Biomedical subjects

M Geffard

Publications and source records attributed to M Geffard.

At least 163 records · Page 9Linked to original sources

PubMed1987

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells.

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.

Animals↗

PubMed1987

An ultrastructural study of GABA-immunoreactive neurons and terminals in the septum of the rat.

The fine structure and types of contact made by GABAergic elements in the septal nuclei were studied at the electronmicroscopic level by means of peroxidase immunocytochemistry, using anti-GABA antibodies. Observations were made on normal and colchicine-injected rats. GABA-immunoreactivity was distributed within somata, dendrites, axonal varicosities and terminals, and myelinated axons. The peroxidase reaction product was diffuse in the cytoplasm; cytoplasmic organelles were generally devoid of immunoreactivity, while showing a strong reaction on the outer surface of their membrane. GABA-immunoreactive (GABA-I) neurons were small (10 microns on average) to medium (20 microns) in size, with round or multipolar cell bodies. Additionally, labeled large (30 microns) cells were observed within the myelinated fibers of the medial septal nucleus after intraseptal administration of colchicine. No difference in the ultrastructural features and distribution of the immunoreactivity of the 2 kinds of cell was noticed, except for a higher number of synaptic contacts on large neurons of the medial septum. GABA-I cells of the medial and lateral nuclei received synapses on their soma and dendrites, made by both immunonegative and GABA-I terminals. Nonimmunoreactive boutons contacting GABA-I cell bodies were of 2 types: those containing small, clear synaptic vesicles and those that additionally contained large dense vesicles. Synaptic vesicles of GABA-I boutons were rarely labeled internally, but showed varying electron densities. Synapses made by GABA-I boutons on GABA-I or unlabeled somata and dendrites were always of symmetrical type. Synapses made by non-GABA-I boutons on GABA-I cells were either symmetrical or asymmetrical.

Animals↗

PubMed1987

Suppression of oscillatory activity in crustacean pyloric neurons: implication of GABAergic inputs.

Generation of rhythmic pyloric motor output in the crustacean stomatogastric ganglion results from synaptic connections and cellular properties of a 14-cell network of pyloric neurons. These cellular properties are under the influences of modulatory inputs, which act, for the most part, in an activating mode, i.e., they enhance the bursting properties of the pyloric neurons and/or their ability to express their regenerative properties. Here we attempt to demonstrate that the pyloric motor output is also under the control of suppressive afferent inputs that are able to stop the pyloric rhythm in a long-lasting manner. Immunohistochemistry, using GABA antibodies, indicates that GABAergic-like fibers are present in both the stomatogastric ganglion and its afferent nerve. Bath-applied GABA suppresses spontaneous pyloric rhythmic activity. This is due to an inability of the pyloric pacemakers to express their bursting properties. The suppressive effect of GABA is blocked by picrotoxin and mimicked by muscimol. Isolating the pyloric neurons from all descending spiking influences with tetrodotoxin demonstrates that exogenously applied GABA acts directly on the pyloric neurons. To confirm the existence of a physiological suppressive system for the pyloric motor pattern, we show that the stimulation of an afferent nerve, known to contain GABA-like fibers, also causes the cessation of rhythmic activity and the inability of the pyloric neurons to express their bursting properties.

Animals↗

PubMed1986

Membrane labeling of the cortex of 'aging' rats by anti-fatty acid antibodies.

The cortex of 'aging' rat brain exhibits no significant difference in the lipid and fatty acid composition in comparison with that of control rat brain, except for a lowered value of the cholesterol/phospholipids ratio. However, an antiserum raised against oleic acid, specifically labels neurones of the cortex of the 'aging' rat mainly within layers IV and V. An electron microscopical study revealed that immunoreactivity was associated with cytoplasmic vesicular inclusions (lipofuscin) and with membranes. Thus, these anti-fatty acid antibodies may help in the evaluation of local modifications of membranes which are not predictable on the basis of biochemical analysis of lipids.

Aging↗

PubMed1986

Immunological similarities between an experimental autoimmune myasthenia gravis model and human myasthenia gravis.

The induction of experimental autoimmune myasthenia gravis (EAMG) in rabbits after immunization with an acetylcholine (ACh) conjugate was found to possess immunological similarities with human myasthenia gravis. Anti-ACh antibodies, present in human sera, recognized the antigenic determinant, glutarylcholine, used to raise anti-ACh antibodies in rabbits. Identification of anti-anti-ACh antibodies in MG patients enabled us to test for recognition of the anti-ACh antibodies present in rabbit sera. The reverse, the recognition of rabbit auto-anti-anti-ACh antibodies by human anti-ACh antibodies was also tested and found to be specific.

Acetylcholine↗

PubMed1986

Glycine neurons in the brain and spinal cord. Antibody production and immunocytochemical localization.

Antibodies were raised against glycine and they were specific for immunocytochemistry. Obtained from rabbits immunized with glycine conjugated to glutaryled protein-carriers, antisera were then purified by adsorption on the various glutaraldehyde-conjugated protein-carriers. Using a modified ELISA method, their specificity was determined in competition experiments between conjugated glycine and either non-conjugated glycine or other conjugated amino acids or derivatives, preincubated with anti-glycine antibodies. Calculated at half-displacement, the resulting cross-reactivity ratios showed conjugated glycine to be the best recognized compound. By revealing the presence of the majority of the glycine-containing cell bodies in the brainstem and spinal cord, immunocytochemical applications of glycine antibodies confirmed their use as specific tools for a better understanding of the role of glycine in the central nervous system.

Animals↗

PubMed1986

One perfusion mixture for immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rat brain.

A perfusion mixture composed of glutaraldehyde and allyl-alcohol at pH 12 is compatible for the fixation and immunocytochemical detection of noradrenaline, dopamine, serotonin and acetylcholine in the same rodent central nervous system. A technical description of this procedure is provided to facilitate the replication of this immunocytochemical approach with these antibodies.

Acetylcholine↗

PubMed1986

Antibodies to histamine. Specificity studies and radioimmunological assay.

Antibodies against conjugated histamine were raised in rabbits. This amine was coupled to different protein carriers by a bifunctional agent, hexamethylene diisocyanate. The specificity of the antibodies was determined with radioimmunological tests in equilibrium dialysis using an iodinated ligand: 125I-labelled histamine-hexamethylene diisocyanate-glycyl-tyrosine. The latter mimicked the antigenic determinant present in immunogens. Competition experiments were established between the radiolabelled ligand and conjugated histamine, conjugated analogs or unconjugated histamine. Cross-reactivity ratios and affinity constants were calculated from displacement curves, thereby allowing the antibody site to be characterized. The antibodies were found to be highly specific and were used for the assay of histamine in biological samples. For this, polystyrene beads coated with purified antiserum were used to establish a simple and reproducible test.

Animals↗

PubMed1986

Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior.

Embryonic dopaminergic neurons were implanted in the form of a cellular suspension in the nucleus accumbens previously deprived of its dopaminergic innervation by a local injection of 6-hydroxydopamine. The graft provided a dopaminergic reinnervation to the nucleus accumbens, the anteromedial striatum, the anteromedial frontal cortex and also, in some cases, of the septum. The pattern of reinnervation was specific for each structure and similar to the innervation provided by mesocorticolimbic dopaminergic neurons to these same structures in the normal animal. The graft restored the locomotor stimulatory action of amphetamine which was abolished in the lesioned controls. Hoarding behavior, which was disrupted following the lesion, was not reinstated by the graft alone. However, if the grafted neurons were stimulated by a small dose (0.2 mg/kg, i.p.) of (+)-amphetamine, hoarding reappeared in the grafted animals, while the same dose of amphetamine had no effect in the lesioned controls.

Animals↗

PubMed1986

Transplantation of dissociated foetal serotonin neurons into the transected spinal cord of adult rats.

Twenty adult rats underwent a complete section of the spinal cord at the lower thoracic level. One week later, 15 of them received a cell suspension obtained from raphe nuclei of 14-day-old foetuses into the distal fragment of the spinal cord. They were sacrificed after survival periods of 10-60 days, and vibratome sections of the spinal cord were processed for immunocytochemical detection of serotonin (5-HT). The control, non-transplanted animals showed a total absence of 5-HT immunoreactivity below the section, whereas the transplanted rats showed many immunoreactive 5-HT perikarya in the graft region, some at a distance of up to 10 mm, and a progressive innervation of the whole grey matter extending at least over 20 mm from the graft site.

Animals↗

PubMed1986

Detection of anti-acetylcholine antibodies in myasthenic patients.

Antibodies, directed against an acetylcholine (ACh) conjugate, were found in the sera of myasthenic patients by using a modified enzyme-linked immunosorbent assay method. Binding of sera from control patients to the conjugate was extremely low. From competition experiments, done with compounds close to ACh, a good antibody specificity for the ACh conjugate was demonstrated in the sera of myasthenic patients. Laboratory tests will be done to help a more precise diagnostic of the disease to be given. These results may also be of value in further understanding of the process of the disease itself.

Acetylcholine↗

PubMed1986

Specific antibodies against aspartate and their immunocytochemical application in the rat brain.

An immunological approach to visualize aspartate in the rat brain was attempted by raising antibodies against this acidic amino acid. Using an adapted ELISA method, their specificity was tested by competition experiments between aspartate conjugated via glutaraldehyde to various protein-carriers and either non-conjugated aspartate or conjugated amino acids, preincubated with anti-aspartate antibodies. Their titer and specificity were found high enough to allow their use in immunocytochemistry which demonstrated the presence of a large number of aspartate-containing cell-bodies in many areas of the brain.

Animals↗

PubMed1986

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies.

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammon's horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular layer. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristic pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.

Animals↗

PubMed1986

Specific detection of noradrenaline in the rat brain by using antibodies.

Noradrenaline (NA) was conjugated to different protein carriers with glutaraldehyde. During the catecholamine conjugate synthesis, precautions were taken in order to preserve the structure of NA. Anti-NA antibodies were raised in rabbits and tested using a modified enzyme-linked immunosorbent assay and immunocytochemistry. The most immunoreactive antigen was found to be the reduced NA conjugate. The octopamine conjugate was recognized by the NA antiserum 16 X less than the reduced NA conjugate. The other conjugates, dopamine, L-dihydroxyphenylalanine and adrenaline were poorly recognized by the anti-NA antibodies. A good correlation was found between results obtained using the in vitro enzyme-linked immunosorbent assay and immunocytochemistry.

Animals↗

PubMed1986

Regional specialization of the rat retina: catecholamine-containing amacrine cell characterization and distribution.

The distribution of catecholaminergic amacrine cells has been investigated in rats by means of immunohistochemical labelling of wholemounted retinas. Two groups of catecholamine-containing cells could be distinguished on the basis of their catecholamine and biosynthetic enzyme content. Both groups could be stained with an anti-tyrosine hydroxylase (TH) antiserum. The first group was composed of large, strongly TH-immunoreactive stellate amacrine cells, located principally in the innermost row of the inner nuclear layer (INL) and sending processes to the outermost sublamina of the inner plexiform layer (IPL). Some were displaced in the IPL or in the ganglion cell layer (GCL). This first group of cells can be regarded as dopaminergic since they were also stained by an anti-dopamine (DA) antiserum. The second group was composed of small, weakly TH-positive cell bodies, located slightly more sclerad within the INL. Their processes were usually not labelled with anti-TH. Identical cells could be better visualized with an anti-phenylethanolamine-N-methyltransferase (PNMT) antiserum. Their processes were observed in the middle sublamina of the IPL. A great number of these cells were displaced in the GCL. They could be regarded as epinephrine cells. Concerning the density and distribution throughout the retina a striking difference was observed between the superior and inferior halves of the retina, whereas a lower difference was observed between the nasal and temporal regions. Almost all the PNMT-immunoreactive cells were located throughout the upper retina, whereas the DA-cells were especially concentrated in the upper temporal quadrant. The distribution of the DA cells parallels that of the ganglion cells whose density is also maximal in the upper temporal retina.

Animals↗

PubMed1986

The dopaminergic innervation of the goldfish pituitary. An immunocytochemical study at the electron-microscope level using antibodies against dopamine.

The dopaminergic innervation of the goldfish pituitary gland was studied by immunocytochemistry at the electron-microscope level using highly specific antibodies against dopamine coupled to bovine serum albumin with glutaraldehyde. A satisfactory preservation of the tissue was achieved after immersion in 5% glutaraldehyde in phosphate buffer containing sodium metabisulfite to prevent oxidation of the endogenous dopamine. The immunocytochemical procedure was performed on Vibratome sections using the preembedding method. Immunoreactivity was restricted to part of the neurosecretory type-B fibers (diameter of the secretory vesicles lower than 100 nm) in which it was found to occupy the whole cytoplasm. Labeled fibers were observed within the neurohypophysis in the different parts of the gland and in the adenohypophyseal tissue where immunoreactive profiles were detected in close apposition to the different cell types. These data are in agreement with previous results obtained by means of radioautography and further support a role for dopamine in the neuroendocrine regulation of pituitary functions in teleosts.

Animals↗

PubMed1986

Comparison of serotonin and 5-methoxytryptamine immunoreactivity in rat raphe nuclei.

We studied the immunoreactivity of 5-methoxytryptamine (MT) and 5-hydroxytryptamine (HT) in the raphe region of rats using specific polyclonal antibodies and the peroxidase/anti-peroxidase (PAP) technique. Overall, the patterns of the specific staining for these two antibodies were found to be the same in this region of the rat brain. The staining reaction was considerably less intense for MT than for HT. Specificity tests were performed using HT, MT and tryptamine (T) conjugates at concentrations of 5 X 10(-8) M for antibodies to HT and 2.5 X 10(-9) M for antibodies to MT. Although the distribution of HT-like and MT-like immunoreactivity broadly overlapped, the results obtained from adsorption-specificity tests confirmed the presence of specific MT staining in the rat raphe.

5-Methoxytryptamine↗

PubMed1986

Effects of long-term ethanol consumption on GABAergic neurons in the mouse hippocampus: a quantitative immunocytochemical study.

The effects of 6 months' ethanol consumption by mice on hippocampal GABAergic neurons were investigated by means of an immunocytochemical method using GABA antibodies. Although ethanol treatment did not modify body or brain weights in our experimental conditions, two differences were observed in ethanol-treated mice, as compared to controls: a decrease in the labelling intensity of immunopositive neurons and fibers in the dorsal and the ventral parts of the hippocampus; and a decrease in the number of immunopositive neurons. This neuronal loss was statistically significant in the ventral hippocampus only, where it reached about 25% in the stratum radiatum. It is concluded that chronic ethanol consumption leads to a decrease in GABA content of hippocampal neurons and to a loss of GABAergic neurons, mostly in the ventral part of the hippocampus. These alterations in GABAergic transmission could be related to the well known functional deficits observed in chronic alcoholism.

Alcoholism↗