Biomedical subjects
Publications and source records attributed to M Geffard.
The combination of several analytical methods, i.e. chemical analysis (high performance liquid chromatography), biochemical analysis (radioimmunoassay) and immunohistochemistry, has shown that a single neuron can contain two 'classical' neurotransmitters.
Specificity of anti-serotonin antibodies to be assayed for immunocytochemical studies was tested in vitro by using a radiolabelled ligand mimicking the hapten structure. A good immunorecognition was found after coupling [3H]HT to a lysine with formaldehyde. Competition experiments by equilibrium dialysis were then carried out between [3H]HT-F-ALM and both conjugated and unconjugated indoles. The cross-reactivity ratios showed that the antibody recognition, though less specific than expected, was found suitable for immunocytochemical applications.
Three morphofunctional techniques for the detection of biogenic monoamines have been used in order to find evidence for the presence of dopaminergic neurons in the preoptic region of the goldfish. The formaldehyde-induced fluorescence technique and the immunohistochemical demonstration of tyrosine hydroxylase allowed the detection of cell bodies containing catecholamines in the ventral and lateral walls of the preoptic recess of the goldfish. Specific antibodies indicated that at least part of these perikarya contain dopamine. Evidence for the projection of these neurons to the pituitary are given. These results support the assumption that dopamine, originating from the preoptic region, may act as a gonadotrophin release-inhibiting factor in goldfish.
Dopamine was coupled to bovine serum albumin (BSA) with glutaraldehyde, precautions were taken in order to preserve the catechol ring. After injection of this immunogen into rabbits, anti-dopamine antibodies were obtained and tested using radioimmunochemical binding studies and adsorption to catecholamine covered sepharose beads. A good correlation was found between the results of the different test systems, allowing us to visualize dopamine specifically in glutaraldehyde-fixed rat brains.
As part of a follow-up study to our previous investigation of the catecholaminergic neurosecretory cells in the brain of adult female locusts (Locusta migratoria migratorioides) by means of the formaldehyde-induced fluorescence method, we have attempted to specify the identity of the amines present in these cells by an immunohistological technique. Using a recently developed anti-dopamine serum, we have demonstrated that the majority of the catecholaminergic median neurosecretory cells contain dopamine. Moreover, dopamine is present in some cell bodies of other zones of the brain, i.e. the median subocellar neurosecretory cells, perikarya in external areas of the protocerebrum, below the calyces, around the pedunculus, in the optic lobes (between the lobula and the medulla, between the medulla and the lamina), and in external zones of the tritocerebrum. Among the structured neuropils, which were particularly fluorescent in the formaldehyde-induced fluorescence method, only the pedunculus, the posterior part of the central body, the external zones of the alpha- and beta lobes and the proximal part of the lamina contain little dopamine.
Antibodies against dopamine and p-tyramine were raised in rabbits. The two catecholamines were conjugated to albumin by glutaraldehyde. The specificity of the antibodies was established by equilibrium dialysis competition experiments using an immunoreactive tritiated derivative synthesized by coupling dopamine or p-tyramine to N-alpha-acetyl-L-lysine N-methylamide with glutaraldehyde. Hence, these radiolabelled ligands mimicked the antigenic determinant of conjugated immunogens. A comparison of the data obtained showed the high specificity of each antiserum for its hapten coupled by glutaraldehyde. The anti-dopamine antibodies recognized dopamine-glutaraldehyde but not p-tyramine-glutaraldehyde. The opposite occurred for the anti-p-tyramine antibodies. A slight modification of the molecular structure provided the opportunity for a specific response against that molecule. But this difference was more important when related to the hapten region where the antibody affinity was maximal. The cross-reactivity was observed to be more important dopamine and p-tyramine than between dopamine and noradrenaline on the one hand and between p-tyramine and dopamine than p-tyramine and octopamine on the other hand.
The dopaminergic innervation of the rat lateral septum has been investigated at ultrastructural level by immunocytochemistry using the unlabelled peroxidase-anti-peroxidase method with anti-dopamine antibodies. The specificity of the reaction has been carefully checked by immunological and histochemical controls. A strong immunoreaction was observed in fibres of the lateral septum as well as in their cells of origin in the ventral tegmental area. In the lateral septum, dopamine-immunoreactive fibres were localized in two distinct areas. A first area, located ventrally in the anterior part of the septum was characterized by a high density of immunoreactive varicosities with barely visible intervaricose segments. A more dorsal area, extending throughout the anteroposterior region of the septum, was characterized by immunoreactive fibres in pericellular arrangements. Electron microscopic observations revealed no difference in the ultrastructure of dopamine-immunoreactive profiles in the different areas. Reaction product was found in vesicles, linked to microtubules and in the cytoplasm. Three types of vesicles were seen: (i) small vesicles (30-50 nm) with varying intensity of immunoreaction, filling up the varicosities; (ii) rare large clear vesicles (50-80 nm) with no internal immunoreaction; (iii) very rare large dense vesicles (50-100 nm) with a strong dopamine immunoreactivity. Labelled profiles were observed in clearly defined asymmetrical synaptic contacts with somata and dendrites. Due to the lack of previous work dealing with the use of anti-dopamine antibodies for electron microscope immunocytochemistry, our observations are compared to previous data obtained by more indirect labelling techniques.
Antibodies against gamma-aminobutyric acid (GABA)-glutaraldehyde-lysine were obtained by using a procedure based upon (i) a high yield of coupling of GABA to protein carriers, (ii) the reduction of the resulting immunoreactive double bonds, and (iii) a protocol of alternative immunizations using different immunogens having in common only the GABA-glutaraldehyde-lysine segment. This strategy led to the use of the resulting GABA antiserum without further purification. Specificity controls have been carried out with a radiolabeled ligand, [3H]GABA-glutaraldehyde- prolylphenylalanyl -lysine , which mimicked the structure of the immunogen and the fixed hapten in the tissue. Displacement curves showed that the nearest coupled analogs, beta-alanine and glycine, cross-react poorly with GABA, requiring 175-fold or 795-fold higher concentrations, respectively. Immunocytochemical results indicated that the localization obtained with this GABA antiserum largely corresponds with that reported after glutamate decarboxylase immunocytochemistry. The approach may have general applicability to other small molecules such as amino acids.
Two classes of anti-3,4- dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross-linking agent. However, anti-dopamine-glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of anti-dopamine-glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N-alpha-acetyl-L-lysine N-methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.
Using two immunocytochemical procedures (i.e., immunofluorescence and the unlabeled peroxidase-antiperoxidase method), the localization of a serotonin(HT)-like and of a N-acetylserotonin (aHT)-like immunoreactivity in the pineal organ of the pike was studied during winter. It was shown that immunostaining was exclusively restricted to the cells of the receptor line (CRL = typical and modified photoreceptors). The intensity of the reactions varied through the light-dark cycle, HT-like immunoreactivity being high during the photophase and low during the scotophase. In contrast, aHT-like immunoreactivity was highest at the beginning of the scotophase. HT and aHT-like immunoreactivities were detected in all cell types of the pineal epithelium after administration of a monoamine oxidase inhibitor. Up to now, only HT immunoreactivity could be localized at the ultrastructural level. In a number of typical and modified photoreceptors, a HT-positive staining seemed to be confined within the hyaloplasm of the inner segment, particularly with that of the perikaryon and basal pedicle. Our previous and present results strongly suggest that indole compounds, which are involved in the regulation of various neuroendocrine processes in fish, are synthetized within the CRL. Taking into account that the CRL of the pike are also photosensitive, it appears more and more likely that they are photoneuroendocrine cells involved in mediating the effects of the photoperiod on various physiological and behavioral processes.
An antibody against dopamine was raised in Rabbits by means of an immunogen constituting dopamine coupled to bovine serum albumin by formaldehyde. These antibodies were applied to frozen sections of Goldfish (Carassius auratus) brain fixed by 4% paraformaldehyde. The localisation of immunoreactive structures corresponded to regions rich in dopamine detected by histofluorescence. The specificity of this antiserum tested with the aid of dopamine analogues coupled with a carrying protein different from that of the antibody, indicates its validity as a method for the detection of dopaminergic neurons.
Synthetic arginine vasotocin (AVT) was infused into rat brains either by intraventricular administration or by local infusion on the pineal body. Subsequently, exploratory behavior was analyzed in a hole board. The behavioral effects induced by this peptide were dependent on the time of day, i. e. the light or the dark phase. High intraventricular doses (0.4 microgram) administered during the light phase altered exploratory activity such that the number of hole visits was increased, while the duration of each visit was decreased; lower doses produced no effect. In contrast, during the dark phase peripineal infusion of AVT (10(-4) pg) attenuated the number of hole visits and increased the mean duration of the visits. The strongest effects were obtained with peripineal applications during the dark phase. This treatment also resulted in significantly lowered levels of pineal melatonin.
Circadian variations in melatonin content have been studied in the pineal organ, retina and plasma of adult pigeons using radioimmunoassay. A clear-cut peak of melatonin was observed in the pineal organ at middark; this middark peak was of larger amplitude in July than in November. A nocturnal rise in melatonin concentration was also observed in the retina and plasma. We also used an immunocytochemical technique revealing the positive reactions of a melatonin-like compound in the photoreceptor cell layer (inner segments) of the pigeon's retina. Up to now, no melatonin storage has been visualized in the pineal organ of the pigeon.
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Although their body weights were decreased by about 77% and their brain weights by about 30%, high-affinity [3H] muscimol binding to a cerebral membrane fraction was not altered in hereditary pituitary dwarf mice. Marked changes in the level of pituitary growth-associated hormones do not appear to be associated with a change in cerebral GABA-receptors.
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The effect of subcutaneously injected DT gamma E (beta-endorphin, (beta E)2-17) on the pineal melatonin level was compared with that of closely related peptides and the neuroleptic drug haloperidol. As found previously, DT gamma E (3 ng/rat and 300 ng/rat) increased the melatonin levels. Similar doses of DT alpha E (beta E 2-16), DT beta E (beta E 2-31), gamma E (beta E 1-17), alpha E (beta E 1-16) and beta E failed to significantly change the melatonin levels in both the dark and the light phase. Haloperidol in a dose of 300 ng/rat exhibited a similar effect as DT gamma E.