Biomedical subjects

M Geffard

Publications and source records attributed to M Geffard.

At least 19 recordsLinked to original sources

PubMed2000

Catecholamine degradation by monoamine oxidase in locus coeruleus neurons of the rat. An immunohistochemical study.

We examined by immunohistochemistry the effects of monoamine oxidase (MAO) inhibition on the content of dopamine (DA) and noradrenaline (NA) in locus coeruleus (LC) neurons of the rat. In normal rats, clusters of DA- and NA-immunopositive neurons were identified in the LC. Rats treated with intraperitoneal injections of pargyline, an MAO inhibitor, showed significantly stronger DA- and NA-staining intensities in LC neurons compared to normal rats. In LC noradrenergic neurons, it is believed that DA is formed in the cytoplasm and then transported into the storage vesicles where it is converted to NA, and the secreted NA is recycled by a reuptake mechanism and transported back into storage vesicles via the cytoplasm. Furthermore, LC neurons of the rat have been shown to contain DA- and NA-degrading MAO activities on the outer membranes of the mitochondria. Therefore, our findings suggest that endogenous MAO degrades not only part of the DA formed in the cytoplasm of LC neurons, but also part of the secreted NA that has been transported back into the cytoplasm.

Animals↗

PubMed2000

Distribution of dopamine-immunoreactive fibers in the rat brainstem.

We describe the distribution of axons immunoreactive for dopamine in pons and medulla oblongata of rat under normal conditions or after inhibition of monoamine oxidase or dopamine beta-hydroxylase. In the pons of non-treated animal, fairly dense plexuses of dopamine-immunoreactive varicose fibers were found in the locus coeruleus, dorsal parabrachial and dorsal raphe nuclei, central gray and reticular formation dorsal to the superior olive. In the medulla oblongata, the immunoreactive fibers were abundant in the dorsal vagal complex, lateral paragigantocellular nucleus, midline raphe nuclei and spinal trigeminal nucleus. Monoamine oxidase inhibition made it possible to increase the intensity of immunoreactivity and consequently the number of labeled fibers in these areas, indicating that dopamine is perpetually oxidized by monoamine oxidase, and consequently in low concentration under normal conditions. Sparse dopamine-immunoreactive fibers were observed in the pontine gray, motor trigeminal nucleus, inferior olive and major axon bundles such as the dorsal and ventral tegmental bundles, where numerous noradrenergic fibers have been reported. In axons of these areas, intense dopamine-immunoreactivity was seen only after inhibition of dopamine-beta-hydroxylase. It appears that dopamine is released and oxidized in response to autonomic changes such as hypoxia, hemorrhage, and cardiovascular variation in the caudal brainstem, as we have described elsewhere.

Animals↗

PubMed1999

Anatomical and functional reconstruction of the nigrostriatal system in vitro: selective innervation of the striatum by dopaminergic neurons.

To study development of the nigrostriatal pathway in an in vitro model system, organotypic slices obtained from rat pups (P4) and containing the striatum and the cortex were grown together with apposed embryonic (E13.5) mesencephalic blocks according to the static slice culture method of Stoppini et al. (1991; J. Neurosci. Methods 37:173-182). Under these conditions, mesencephalic dopaminergic (DA) fibers rapidly grow through the slice, preferentially its striatal portion. This innervation provides a true synaptic innervation to the striatum, as shown by the presence of DA terminals on striatal neurons. DA fibers are able to exert a functional influence, as seen by their ability to modulate c-Fos expression in striatal neurons in the same way as in vivo. Thus, blockade, under basal conditions, of the effect of spontaneously released dopamine by the D2 receptor antagonist haloperidol leads to the activation of c-Fos expression in the striatum. Furthermore, stimulation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striatal innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seems to be specific for DA neurons; no selectivity could be observed when noradrenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reveal any oriented outgrowth of DA fibers from the mesencephalon, suggesting that the selective innervation observed in the organotypic slices results from some contact-dependent, presumably adhesive interactions rather than from the presence of some diffusible substance orienting the growth of DA fibers towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the selective striatal innervation are not the same as those determining the adhesion of the DA neurons. These results show that cocultures of cortex-striatum and mesencephalic slices result in a system that displays a number of the morphological and functional traits of the normal nigrostriatal system and that can be relied on as a good in vitro model of in vivo development.

Animals↗

PubMed1999

Detection of nitrosylated epitopes in Trypanosoma brucei gambiense by polyclonal and monoclonal anti-conjugated-NO-cysteine antibodies.

Activated macrophages with the Calmette/Guérin bacillus (BCG) have a cytotoxic/cytostatic effect on the extracellular parasite, Trypanosoma brucei gambiense. This effect was inhibited when the NO-synthase inhibitor NG-monomethyl-L-arginine (NMMA; 0.5 mM) was added to the culture media. Using an immunocytochemical method with rabbit polyclonal or mouse monoclonal antibodies directed against conjugated nitroso-epitopes (anti-conjugated-NO-cysteine), nitrosylated antigens were visualized in fixed trypanosomes. These results suggest that NO was synthesized by the activated macrophages and that it reacted with some parasitic proteins containing cysteine. The release of NO bound to parasitic proteins may cause the killing of trypanosomes. The immunoreactivity was positive when the trypanosomes were obtained from the supernatant of the BCG-activated macrophages that contains BSA (4 mg/mL). In contrast, the parasites cocultured with non-activated macrophages remained completely viable, and, the immunoreactivity was completely negative.

Animals↗

PubMed1998

Dopamine synthesizing enzymes in paraventricular hypothalamic neurons of the human and monkey (Macaca fuscata).

Using immunohistochemistry, we demonstrated that paraventricular hypothalamic neurons immunoreactive for tyrosine hydroxylase (TH) were not immunopositive for the second step catecholamine synthesizing enzyme L-amino acid decarboxylase (AADC) in the human and monkey Macaca fuscata. In the latter species, they were not immunoreactive for dopamine. It is most likely that primate paraventricular TH-containing neurons do not synthesize dopamine.

Adult↗

PubMed1997

Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies.

Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi. This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule. Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate. This conjugate was nitrosylated using sodium nitrite as a NO donor. Binding of NO to the conjugated haptens was assayed using spectrophotometry. It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond. Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates. Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds. The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound. These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T. musculi. Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.

Acetylcysteine↗

PubMed1997

Quantitative and morphometric data indicate precise cellular interactions between serotonin terminals and postsynaptic targets in rat substantia nigra.

We have quantified the density of serotonin axonal varicosities, their synaptic incidence and their distribution among potential targets in the pars reticulata and pars compacta of the rat substantia nigra. Serotonin axonal varicosities, counted at the light microscopic level following in vitro [3H]serotonin uptake and autoradiography, amounted to 9 x 10(6)/mm3 in the pars reticulata and 6 x 10(6)/mm3 in the pars compacta, among the densest serotonin innervations in brain. As determined at the electron microscopic level following immunolabelling for serotonin, virtually all serotonin varicosities in the pars reticulata and 50% of those in the pars compacta formed a synapse, essentially with dendrites. The combination of serotonin immunocytochemistry with tyrosine hydroxylase immunolabelling of dopamine neurons reveals that 20% of the serotonin synaptic contacts in the pars reticulata are on dopamine dendrites and 6% are on a type of unlabelled dendrite characterized by its peculiarly high cytoplasmic content of microtubules. The comparison of the diameter of the dendritic profiles that were in synaptic contact with serotonin-immunoreactive varicosities with the diameter of all other dendritic profiles of the same type suggests that serotoninergic varicosities innervate dopamine dendrites uniformly along their length, whereas they tend to contact microtubule-filled dendrites in more proximal regions and the other, unidentified dendrites in more distal regions. Furthermore, the size of the serotonin-immunoreactive varicosities and of their synaptic junctions is significantly smaller on dopamine dendrites and larger on microtubule-filled dendrites than on other, unidentified dendrites, indicating that the nature of the postsynaptic target is an important determinant of synaptic dimensions. These data should help to clarify the role of serotonin in the nigral control of motor functions. They indicate that this dense serotonin input to the substantia nigra is very precisely organized, acting through both "non-junctional" and "junctional" modes of neurotransmission in the pars compacta, which projects to the neostriatum and the limbic system, whereas the predominant mode of serotonin transmission appears to be of the "junctional" type in the pars reticulata, where serotonin can finely control the motor output of the basal ganglia by acting on the GABA projection neurons either directly or through the local release of dopamine by dopaminergic dendrites. The data also raise the possibility that the postsynaptic targets have trophic retrograde influences on serotoninergic terminals.

Animals↗

PubMed1996

Anatomy and targets of Dorsal Unpaired Median neurones in the Terminal Abdominal Ganglion of the male cockroach Periplaneta americana L.

The morphology of the Dorsal Unpaired Median (DUM) neurones in the Terminal Abdominal Ganglion (TAG) of the adult male cockroach Periplaneta americana were described based on wholemount preparations and paraffin sections and by using anterograde and retrograde cobalt mapping, octopamine-like immunohistochemistry, and double immunofluorescence technique with both conjugated gamma-aminobutyric acid (GABA) and octopamine antisera. Among 60 +/- 6 neurones with large somata (diameter 40 to 60 microns) on the dorsal midline surface of the TAG that were stained with toluidine blue, about 36 efferent DUM neurones exhibited octopamine-like immunoreactivity. The DUM neurones were arranged in three clusters (anterior, median and posterior) corresponding to the 7th-11th abdominal ganglia of the fused TAG. Anterior efferent DUM neurones with one, two, and four pairs of lateral neurites entered segmental nerves VIIB; VIIB and phallic nerves; IXB and phallic nerves; VIIIA, IXA, X, and IX, respectively. Three octopamine-like immunoreactive DUM neurones innervating heart chambers via segmental nerves (VIIA, VIIIA, and IXA) in the last abdominal segments occurred within abdominal ganglia 7, 8, and 9. Together with octopamine-like immunoreactive efferent DUM neurones, GABA-like immunoreactive dorsal midline neurones with small somata (10 to 20 microns) also occurred within the median group. The spatial distribution of DUM neurones in the TAG suggested that they had their origins in the median neuroblast, as for DUM neurones in the grasshopper.

Abdomen↗

PubMed1996

Electron-microscopic study of dopaminergic structures in the medial subdivision of the monkey nucleus accumbens.

The medial subdivision of the monkey nucleus accumbens (NAC) is rich in dopamine (DA) and peptides. In the present investigation the mode of DA transmission in the medial subdivision was studied morphologically by light- and electron-microscopic immunocytochemistry using a monoclonal antibody raised against dopamine. The medial subdivision showed extremely dense accumulation of thick DA-immunoreactive varicose fibers. Electron-microscopic observation of single sections revealed that DA afferents had a relatively high incidence (33.2%) of asymmetric junctions in this area. Approximately 50% of the targets were dendritic shafts, 44.2% dendritic spines, and 5.1% somata. Some DA axons showed terminal profiles en passant within the synaptic complex, some of which showed synaptic triads. The unique ultrastructural features of DA terminals in the medial NAC indicate the existence of specific styles of DA transmission in the limbic structure.

Animals↗

PubMed1996

GABA uptake and phenotypic characteristics of the subcommissural ependymocytes of the semi-desertic rodent, Meriones shawi: correlation with serotoninergic innervation.

Many studies have emphasized species differences in the serotoninergic innervation and phenotypic characteristics of the subcommissural organ in mammals. The post-natal distribution patterns of serotonin-containing fibers, the onset of gamma-aminobutyric acid uptake, and glial markers have been studied in the subcommissural organ of the semi-desertic rodent, Meriones shawi, by using immunohistochemical and autoradiographic techniques. Abundant serotoninergic fibers can be observed in the subcommissural organ of the newborn Meriones, some of them running among the ependymocytes and reaching the apical part of this organ. During the first 2 post-natal weeks of development, the subcommissural organ displays a progressive increase of serotonin fiber density throughout the organ, including the apical part. The existence of a dense serotonin-containing basal plexus concomitantly with a high apical innervation in this organ is a specific characteristic of Meriones. Ependymocytes of this organ have the ability to take up gamma-aminobutyric acid at birth. This uptake decreases and completely disappears from the 2nd week. The reappearance of gamma-aminobutyric acid accumulation in ependymocytes of the adult subcommissural organ after destruction of the serotonin innervation by a neurotoxin (5-7 dihydroxytryptamine) suggests an inhibitory effect of the serotonin innervation on this accumulation. Immunohistochemical studies of the phenotype of the ependymocytes with respect to glial markers during ontogeny show the transitory expression of glial fibrillary acidic protein, the presence of vimentin and the absence of S100 protein expression. No correlation has been found between the serotonin innervation and the expression of the glial markers.

Animals↗

PubMed1996

Circulating antibodies directed against conjugated fatty acids in sera of patients with multiple sclerosis.

Using an adapted ELISA assay, we have tested sera from multiple sclerosis (MS) patients for antibodies directed against ten fatty acids conjugated to bovine serum albumin. In serum samples from 68 MS patients and 20 patients suffering from rheumatoid arthritis (RA), a significant antibody titer elevation to the ten tested fatty acids was found when compared to sera of 40 healthy subjects and 82 patients with other neurological and autoimmune diseases. G-200 purified IgM of MS patients reacted specifically with the aliphatic chains with an avidity of 3 x 10(-7) M. These results suggest that in MS and RA, autoepitopes on cell membranes that are normally hidden from the immune system become immunogenic. This may arise because of previous membrane disruption by oxidative processes.

Adult↗

PubMed1995

L-DOPA is converted to dopamine in serotonergic fibers of the striatum of the rat: a double-labeling immunofluorescence study.

The aim of the present study is to examine whether serotonergic terminals of the rat brain are induced to contain dopamine by L-DOPA administration. In rats that received intraperitoneally L-DOPA plus a peripheral decarboxylase inhibitor, we showed by use of a double-labeling immunofluorescence method that dopamine was localized in serotonergic fibers of the striatum and cerebral cortex as well as in serotonergic cell bodies of the midbrain raphe nuclei. In normal rats, no dopamine was detected in the serotonergic fibers or cell bodies. The finding suggests that a part of the administered L-DOPA may enter the serotonergic terminals and be converted to dopamine in the rat brain.

Animals↗

PubMed1995

Identification of a benzo[a]pyrene-like binding protein involved in circulating immune complexes of patients with mammary tumors.

Anti-benzo[a]pyrene (B[a]P)-like autoantibodies (autoAb) have been characterized in sera of patients with epithelial tumors. Circulating immune complexes (CIC) from these sera have been analysed after polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions. Immunoblotting was performed using a monoclonal antiidiotypic antibody (Ab), internal image of conjugated B[a]P called AIB1 and anti-human immunoglobulins (Ig). An immunoreactivity was seen only with AIB1 Ab, suggesting the presence of a 'B[a]P-like' binding protein. Additional studies showed that this immunoreactivity is not associated with an 18- to 20-kDa protein previously identified in the same CIC.

Antigen-Antibody Complex↗

PubMed1995

Molecular detection of methionine in rat brain using specific antibodies.

In order to study the localization of methionine in rat brain, an immunological approach was developed by raising antibodies directed against this amino acid. Methionine was conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) via glutaraldehyde. The conjugates were then reduced by sodium borohydride and injected alternately into rabbits. Antibody affinity and specificity were evaluated using an adapted ELISA method, by competition experiments between conjugated methionine and related conjugated compounds, pre-incubated with anti-methionine antibodies diluted at 1/20,000. The resulting cross-reactivity ratios, calculated at half-displacement, showed that glutaraldehyde-methionine conjugate (methionine-G-BSA) was the best recognized compound. Non-reduced methionine conjugate (methionine=G=BSA) and the related-conjugated molecules such as homocysteine, homocysteic acid, cysteine, cystathionine and glutamate were not recognized at all. Antibodies to methionine were directed against a glutaraldehyde-methionine epitope and their very high affinity and specificity made them reliable tools for molecular detection of methionine in rat brain. Using purified antibodies diluted at 1/20,000, motoneurons were found to be the most methionine-immunoreactive cell bodies in glutaraldehyde-fixed rat brain sections.

Animals↗

PubMed1995

Indirect evidence for nitric oxide involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine.

Converging data suggest that nitric oxide (NO) production by cytokine-induced immune cells in demyelinating lesions is involved in multiple sclerosis (MS). High levels of NO may complex to suitable amino acids, causing an immune response against the formed neo-epitopes. By testing MS sera with chemically defined nitroso-amino acids conjugated to carrier protein in ELISA, we observed a significant antibody reaction against the S-nitroso-cysteine epitope. The MS antibody response was exclusively of IgM isotype with an avidity of 8 x 10(-7) M. Sera of all clinical MS forms showed a significantly elevated antibody titer versus sera from healthy subjects or from patients affected with other neurological and autoimmune diseases. The detection of circulating antibodies to a conjugated S-nitroso-cysteine epitope provides indirect evidence for NO involvement in MS.

Adult↗

PubMed1995

Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment in constant darkness.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

Analysis of Variance↗

PubMed1995

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine.

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.

Adrenergic Fibers↗

PubMed1995

Autoantibodies to malondialdehyde-modified epitope in connective tissue diseases and vasculitides.

Malondialdehyde (MDA), a peroxidative end-product released during polyunsaturated fatty acid degradation, reacts strongly with lysine residues of cellular proteins. MDA-modified proteins become immunogenic and may elicit specific autoantibody formation. We hypothesized that systemic diseases in which inflammatory events occur, could be an interesting model for studying oxidative stress. A few studies have suggested that MDA-modified proteins may exist in systemic diseases, and that autoantibodies to MDA-modified structures might reflect this oxidative process. Autoantibodies to MDA-modified epitope(s) were therefore assayed in sera of patients with systemic lupus erythematosus (SLE, n = 29), scleroderma (SCL, n = 11), giant cell arteritis (GCA, n = 11), periarteritis nodosa (PAN, n = 10), rheumatoid arthritis (RA, n = 9), and healthy subjects (HS, n = 32). Significantly increased anti-MDA-modified epitope(s) autoantibodies were found in patients with SLE and also in other systemic diseases such as PAN and SCL. Autoantibodies to MDA-modified epitope(s) were predominantly of IgM isotype, with low levels of IgG and no IgA activity. In SLE, anti-MDA-modified epitope(s) autoantibody titres correlated strongly with systemic lupus activity measure (SLAM, r = 0.702, P = 0.0001), anti-nuclear antigen autoantibodies (ANA, r = 0.4, P = 0.029), IgG anti-cardiolipin (r = 0.558, P = 0.03) and the steroid drug regimen (r = 0.52, P = 0.004). Autoantibodies to MDA-modified epitope(s) may reflect oxidative modifications occurring in systemic diseases, and might be useful as clinical markers of SLE activity if further investigated.

Adult↗