainly in immature stages, while the concentration of TMBr-3 increased with maturation. The two isoforms also had different subcellular distributions. TM-4 was concentrated in the growth cones of cultured neurons and, in vivo, in areas where neurites were growing. Later, when development was complete, TM-4 was restricted to postsynaptic sites in the cerebellar cortex, whereas TMBr-3 was found in the presynaptic terminals. These data suggest that the tropomyosin isoforms have different functions, through their interaction with the actin cytoskeleton. TM-4 may be involved in the motile events of neurite growth and synaptic plasticity, while TMBr-3 could play a role in stabilizing neuronal networks and synaptic functioning.

Amino Acid Sequence↗

PubMed1993

The expression of tropomyosin genes in pure cultures of rat neurons, astrocytes and oligodendrocytes is highly cell-type specific and strongly regulated during development.

Transcripts from the alpha-, beta- and delta-tropomyosin genes were studied during development of pure cultures of rat neurons, astrocytes and oligodendrocytes. The three cell types contained five alpha-tropomyosin messengers, produced using both alternative promoters and splicing; one was specific for mature neurons. The beta-tropomyosin gene is expressed only in astrocytes and the delta-tropomyosin gene in all three cell types, especially in immature cells. Most of the tropomyosin isoforms are highly cell-specific. Their developmental regulation involves either differential expression of genes, in neurons and oligodendrocytes, and/or changes in alternative splicing, in astrocytes, delta-Tropomyosin (TM-4) may be important during the growth of neuronal and glial cell processes, while specialized isoforms such as the neuron-specific alpha-tropomyosin TMBr-3 may be involved in the function or plasticity of the mature cells.

Animals↗

PubMed1992

Calbindin-D28K and ischemic damage of pyramidal cells in rat hippocampus.

An antibody against rat calbindin-D28K, a calcium-binding protein present at high concentration in certain neurons of the central and peripheral nervous systems, was used to determine the progression of the pathological events in the rat hippocampus following experimental cerebral ischemia. Calbindin-D28K immunoreactivity is present in dentate granule cells and in the CA1-CA2 pyramidal cells. CA1 subfield contains a higher proportion of calbindin-D28K-positive pyramidal cells than does the CA2 subfield and CA1 cells are more immunoreactive than the CA2 cells. The pyramidal cells of the CA1 and CA2 subfields are vulnerable to ischemia. The cells in the CA1 became necrotic within 3-4 days after ischemia while those of the CA2 became necrotic within 2 days. There was a concomitant decrease in calbindin-D28K immunoreactivity in the whole hippocampal regio superior after ischemia which peaked 3 days postischemia. The difference in CA2 and CA1 vulnerability seemed to be inversely correlated with the calbindin-D28K contents of the CA2 and CA1 pyramidal cells. The decrease in the calbindin-D28K contents of these neurons was accompanied by cell damage. We therefore suggest that calbindin-D28K is an important factor for the survival of pyramidal cells in the hippocampal formation after ischemia.

Animals↗

PubMed1992

Activated astrocytes, but not pyramidal cells, increase glucose utilization in rat hippocampal CA1 subfield after ischemia.

The local cerebral glucose utilization (CMRglc) in the damaged rat hippocampal CA1 subfield increases 7 days after 10 min of cerebral ischemia. We have used the N-methyl-D-aspartate antagonist (NMDA antagonist) ketamine in rats 7 days after sham operation or cerebral ischemia to determine whether the elevated postischemic CMRglc of the CA1 subfield is due to long-lasting hyperexcitation of surviving or injured neurons, or, alternatively, to the metabolism of other cell types. The autoradiographic data were interpreted with the aid of histochemical analysis of the postischemic hippocampal cell changes. Anesthetic doses of ketamine significantly reduced the CMRglc in the CA1 strata oriens, pyramidale and radiatum of sham-operated rats, while the postischemic increases in CMRglc in these hippocampal CA1 strata were not affected by ketamine. In addition, there were ketamine-induced increases in the CMRglc of the CA1 stratum lacunosum moleculare of both sham-operated and postischemic rats. The immunoreactivity of the microtubule-associated protein 2 (MAP2), a postsynaptic protein marker, was decreased markedly in the CA1 subfield in postischemic rats, while the presynaptic protein marker, synaptophysin, remained the same in sham-operated and postischemic rats. The glial fibrillary acidic protein (GFAP) immunoreactivity of astrocytes raised markedly in the ischemically damaged CA1 subfield. Although it could be demonstrated that presynaptic terminals remain intact in the postischemic damaged CA1 subfield, the lacking ketamine effect on CA1 pyramidal neurons indicated that the increase in CMRglc in this brain area is not due to postsynaptic neural hyperexcitation, but probably has to be attributed to astrocytes activated by neuronal damage.

Animals↗

PubMed1991

Actin-depolymerizing factor (ADF) in the cerebellum of the developing rat: a quantitative and immunocytochemical study.

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.

Actin Depolymerizing Factors↗

PubMed1991

Distribution of gelsolin in the retina of the developing rabbit.

The distribution of gelsolin, a calcium-dependent actin-severing and capping protein, in the retina of the developing and adult rabbit was studied. Gelsolin immunoreactivity was found in the photoreceptors and ganglion cells, where it may have a role in neuronal morphogenesis. Only the inner segment of the photoreceptors retained a high gelsolin content in the adult retina, perhaps because the attached outer segment is continuously renewed throughout life. Gelsolin, which is a major component of the rabbit brain oligodendrocytes, was also found in the myelin of the medullary ray region of the rabbit retina. Müller cells in all regions of the rabbit retina also contain gelsolin from early in development to adulthood. Since one of the functions of these cells is to ensheath neuronal elements in the inner plexiform and optic fiber layers, we suggest that gelsolin may play the same role in Müller cells as it does in oligodendrocytes, i.e., sheath formation via its calcium-dependent action on the actin microfilament networks.

Animals↗

PubMed1991

Profilin and profilin mRNA in the cerebellum of the developing rat.

Profilin and profilin mRNA concentrations were determined in the cerebellum of the developing rat. Profilin represented nearly 0.1% of total Triton-soluble protein both at birth and at the end of the developmental period. During brain maturation, the profilin concentration was regulated at the transcriptional level. A transient increase of profilin mRNA and profilin was observed at the end of the first postnatal week, a period of intense cell proliferation, neuritic outgrowth and synaptogenesis. We propose that profilin may act by forming, during the critical period of cerebellar development, a reserve pool of monomeric actin that can easily be mobilized in cell regions such as growth cones or synaptic junctions where filamentous actin is highly concentrated.

Actins↗